Transmembrane proteins and related compositions and uses

Abstract

Provided herein are transmembrane proteins containing a cytokine or a functional portion thereof, such as an interleukin or a functional portion thereof. In some aspects, the disclosure further relates to engineered cells and compositions comprising the transmembrane proteins and methods for their administration to subjects. In some embodiments, the cells engineered to contain the transmembrane protein, such as immune cells, further contain a genetically engineered recombinant receptor, such as a chimeric antigen receptor (CAR). In some embodiments, features of the transmembrane proteins, engineered cells, and methods and uses thereof provide for improved treatment of diseases or disorders.

Description

TRANSMEMBRANE PROTEINS AND RELATED COMPOSITIONS AND USESCross-Reference to Related Applications

[0001] This application claims priority to United States Provisional Application No. 63 / 551941, filed February 9, 2024 and United States Provisional Application No. 63 / 733187, filed December 12, 2024, the entire contents of which is incorporated by reference herein.Field

[0002] The present disclosure relates in some aspects to transmembrane proteins containing a cytokine or a functional portion thereof, such as an interleukin or a functional portion thereof. In some aspects, the disclosure further relates to engineered cells and compositions comprising the cells, and methods for their administration to subjects. In some embodiments, the cells engineered to contain the transmembrane protein, such as immune cells, further contain a genetically engineered recombinant receptor, such as a chimeric antigen receptor (CAR). In some embodiments, features of the transmembrane proteins, engineered cells, and methods provide for improved treatment of diseases or conditions, such as by reducing adverse effects of cytokine therapy and / or increasing the persistence and / or activity of cell therapy.Background

[0003] Survival and proliferation of cells, such as immune cells, requires stimulation by cytokines. While some protocols for providing cell therapy to a subject rely on administration of cytokines to a subject, such approaches can result in considerable side effects. Improved strategies are needed to promote the surv ival, proliferation, and / or activity of immune cells, including to increase the efficacy of cell therapy treatment. Provided are transmembrane proteins, cells, compositions, and methods that meet such needs.Incorporation by Reference of Material in Sequence Listing File

[0004] This application incorporates by reference the material contained in the Sequence Listing XML file being submitted concurrently herewith: File name: NKT.099WO_ST26.xml; created on February’ 5, 2025 and is approximately 73, 973 bytes in size.Summary

[0005] Provided herein are transmembrane proteins comprising a cytokine or a functional portion thereof and a transmembrane domain. In some embodiments, the transmembrane protein comprises: (a) an extracellular domain comprising an interleukin; and (2) a transmembrane domain. Also provided herein are transmembrane proteins comprising: (a) an extracellular domain comprising an interleukin or a functional portion thereof; and (2) a transmembrane domain comprising a transmembrane portion of a receptor. In some embodiments, the transmembrane portion of a receptoris a fragment of interleukin- 15 receptor alpha (IL15Ra) or a fragment of CD25 (also known as interleukin-2 receptor alpha; IL2Ra).

[0006] Also provided herein is a transmembrane protein comprising: (a) an extracellular domain comprising an interleukin; and (2) a transmembrane domain comprising a fragment of interleukin- 15 receptor alpha (IL15Ra).

[0007] Also provided herein is a transmembrane protein comprising: (a) an extracellular domain comprising an interleukin; and (b) a transmembrane domain comprising a fragment of CD25.

[0008] In some embodiments, the interleukin is interleukin- 15 (IL15) or interleukin-2 (IL2).

[0009] Also provided herein is a transmembrane protein comprising: (a) an extracellular domain comprising interleukin- 15 (IL 15); and (b) a transmembrane domain comprising a fragment of interleukin- 15 receptor alpha (IL15Ra).

[0010] Also provided herein is a transmembrane protein comprising: (a) an extracellular domain comprising interleukin- 15 (IL 15); and (b) a transmembrane domain comprising a fragment of CD25.

[0011] In some embodiments. IL 15 is full-length IL15. In some embodiments. IL 15 is human IL 15. In some embodiments, IL 15 is full-length human IL15. In some embodiments, IL 15 comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:25. In some embodiments, IL15 comprises the amino acid sequence of SEQ ID NO:25.

[0012] In some embodiments, IL 15 is a mutated and / or truncated form of IL15. In some embodiments, IL 15 is a mutated form of IL15. In some embodiments, the mutated form of IL 15 is a mutation of human IL 15. In some embodiments, IL 15 is a truncated form of IL 15. In some embodiments, the truncated form of IL 15 is a truncation of human IL 15. In some embodiments, IL 15 comprises an amino acid substitution. In some embodiments, IL 15 comprises a K86R substitution. In some embodiments, IL 15 comprises a N112A substitution. In some embodiments, IL 15 comprises a K86R substitution and a N112A substitution. In some embodiments, IL 15 comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:46. In some embodiments, IL15 comprises the amino acid sequence of SEQ ID NO:46.

[0013] Also provided herein is a transmembrane protein comprising: (a) an extracellular domain comprising interleukin-2 (IL2); and (b) a transmembrane domain comprising a fragment of interleukin- 15 receptor alpha (IL15Ra).

[0014] Also provided herein is a transmembrane protein comprising: (a) an extracellular domain comprising interleukin-2 (IL2); and (b) a transmembrane domain comprising a fragment of CD25.

[0015] In some embodiments. IL2 is full-length IL2. In some embodiments, IL2 is human IL2. In some embodiments, IL2 is full-length human IL2. In some embodiments. IL2 comprises anamino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:44. In some embodiments, IL2 comprises the amino acid sequence of SEQ ID NO:44.

[0016] In some embodiments, IL2 is a mutated and / or truncated form of IL2. In some embodiments, IL2 is a mutated form of IL2 comprising an amino acid substitution. In some embodiments, the mutated form of IL2 is a mutation of human IL2. hi some embodiments, IL2 is a truncated form of IL2. In some embodiments, the truncated form of IL2 is a truncation of human IL2. In some embodiments, IL2 comprises an amino acid substitution. In some embodiments, IL2 comprises a L80F substitution, a R81D substitution, a L85V substitution, a I86V substitution, a I92F substitution, or any combination thereof. In some embodiments, IL2 comprises a L80F substitution. In some embodiments. IL2 comprises a R81D substitution. In some embodiments. IL2 comprises a L85V substitution. In some embodiments, IL2 comprises a I86V substitution. In some embodiments, 1L2 comprises a 192F substitution. In some embodiments, IL2 comprises a L80F substitution, a R81D substitution, a L85V substitution, a I86V substitution, and a I92F substitution. In some embodiments. IL2 comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:45. In some embodiments. IL2 comprises the amino acid sequence of SEQ ID NO:45.

[0017] In some embodiments, the extracellular domain comprises a stalk. In some embodiments, the extracellular domain comprises a IL15Ra stalk. In some embodiments, the IL15Ra stalk of the extracellular domain is C-terminal to IL15. In some embodiments, the extracellular domain does not comprise the IL15Ra sushi domain or a functional fragment thereof. In some embodiments, the extracellular domain does not comprise SEQ ID NO:61. In some embodiments, the IL15Ra stalk comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:35. In some embodiments, the IL15Ra stalk comprises the amino acid sequence of SEQ ID NO:35.

[0018] In some embodiments, the fragment of IL15Ra comprises a IL15Ra transmembrane region. In some embodiments, the fragment of IL15Ra comprises a IL15Ra intracellular region. In some embodiments, the fragment of IL15Ra comprises a IL15Ra transmembrane region and a IL15Ra intracellular region. In some embodiments, the IL15Ra transmembrane region comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:36. In some embodiments, the IL15Ra transmembrane region comprises the amino acid sequence of SEQ ID NO:36. In some embodiments, the IL15Ra intracellular region comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:37. In some embodiments, the IL15Ra intracellular region comprises the amino acid sequence of SEQ ID NO:37. In some embodiments, the IL15Ra transmembrane region comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:36, and the IL15Ra intracellular region comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:37. In some embodiments, the IL15Ratransmembrane region comprises the amino acid sequence of SEQ ID NO:36, and the IL15Ra intracellular region comprises the amino acid sequence of SEQ ID NO:37. In some embodiments, the fragment of IL15Ra comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:38. In some embodiments, the fragment of IL15Ra comprises the ammo acid sequence of SEQ ID NO:38. In some embodiments, the transmembrane protein comprises from N-terminus to C-terminus, IL15, a linker, a IL15Ra stalk, a IL15Ra transmembrane region, and a IL15Ra intracellular region. In some embodiments, the transmembrane protein comprises, from N-terminus to C-terminus: IL15, a linker, and SEQ ID NO:39.

[0019] In some embodiments, the extracellular domain comprises a linker between the interleukin and the IL15Ra stalk. In some embodiments, the extracellular domain comprises, from N- terminus to C-terminus: (I) the interleukin; (ii) a linker, and (iii) the IL15Ra stalk. In some embodiments, the extracellular domain comprises, from N-terminus to C-tenninus: (I) 1L15; (ii) a linker, and (iii) the IL15Ra stalk. In some embodiments, the extracellular domain comprises, from N- terminus to C-terminus: (i) SEQ ID NO:25; (ii) a linker; and (iii) SEQ ID NO:35. In In some embodiments, the extracellular domain consists of, from N-terminus to C-terminus: (i) SEQ ID NO:25; (ii) a linker; and (iii) SEQ ID NO:35. In some embodiments, the linker comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:21. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:21.

[0020] In some embodiments, the fragment of IL15Ra comprises a IL15Ra transmembrane region and a IL15Ra intracellular region. In some embodiments, the IL15Ra transmembrane region comprises the amino acid sequence of SEQ ID NO:36. In some embodiments, the IL15Ra intracellular region comprises the amino acid sequence of SEQ ID NO:37. In some embodiments, the IL15Ra transmembrane region comprises the amino acid sequence of SEQ ID NO:36, and the IL15Ra intracellular region comprises the amino acid sequence of SEQ ID NO:37. In some embodiments, the transmembrane domain comprising the fragment of IL15Ra comprises the amino acid sequence of SEQ ID NO:38.

[0021] Also provided herein is provided herein is a transmembrane protein comprising: (a) an extracellular domain comprising, from N-terminus to C-terminus: IL15; a linker; and an interleukin- 15 receptor alpha (IL15Ra) stalk comprising the amino acid sequence of SEQ ID NO:35; and (b) a transmembrane domain comprising a fragment of IL15ra, the fragment of IL15Ra comprising, from N-terminus to C-terminus, a IL15Ra transmembrane region comprising the amino acid sequence of SEQ ID NO:36 and a IL15Ra intracellular region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO:38.

[0022] Also provided herein is provided herein is a transmembrane protein comprising: an extracellular domain comprising, from N-terminus to C-terminus: IL15 ; a linker; and the amino acid sequence of SEQ ID NO:39.

[0023] In some embodiments, IL15 is full-length IL15. In some embodiments, IL15 is human IL15. In some embodiments, IL15 comprises the amino acid sequence of SEQ ID NO:25. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:21. In some embodiments, the extracellular domain does not comprise the IL15Ra sushi domain or a functional fragment thereof. In some embodiments, the extracellular domain does not comprise SEQ ID NO:61.

[0024] Also provided herein is a transmembrane protein comprising interleukin- 15 (IL15) and a fragment of interleukin- 15 receptor alpha (IL15Ra), wherein the transmembrane protein does not comprise the IL15Ra sushi domain or a functional fragment thereof. In some embodiments, the transmembrane protein does not comprise SEQ ID NO:61.

[0025] In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:47. In some embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:47. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:51. In some embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:51.

[0026] In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:54. In some embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:54. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:57. In some embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:57.

[0027] In some embodiments, the extracellular domain comprises a stalk. In some embodiments, the extracellular domain comprises a CD25 stalk. In some embodiments, the CD25 stalk comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:29. In some embodiments, the CD25 stalk comprises the amino acid sequence of SEQ ID NO:29.

[0028] In some embodiments, the fragment of CD25 comprises a CD25 transmembrane region. In some embodiments, the fragment of CD25 comprises a CD25 intracellular region. In some embodiments, the fragment of CD25 comprises a CD25 transmembrane region and a CD25 intracellular region. In some embodiments, the CD25 transmembrane region comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:31. In some embodiments, the CD25 transmembrane region comprises the amino acid sequence of SEQ ID NO:31. In some embodiments, CD25 intracellular region comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:32. In some embodiments. CD25 intracellular region comprises the amino acid sequence of SEQ ID NO:32. In some embodiments, the CD25 transmembrane region comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:31, and the CD25intracellular region comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:32. In some embodiments, the CD25 transmembrane region comprises the amino acid sequence of SEQ ID NO:31, and the CD25 intracellular region comprises the amino acid sequence of SEQ ID NO:32. In some embodiments, the fragment of CD25 comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:33. In some embodiments, the fragment of CD25 comprises the amino acid sequence of SEQ ID NO:33.

[0029] In some embodiments, the extracellular domain comprises a linker between the interleukin and the CD25 stalk. In some embodiments, the extracellular domain comprises, from N- terminus to C-terminus: (i) the interleukin; (ii) a linker; and (iii) the CD25 stalk. In some embodiments, the linker comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:21. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:21.

[0030] In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:34. In some embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:34. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:50. In some embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:50.

[0031] In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:53. In some embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:53. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:56. In some embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:56.

[0032] Also provided herein is a transmembrane protein comprising: (a) an extracellular domain comprising the amino acid sequence of SEQ ID NO:25; and (b) a transmembrane domain comprising the amino acid sequence of SEQ ID NO:58. In some embodiments, the extracellular domain further comprises a stalk comprising the amino acid sequence of SEQ ID NO:29.

[0033] Also provided herein is a transmembrane protein comprising: (a) an extracellular domain comprising the amino acid sequence of SEQ ID NO:25; and (b) a transmembrane domain comprising the amino acid sequence of SEQ ID NO:38. In some embodiments, the extracellular domain further comprises a stalk comprising the amino acid sequence of SEQ ID NO:35.

[0034] Also provided herein is a transmembrane protein comprising: (a) an extracellular domain comprising the amino acid sequence of SEQ ID NO:44: and (b) a transmembrane domain comprising the amino acid sequence of SEQ ID NO:58. In some embodiments, the extracellular domain further comprises a stalk comprising the amino acid sequence of SEQ ID NO:29.

[0035] Also provided herein is a transmembrane protein comprising: (a) an extracellular domain comprising the amino acid sequence of SEQ ID NO:44; and (b) a transmembrane domain comprising the amino acid sequence of SEQ ID NO:38. In some embodiments, the extracellular domain further comprises a stalk comprising the amino acid sequence of SEQ ID NO:35.

[0036] Provided herein is a nucleic acid sequence encoding the transmembrane protein according to any one of the embodiments described herein.

[0037] Provided herein is a polynucleotide comprising: (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising: (i) an extracellular antigenbinding domain; (ii) a transmembrane region; and (iii) an intracellular signaling region; and (b) a second nucleic acid sequence encoding the transmembrane protein according to any one of the embodiments described herein.

[0038] In some embodiments, the nucleic acid sequences encoding the CAR and the transmembrane protein are separated by a nucleic acid sequence encoding a ribosomal skip element. In some embodiments, the ribosomal skip element is a 2A peptide. In some embodiments, the ribosomal skip element is a T2A peptide. In some embodiments, the ribosomal skip element comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:23. In some embodiments, the ribosomal skip element comprises the amino acid sequence of SEQ ID NO:23.

[0039] In some embodiments, the extracellular antigen-binding domain binds to an antigen expressed by cells of a disease or condition. In some embodiments, the disease or condition comprises a cancer, an infectious disease, or an autoimmune disease. In some embodiments, the disease or condition comprises a cancer. In some embodiments, the disease or condition comprises an infectious disease. In some embodiments, the disease or condition comprises an autoimmune disease.

[0040] In some embodiments, the CAR does not comprise interleukin- 15 receptor alpha (IL15Ra) or a fragment thereof. In some embodiments, the transmembrane region of the CAR docs not comprise IL1 Ra or a fragment thereof. In some embodiments, the transmembrane region of the CAR comprises a CD8 alpha (CD8a) transmembrane protein or a CD28 transmembrane protein. In some embodiments, the transmembrane region of the CAR comprises a CD8 alpha (CD8a) transmembrane protein. In some embodiments, the transmembrane region of the CAR comprises a CD28 transmembrane protein.

[0041] In some embodiments, the intracellular signaling region of the CAR comprises a costimulatory signaling domain. In some embodiments, the intracellular signaling region of the CAR comprises a CD3zeta domain. In some embodiments, the intracellular signaling region of the CAR comprises a co-stimulatory signaling domain and a CD3zeta domain. In some embodiments, the costimulatory signaling domain of the CAR comprises a CD28 protein, an ICOS protein, a CD27 protein, a 4-1BB protein, an 0X40 protein, or a CD40L protein. In some embodiments, the costimulatory signaling domain of the CAR comprises a CD28 protein. In some embodiments, the co-stiniulatory signaling domain of the CAR comprises an ICOS protein. In some embodiments, the costimulatory signaling domain of the CAR comprises a CD27 protein. In some embodiments, the costimulatory signaling domain of the CAR comprises a 4-1BB protein. In some embodiments, the costimulatory signaling domain of the CAR comprises an 0X40 protein. In some embodiments, the costimulatory signaling domain of the CAR comprises a CD40L protein.

[0042] Provided herein is a polypeptide encoded by the polynucleotide according to any one of the embodiments described herein. Also provided herein is a vector comprising the nucleic acid sequence according to any one of the embodiments described herein. Also provided herein is a vector comprising the polynucleotide according to any one of die embodiments described herein. In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is a gamma retroviral vector. In some embodiments, the vector is a lentiviral vector.

[0043] Provided herein is a cell expressing the transmembrane protein according to any one of the embodiments described herein. Also provided herein is a cell expressing the nucleic acid sequence according to any one of the embodiments described herein. Also provided herein is a cell expressing the polynucleotide according to any one of the embodiments described herein. Also provided herein is a cell expressing the polypeptide according to any one of the embodiments described herein. Also provided herein is a cell expressing the vector according to any one of the embodiments described herein.

[0044] Provided herein is a cell expressing the transmembrane protein according to any one of the embodiments described herein and a recombinant receptor. In some embodiments, the recombinant receptor comprises a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, the recombinant receptor comprises a TCR. In some embodiments, the recombinant receptor comprises a CAR. In some embodiments, the recombinant receptor comprises a chimeric antigen receptor (CAR) comprising: (i) an extracellular antigen-binding domain; (ii) a transmembrane region; and (iii) an intracellular signaling region.

[0045] Provided herein is a cell expressing: (a) a chimeric antigen receptor (CAR) comprising: (i) an extracellular antigen binding domain; (ii) a transmembrane region; and (iii) an intracellular signaling region; and (b) the transmembrane protein according to any one of the embodiments described herein.

[0046] In some embodiments, the CAR does not comprise interleukin- 15 receptor alpha (IL15Ra) or a fragment thereof. In some embodiments, the transmembrane region of the CAR does not comprise IL15Ra or a fragment thereof.

[0047] In some embodiments, the cell is an immune cell. In some embodiments, the cell is a dendritic cell, macrophage, monocyte, T cell, natural killer (NK) cell. NK T cell, or B cell. In some embodiments, the cell is a dendritic cell. In some embodiments, the cell is a macrophage. In some embodiments, the cell is a monocyte. In some embodiments, the cell is a natural killer (NK) cell or a T cell. In some embodiments, the cell is a natural killer (NK) cell. In some embodiments, the cell is aprimary NK cell obtained from a donor. In some embodiments, the cell is a T cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is obtained from peripheral blood mononuclear cells (PBMCs). In some embodiments, the cell is obtained from cord blood (CB). In some embodiments, the cell is differentiated from a pluripotent stem cell (PSC). In some embodiments, the PSC is an induced PSC (iPSC). In some embodiments, the cell is derived from a stimulatory cell line. In some embodiments, the cell is an immortalized cell. In some embodiments, the cell is a K562 cell. In some embodiments, the cell is a NK92 cell.

[0048] Provided herein is a composition comprising a plurality of the cells according to any one of the embodiments described herein. Also provided herein is use of a composition comprising a plurality of the cells according to any one of the embodiments described herein, for expanding a population of immune cells.

[0049] Provided herein is a composition comprising a plurality of the cells according to any one of the embodiments described herein. In some embodiments, the plurality of cells comprises immune cells. In some embodiments, the plurality of cells comprises dendritic cells, macrophages, monocytes, T cells, natural killer (NK) cells. NK T cells. B cells, or any combination thereof. In some embodiments, the plurality of cells comprises dendritic cells. In some embodiments, the plurality of cells comprises macrophages. In some embodiments, the plurality of cells comprises monocytes. In some embodiments, the plurality’ of cells comprises natural killer (NK) cells and / or T cells. In some embodiments, the plurality- of cells comprises NK cells. In some embodiments, the plurality of cells comprises T cells. In some embodiments, the plurality of cells comprises NK and T cells. In some embodiments, the plurality of cells comprises B cells. In some embodiments, the composition comprises a pharmaceutically acceptable excipient.

[0050] Provided herein is use of the transmembrane protein according to any one of the embodiments described herein in the manufacture of a medicament for treatment of a subject having or suspected of having a disease or condition. Also provided herein is use of the nucleic acid sequence according to any one of the embodiments described herein in the manufacture of a medicament for treatment of a subject having or suspected of having a disease or condition. Also provided herein is use of the polynucleotide according to any one of the embodiments described herein in the manufacture of a medicament for treatment of a subject having or suspected of having a disease or condition. Also provided herein is use of the polypeptide according to any one of the embodiments described herein in the manufacture of a medicament for treatment of a subject having or suspected of having a disease or condition. Also provided herein is use of the vector according to any one of the embodiments described herein in the manufacture of a medicament for treatment of a subject having or suspected of having a disease or condition. Also provided herein is use of the cell according to any one of the embodiments described herein in the manufacture of a medicament for treatment of a subject having or suspected of having a disease or condition. Also provided herein is use of the composition according to any one of the embodiments described herein in the manufacture of amedicament for treatment of a subject having or suspected of having a disease or condition. Also provided herein is the use of any one of the embodiments described herein as a medicament.

[0051] Provided herein is use of the composition according to any one of the embodiments described herein for the treatment of a subject having or suspected of having a disease or condition.

[0052] Provided herein is a method of treating a subject having or suspected of having a disease or condition, the method comprising administering to the subject having or suspected of having the disease or condition the composition according to any one of the embodiments described herein.

[0053] In some embodiments, the disease or condition comprises a cancer, an infectious disease, or an autoimmune disease. In some embodiments, the disease or condition comprises a cancer. In some embodiments, the disease or condition comprises an infectious disease. In some embodiments, the disease or condition comprises an autoimmune disease. In some embodiments, the cells are allogeneic to the subject.

[0054] Provided herein is a method of producing genetically engineered cells, the method comprising introducing into a plurality of cells a polynucleotide comprising a nucleic acid sequence encoding the transmembrane protein according to any one of the embodiments described herein. Also provided herein is a method of producing genetically engineered cells, the method comprising introducing into a plurality of cells a nucleic acid sequence according to any one of the embodiments described herein. Also provided herein is a method of producing genetically engineered cells, the method comprising introducing into a plurality of cells a polynucleotide according to any one of the embodiments described herein. Also provided herein is a method of producing genetically engineered cells, the method comprising introducing into a plurality of cells a vector according to any one of the embodiments described herein. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising: (i) an extracellular antigen-binding domain; (ii) a transmembrane region; and (iii) an intracellular signaling region. In some embodiments, the nucleic acid sequences encoding the transmembrane protein and the CAR are separated by a nucleic acid sequence encoding a ribosomal skip element.

[0055] In some embodiments, the plurality of cells comprises immune cells. In some embodiments, the plurality of cells comprises dendritic cells, macrophages, monocytes, T cells, natural killer (NK) cells. NK T cells, B cells, or any combination thereof. In some embodiments, the plurality of cells comprises dendritic cells. In some embodiments, the plurality of cells comprises macrophages. In some embodiments, the plurality of cells comprises monocytes. In some embodiments, the plurality of cells comprises natural killer (NK) cells and / or T cells. In some embodiments, the plurality of cells comprises NK cells. In some embodiments, the plurality of cells comprises T cells. In some embodiments, the plurality of cells comprises NK and T cells. In some embodiments, the plurality of cells comprises B cells. In some embodiments, the plurality of cells is obtained from peripheral blood mononuclear cells (PBMCs). In some embodiments, the plurality ofcells is obtained from cord blood (CB). In some embodiments, the plurality of cells is differentiated from pluripotent stem cells (PSCs). In some embodiments, the PSCs are induced PSCs (iPSCs). In some embodiments, the plurality of cells is derived from a stimulatory' cell line. In some embodiments, the plurality of cells comprises immortalized cells. In some embodiments, the plurality of cells comprises K562 cells. In some embodiments, the plurality of cells comprises NK92 cells.

[0056] Provided herein is a composition comprising the genetically engineered cells produced by the method according to any one of the embodiments described herein.

[0057] Provided herein is a method of increasing the persistence of a cell comprising introducing into the cell a polynucleotide encoding the transmembrane protein according to any one of the embodiments described herein. Also provided herein is a method of increasing the persistence of a cell comprising introducing into the cell a nucleic acid sequence according to any one of the embodiments described herein. Also provided herein is a method of increasing the persistence of a cell comprising introducing into the cell a polynucleotide according to any one of the embodiments described herein. Also provided herein is a method of increasing the persistence of a cell comprising introducing into the cell a vector according to any one of the embodiments described herein. In some embodiments the method increases the persistence of the cell, as compared to a cell in which the polynucleotide encoding the transmembrane protein has not been introduced. In some embodiments, the method increases the persistence of the cell in the absence of interleukin-2 (IL2), as compared to a cell in the presence of IL2. In some embodiments, the persistence is in vitro persistence. In some embodiments, the persistence is ex vivo persistence. In some embodiments, the persistence is in vivo persistence.

[0058] Provided herein is a method of increasing the proliferation of a cell comprising introducing into the cell a polynucleotide encoding the transmembrane protein according to any one of the embodiments described herein. Also provided herein is a method of increasing the proliferation of a cell comprising introducing into the cell a nucleic acid sequence according to any one of the embodiments described herein. Also provided herein is a method of increasing the proliferation of a cell comprising introducing into the cell a polynucleotide according to any one of the embodiments described herein. Also provided herein is a method of increasing the proliferation of a cell comprising introducing into the cell a vector according to any one of the embodiments described herein. In some embodiments the method increases the proliferation of the cell, as compared to a cell in which the polynucleotide encoding the transmembrane protein has not been introduced. In some embodiments, the method increases the proliferation of the cell in the absence of interleukin-2 (IL2), as compared to a cell in the presence of IL2. In some embodiments, the proliferation is in vitro proliferation. In some embodiments, the proliferation is ex vivo proliferation. In some embodiments, the proliferation is in vivo proliferation.

[0059] Provided herein is a method of increasing the IL 15 signaling of a cell, the method comprising introducing into the cell a polynucleotide encoding the transmembrane protein accordingto any one of the embodiments described herein. Also provided herein is a method of increasing the IL 15 signaling of a cell, the method comprising introducing into the cell a nucleic acid sequence according to any one of the embodiments described herein. Also provided herein is a method of increasing the IL 15 signaling of a cell, the method comprising introducing into the cell a polynucleotide according to any one of the embodiments described herein. Also provided herein is a method of increasing the IL 15 signaling of a cell, the method comprising introducing into the cell a vector according to any one of the embodiments described herein. Also provided herein is a method of increasing the IL 15 signaling of a cell, the method comprising genetically engineering the cell to express the transmembrane protein according to any one of the embodiments described herein. In some embodiments, the method increases the IL 15 signaling of the cell, as compared to a cell in which the polynucleotide encoding the transmembrane protein has not been introduced. In some embodiments, the method increases the IL 15 signaling of the cell in the absence of interleukin-2 (IL2). as compared to a cell in the presence of IL2.Also provided herein is a method of increasing the IL2 signaling of a cell, the method comprising introducing into the cell a polynucleotide encoding the transmembrane protein according to any one of the embodiments described herein. Also provided herein is a method of increasing the IL2 signaling of a cell, the method comprising genetically engineering the cell to express the transmembrane protein according to any one of the embodiments described herein.

[0060] Also provided herein is a method of increasing the cytotoxicity of a cell, the method comprising introducing into the cell a polynucleotide encoding the transmembrane protein according to any one of the embodiments described herein. Also provided herein is a method of increasing the cytotoxicity of a cell, the method comprising introducing into the cell a nucleic acid sequence according to any one of the embodiments described herein. Also provided herein is a method of increasing the cytotoxicity of a cell, the method comprising introducing into the cell a polynucleotide according to any one of the embodiments described herein. Also provided herein is a method of increasing the cytotoxicity of a cell, the method comprising introducing into the cell a vector according to any one of the embodiments described herein. Also provided herein is a method of increasing the cytotoxicity of a cell, the method comprising genetically engineering the cell to express the transmembrane protein according to any one of tire embodiments described herein

[0061] In some embodiments, the cell is an immune cell. In some embodiments, the plurality of cells comprises dendritic cells, macrophages, monocytes, T cells, natural killer (NK) cells, NK T cells, B cells, or any combination thereof. In some embodiments, the plurality of cells comprises dendritic cells. In some embodiments, the plurality of cells comprises macrophages. In some embodiments, the plurality of cells comprises monocytes. In some embodiments, the immune cell is a natural killer (NK) cell or a T cell. In some embodiments, the immune cell is a NK cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a B cell.Brief Description of The Drawings

[0062] The descriptions of the figures below are related to experiments and results that represent non-limiting embodiments of the inventions disclosed herein.

[0063] Figure 1A shows non-limiting examples of membrane-bound interleukin (mbIL) constructs.

[0064] Figure IB shows non-limiting examples of constructs for bicistronic expression of both a chimeric antigen receptor (CAR) and a membrane-bound interleukin (mbIL).

[0065] Figures 2A and 2B depict cell surface expression of a chimeric antigen receptor (CAR) and interleukin- 15 (IL15) or IL15R-alpha (IL15Ra), respectively, by 293FT cells expressing a CAR and the indicated membrane-bound interleukin- 15 (mbIL15) construct.

[0066] Figures 3A and 3B depict cell surface expression of a chimeric antigen receptor (CAR) and interleukin-2 (IL2) or IL15R-alpha (IL15Ra). respectively, by 293FT cells expressing a CAR and the indicated membrane-bound interleukin-2 (mbIL2) construct.

[0067] Figure 4 shows cell surface expression of a chimeric antigen receptor (CAR) and interleukin- 15 (IL 15) by Jurkat cells expressing a CAR and the indicated membrane-bound interleukin- 15 (mbIL 15) construct.

[0068] Figure 5 shows cell surface expression of a chimeric antigen receptor (CAR) and interleukin-2 (IL2) by Jurkat cells expressing a CAR and the indicated membrane-bound interleukin-2 (mbIL2) construct.

[0069] Figure 6A shows cell surface expression of a chimeric antigen receptor (CAR) and interleukin-2 (IL2) by NK-92 cells expressing a CAR and the indicated membrane-bound interleukin- 2 (mbIL2) or membrane-bound interleukin- 15 (mbIL 15) construct.

[0070] Figure 6B shows cell surface expression of a chimeric antigen receptor (CAR) and CD56 by NK-92 cells expressing a CAR and the indicated membrane-bound interleukin-2 (mbIL2) or membrane-bound interleukin- 15 (mbIL15) construct approximately three weeks after IL2 withdrawal from culture media.

[0071] Figure 7 shows cell surface expression of a chimeric antigen receptor (CAR) and CD56 by primary natural killer (NK) cells expressing a CAR and the indicated membrane-bound interleukin-2 (mbIL2) or membrane -bound interleukin- 15 (mbIL 15) construct.

[0072] Figure 8A shows cell surface expression of a chimeric antigen receptor (CAR) and IL15R-alpha (IL15Ra) by primary' natural killer (NK) cells expressing a CAR and the indicated membrane-bound interleukin-2 (mbIL2) or membrane-bound interleukin- 15 (mbIL15) construct.

[0073] Figure 8B shows cell surface expression of a chimeric antigen receptor (CAR) and interleukin- 15 (IL15; top panels) or interleukin-2 (IL2; bottom panels) by primary natural killer (NK) cells expressing a CAR and the indicated membrane-bound interleukin-2 (mbIL2) or membranebound interleukin- 15 (mbIL 15) construct.

[0074] Figure 9A shows cell surface expression of a chimeric antigen receptor (CAR) and CD56 by primary natural killer (NK) cells expressing a CAR and the indicated membrane-bound interleukin- 15 (mbIL15) construct approximately one week after IL2 withdrawal from culture media.

[0075] Figure 9B shows cell surface expression of a chimeric antigen receptor (CAR) and CD56 by primary natural killer (NK) cells expressing a CAR and the indicated membrane-bound interleukin-2 (mbIL2) construct approximately one week after IL2 withdrawal from culture media.

[0076] Figure 10 shows cell surface expression of a chimeric antigen receptor (CAR) and interleukin-2 (IL2) or interleukin- 15 (IL 15) by primary natural killer (NK) cells expressing a CAR and the indicated membrane-bound interleukin-2 (mbIL2) or membrane -bound interleukin- 15 (mbIL15) construct, respectively, approximately one week after IL2 withdrawal from culture media.

[0077] Figure 11 shows cell surface expression of a chimeric antigen receptor (CAR) and lL15R-alpha (lL15Ra) by primary natural killer (NK) cells expressing a CAR and the indicated membrane-bound interleukin-2 (mbIL2) or membrane-bound interleukin- 15 (mbIL15) construct approximately one week after IL2 withdrawal from culture media.

[0078] Figures 12A and 12B show expression of Bcl2 and Mell, respectively, by primary natural killer (NK) cells expressing a chimeric antigen receptor (CAR) and the indicated membranebound interleukin- 15 (mbIL15) construct one week after IL2 withdrawal.

[0079] Figures 12C and 12D show expression of Bcl2 and Mell , respectively, by primary natural killer (NK) cells expressing a chimeric antigen receptor (CAR) and the indicated membranebound interleukin- 15 (mbIL15) construct two weeks after IL2 withdrawal.

[0080] Figures 13A and 13B show expression of phospho-STAT5 and phospho-S6, respectively, by primary' natural killer (NK) cells expressing a chimeric antigen receptor (CAR) and the indicated membrane-bound interleukin- 15 (mbIL15) construct one week after IL2 withdrawal.

[0081] Figures 13C and 13D show expression of phospho-STAT5 and phospho-S6, respectively , by primary natural killer (NK) cells expressing a chimeric antigen receptor (CAR) and the indicated membrane-bound interleukin- 15 (mbIL15) construct two weeks after IL2 withdrawal.

[0082] Figures 14A and 14B show expression of perforin and granzyme B, respectively, by primary natural killer (NK) cells expressing a chimeric antigen receptor (CAR) and the indicated membrane-bound interleukin- 15 (mbIL15) construct one week after IL2 withdrawal.

[0083] Figures 14C and 14D show expression of perform and granzyme B, respectively, by primary natural killer (NK) cells expressing a chimeric antigen receptor (CAR) and the indicated membrane-bound interleukin- 15 (mbIL15) construct two weeks after IL2 withdrawal.

[0084] Figures 14E and 14F show the percentage of primary natural killer (NK) cells expressing the CD19 chimeric antigen receptor (CAR) and membrane-bound interuleukin-15 (mbIL15). respectively, at 2, 7. and 14 days post-transduction.

[0085] Figure 14G shows the expansion of primary’ natural killer (NK) cells expressing the CD 19 chimeric antigen receptor (CAR) and indicated membrane-bound interuleukin-15 (mbIL15) constructs at 8 days post-transduction.

[0086] Figures 14H and 141 show the cell counts of primary natural killer (NK) cells expressing the indicated mbIL15 construct, alone or in combination with a CD19 chimeric antigen receptor (CAR) at 8 and 15 days post-transduction, in the presence or absence of interleukin-2 (IL2), respectively.

[0087] Figure 14J shows the cytotoxicity of primary' NK cells expressing the indicated mbIL15 construct, alone or in combination with a CD19 chimeric antigen receptor (CAR), against Nalm6 target cells when the NK cells were cultured with target cells at an effector-to-target (E:T) ratio of 1:1.

[0088] Figure 15A shows the percentage of primary natural killer (NK) cells from a representative donor expressing a chimeric antigen receptor (CAR) at various time points following transduction (MOI indicated as 1, 3 or 5).

[0089] Figure 15B shows the mean fluorescence intensity (MFI) of CAR expression by primary natural killer (NK) cells from a representative donor at various time points following transduction (MOI indicated as 1. 3. or 5).

[0090] Figure 15C shows the percentage of primary natural killer (NK) cells from a representative donor expressing a chimeric antigen receptor (CAR) at 18 days post-transduction in the presence or absence of interleukin-2 (IL2) (MOI of 3).

[0091] Figure 15D shows the mean fluorescence intensity (MFI) of CAR expression by primary’ natural killer (NK) cells from a representative donor at 18 days post-transduction in the presence or absence of interleukin-2 (IL2) (MOI of 3).

[0092] Figure 16 shows cell surface expression of a chimeric antigen receptor (CAR) and IL15R-alpha (IL15Ra) by primary' natural killer (NK) cells expressing a CAR and the indicated membrane-bound interleukin- 15 (mbIL15) construct.

[0093] Figure 17A shows the percentage of primary natural killer (NK) cells from a representative donor expressing IL15 at 27 days post-transduction in the presence or absence of interleukin-2 (IL2) (MOI of 3).

[0094] Figure 17B shows the mean fluorescence intensity (MFI) of IL 15 expression by primary natural killer (NK) cells from a representative donor at 27 days post-transduction in the presence or absence of interleukin-2 (IL2) (MOI of 3).

[0095] Figure 18 shows the fold expansion of primary NK cells expressing a CAR and membrane-bound interleukin- 15 (mbIL15) construct at 7 days post-transduction (MOI of 3).

[0096] Figures 19A and 19B show the number of live cells (e6 / mL) at 7, 14. 18, and 27 days post-transduction in the presence or absence of interleukin-2 (IL2). respectively.

[0097] Figure 20A shows the cytotoxicity of primary’ NK cells expressing a CAR and membrane-bound interleukin- 15 (mbIL15) construct against Daudi target cells when the NK cells were cultured with target cells at an effector -to-target (E:T) ratio of 1:2.

[0098] Figure 20B shows the cytotoxicity of primary NK cells expressing a CAR and membrane-bound interleukin- 15 (mbIL15) construct against Daudi target cells when the NK cells were cultured with target cells at an effector-to-target (E:T) ratio of 1:4.

[0099] Figure 21A shows the percentage of primary NK cells from a representative donor expressing a CAR at 21. 27. and 36 days post-transduction in the presence or absence of interleukin-2 (IL2) (MOI of 3).

[0100] Figure 21B shows the percentage of primary NK cells from a representative donor expressing IL15 at 21. 27, and 36 days post-transduction in the presence or absence of interleukin-2 (IL2) (MOI of 3.

[0101] Figure 22 shows the fold expansion of primary NK cells expressing a CAR and membrane-bound interleukin- 15 (mbIL15) construct at 7 days post-transduction.

[0102] Figures 23A and 23B show the number of live cells (e6 / mL) at 7. 13, 21, 28, and 36 days post-transduction in the presence or absence of interleukin-2 (IL2), respectively.

[0103] Figure 24A shows the cytotoxicity’ of primary NK cells expressing a CAR and membrane -bound interleukin- 15 (mbIL15) construct against HL60 target cells when the NK cells were cultured with target cells at an effector-to-target (E:T) ratio of 1 : 1.

[0104] Figure 24B shows the cytotoxicity’ of primary NK cells expressing a CAR and membrane -bound interleukin- 15 (mbIL15) construct against HL60 target cells when the NK cells were cultured with target cells at an effector-to-target (E:T) ratio of 1 :2.Detailed Description

[0105] Provided herein are transmembrane proteins, including those containing an extracellular cytokine or a functional portion thereof and a transmembrane domain. In some embodiments, the cytokine or functional portion thereof and the transmembrane domain are joined (e.g., directly or indirectly) by a stalk. In some embodiments, the cytokine is an interleukin, such as interleukin-2 (IL2) or interleukin- 15 (IL 15), or a functional portion thereof. Thus, in some embodiments, the transmembrane proteins are membrane-bound interleukins. In some embodiments, the transmembrane domain of the transmembrane protein is a transmembrane domain of CD25 (also known as the alpha chain of the interleukin-2 receptor; IL2Ra) or interleukin- 15 receptor alpha (IL15Ra), such as a transmembrane and intracellular region thereof. In some embodiments, the transmembrane protein is a membrane -bound interleukin-2 (mbIL2) or a membrane -bound interleukin- 15 (mbIL15). Also provided arc compositions and engineered cells containing the transmembrane proteins and methods of administering such compositions and cells.

[0106] Administration of various peptides such as cytokines and chemokines can be used to treat diseases and conditions such as cancer. However, certain such treatments, such as those involving systemic administration of IL2, can be associated with toxic side effects including organ dysfunction and death. Such side effects can arise from activity of the molecules at sites other than cells or tissues of tire disease or condition, e.g., cancer cells. Accordingly, current methods may not be entirely satisfactory, and improved safety and efficacy are needed.

[0107] In some embodiments, the transmembrane proteins provided herein (e.g., membrane-bound interleukins) bind to and / or activate a receptor expressed by the same cell expressing the transmembrane protein and / or a different cell. Thus, in some embodiments, the transmembrane protein (e.g., membrane-bound interleukin) binds to and / or activates a receptor expressed on the cell expressing the transmembrane protein to induce cis signaling. In some embodiments, the transmembrane protein (e.g., membrane -bound interleukin) binds to and / or activates a receptor expressed on a different cell than the cell expressing the transmembrane protein to induce trans signaling. The transmembrane protein may induce both cis and trans signaling. In some cases, the signaling effected by the transmembrane protein (e.g., membrane-bound interleukin) increases the proliferation, survival, and / or activation of target cells (e.g., immune cells).

[0108] In some aspects, cells (e.g., immune cells) engineered to express the provided transmembrane proteins exhibit improved survival, proliferation, and / or cytotoxicity against target cells, as compared to cells not expressing a transmembrane protein or expressing alternative transmembrane proteins. In some respects, the provided embodiments may allow for increased survival and expansion of immune cells, including for use in cell therapies.

[0109] In some aspects, the transmembrane protein is a membrane -bound interleukin-2 (mbIL2). In some cases, the transmembrane domain of the mbIL2 is a CD25 transmembrane domain or a IL15Ra transmembrane domain. For example, in some aspects, the transmembrane protein contains (a) an extracellular domain containing IL2 (e.g., full-length IL2) and a CD25 stalk; and (b) a transmembrane domain containing CD25 transmembrane and intracellular signaling regions. Alternatively, in some aspects, the transmembrane protein contains (a) an extracellular domain containing IL2 (e.g., full-length IL2) and a IL15Ra stalk; and (b) a transmembrane domain containing IL15Ra transmembrane and intracellular signaling regions.

[0110] In some cases, the transmembrane protein is a membrane-bound interleukin- 15 (mbIL15). In some cases, the transmembrane domain of the mbIL15 is a CD25 transmembrane domain or a IL15Ra transmembrane domain. For example, in some aspects, the transmembrane protein contains (a) an extracellular domain containing IL15 (e.g., full-length IL15) and a CD25 stalk; and (b) a transmembrane domain containing CD25 transmembrane and intracellular signaling regions. Alternatively, in some aspects, the transmembrane protein contains (a) an extracellular domain containing IL15 (e.g., full-length IL15) and a IL15Ra stalk: and (b) a transmembrane domain containing IL15Ra transmembrane and intracellular signaling regions.

[0111] The development, differentiation, survival, immune responses, and proliferation of immune cells are regulated by a host of cytokines, including IL2, IL4, IL7, IL9, IL 15, and IL21. Cytokines are therefore potential therapeutic targets for modulation of immune responses, including for mediating the survival, expansion, and persistence of immune cells.

[0112] IL2 is a short-chain type I cytokine primarily produced by antigen-activated T cells and binds to the high-affinity IL2 receptor (IL2R). The IL2R has three subunits: IL2Ralpha (IL2Ra; also known as CD25), IL2Rbeta (IL2Rb), and IL2Rgamma (also known as gamma-c or CD 132). Further, there are three classes of IL2 receptors: low-affinity receptors contain only IL2Ra; intermediate-affinity receptors contain IL2Rb and IL2Rg; and high-affinity receptors contain all three subunits (Mitra and Leonard, J Leukocyte Biol (2018) 103(4):643-55; Malek et al., Immunity (2010) 33(2): 153-65). IL2 production is highly regulated and generally restricted to activated CD4+ T cells, though other cells such as activated CD8+ T cells, NK T cells, and dendritic cells have been reported to secrete low levels of IL2. IL2 mainly signals in cis. but can also signal in trans to neighboring cells. IL-2 has a wide range of actions, including the ability to boost the cytolytic activity of natural killer (NK) or lymphokine-activated killer cells, increase the cytolytic activity of tumor-infiltrating lymphocytes (TILs), augment immunoglobulin production by activated B cells, maintain homeostatic proliferation of regulatory T cells (Tregs), act on innate lymphoid cells, and modulate effector T cell differentiation (Mitra and Leonard 2018).

[0113] Use of IL2 as an agonist to bind the IL2R and modulate immune responses therapeutically has been tested for autoimmune disease, transplantations, inflammatory conditions, infections, and cancer. However, its success has been limited due to a short in vivo half-life, toxicity, and its ability to stimulate Tregs. (Bendickova and Fric, J Leukocyte Biol (2020) 108(l):427-37).

[0114] IL 15 is a 14-15 kDa cytokine from the IL2 family and has many functions in regulating adaptive and innate immune responses similar to that of IL2. IL15 signals through the IL2R, including IL2Rb and IL2Rg, but not IL2Ra. IL 15 is primarily secreted from dendritic cells, macrophages, and monocytes. The IL15R-alpha (IL15Ra) is expressed by T cells, NK cells, NK T cells, B cells, dendritic cells, monocytes, and macrophages. After IL15 binds to IL15Ra, the IL15 / IL15Ra complex binds to the IL2R / IL15Rb and IL2R / IL15R-g heterodimer expressed on effector T, B, and NK cells to promote their survival, maturation, and cytotoxicity. The biological function of IL15 is attributed to the mediation of the Sushi domain (1-65 amino acids) contained in the IL15Ra and is responsible for interacting with IL15. Like IL2, challenges associated with therapeutic use of IL15 include low biological potency and short half-life. To overcome these challenges, some IL15 superagonists formed by IL15 and the Sushi domain of soluble IL15Ra have been developed (Zhang et al., International Immunopharmacol (2021) 91:107318). In some aspects, however, a transmembrane protein provided herein does not comprise the Sushi domain of IL15Ra (e.g., does not comprise SEQ ID NO:61). Thus, in some embodiments, the transmembrane protein does not comprise full-length IL15Ra. In particular, it is contemplated herein that a transmembraneprotein incorporating a transmembrane domain derived from IL15Ra, but not the Sushi domain of IL15Ra, may prolong IL15 signaling. For example, the inventors contemplate that, in the absence of being bound to the Sushi domain of IL15Ra, a IL15Ra-derived transmembrane domain may participate in intracellular internalization, thereby increasing IL15-mediated signaling.

[0115] In some embodiments, the transmembrane protein is provided on or in combination with cells encoding a recombinant receptor, such as a chimeric antigen receptor (CAR), that contains an antigen-binding domain (e.g.. antibody or antibody fragment) that provides specificity for a desired antigen, a transmembrane domain, and an intracellular signaling region containing, for example, a T cell activating domain providing a primary activation signal (e.g., CD3zeta). In some embodiments, the antigen-binding domain comprises an extracellular domain of a receptor. In such embodiments, the antigen is a cognate ligand of the receptor.

[0116] In some embodiments, the transmembrane protein and the CAR are encoded by the same polynucleotide and separated by a ribosomal skip element, such that they are bicistronically (separately) expressed by a cell introduced with the polynucleotide. In some embodiments, the transmembrane domain of the transmembrane protein and the transmembrane region of the CAR share less than about 30%, 25%, 20%, 15%. 10%. 5%, or less amino acid sequence identity. In some embodiments, the transmembrane domain of the transmembrane protein and the transmembrane region of the CAR are encoded by nucleic acid sequences that share less than about 30%, 25%, 20%, 15%, 10%, 5%. or less sequence identity. In some aspects, the transmembrane domain of the transmembrane protein is derived from a different protein than the transmembrane region of the CAR. For example, in some embodiments, the transmembrane domain of the CAR is derived from CD8 or CD28, whereas the transmembrane domain of the transmembrane protein does not comprise CD8, CD28, or any portion thereof (e.g., the transmembrane domain of tire transmembrane protein comprises CD25, IL15Ra, or a fragment thereof). It is contemplated herein that such embodiments may reduce the potential for homologous recombination of nucleic acid sequences encoding the transmembrane protein and the CAR, thereby increasing expression of the transmembrane protein and / or the CAR, as compared to embodiments in which the transmembrane domain of the transmembrane protein and the transmembrane region of the CAR are derived from the same protein (e.g., CD8). It is further contemplated herein that, by virtue of the transmembrane protein and the CAR having transmembrane domains derived from different proteins, the potential for heterodimerization of the transmembrane protein and CAR may be reduced. For example, a transmembrane protein and a CAR both having transmembrane domains derived from CD8 may allow for heterodimerization of the transmembrane protein and CAR. By contrast, a transmembrane protein having a transmembrane domain derived from CD25 or IL15Ra and a CAR having a transmembrane domain derived from e.g., CD8 or CD28, reduce the potential for heterodimerization of the transmembrane protein and CAR.

[0117] In some embodiments, the transmembrane protein and the CAR are not bicistronically encoded. In some embodiments, the transmembrane protein and tire CAR are encoded by different polynucleotides, such that they are not bicistronically expressed by a cell introduced with the polynucleotides. For example, in some embodiments, the transmembrane protein is encoded by a first polynucleotide comprised within a first vector, and the CAR is encoded by a second polynucleotide comprised within a second vector.

[0118] In some embodiments, the provided transmembrane proteins and recombinant receptors, when genetically engineered into cells can modulate the activation and / or stimulation of immune cells (e.g.. NK and / or T cells), thereby resulting in genetically engineered cells with improved survival and / or persistence in vivo, such as for use in cell therapy methods. Thus, also provided are cells, such as cells that contain the transmembrane proteins and / or an engineered recombinant receptor, such as described herein.

[0119] In some aspects, the transmembrane proteins and recombinant receptors can be expressed in cells to produce genetically engineered immune cells (e.g., NK and / or T cells), that, when administered to a subject, exhibit one or more properties that are improved compared to a reference cell composition that does not express the transmembrane protein. In some instances, the increased survival, proliferation, and / or cytotoxicity is greater than that effected by a reference composition comprising an engineered cell not expressing the transmembrane protein. Thus, in some cases, properties of administered genetically engineered cells that can be improved or increased compared to administered cells of a reference composition include increased survival, persistence, proliferation, activation, and / or killing of target cells.

[0120] In some embodiments, engineered cells containing the transmembrane proteins exhibit increased survival and / or persistence compared to cells not engineered to compared to cells not containing the transmembrane protein, including in the absence of IL2. In some aspects, the persistence of engineered cells in the absence of IL2 is increased at least or about at least 1.5 -fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, or more.

[0121] In some embodiments, engineered cells containing the transmembrane proteins and a recombinant receptor exhibit increased survival and / or persistence compared to cells not engineered to compared to cells comprising a recombinant receptor but not the transmembrane protein. In some embodiments, a genetically engineered cell with increased persistence exhibits better potency in a subject to which it is administered. In some aspects, the persistence of administered cells is increased at least or about at least 1.5-fold, 2-fold, 3-fold, 4-fold. 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 20-fold, 30-fold. 40-fold. 50-fold, 100-fold, or more. In some aspects, the cytotoxicity of administered cells is increased at least or about at least 1.5-fold. 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold. 8-fold, 9-fold. 10-fold. 20-fold. 30-fold. 40-fold, 50-fold, 100-fold, or more.

[0122] In some embodiments, the degree or extent of persistence of administered cells can be detected or quantified after administration to a subject. For example, in some aspects, quantitative PCR (qPCR) is used to assess the quantity of cells expressing the recombinant receptor (e.g., CAR-expressing cells) in the blood or serum or organ or tissue (e.g., disease site) of the subject. In some aspects, persistence is quantified as copies of DNA or plasmid encoding the receptor, e.g., CAR, per microgram of DNA. or as the number of receptor-expressing, e.g., CAR-expressing, cells per microliter of the sample, e.g., of blood or serum, or per total number of peripheral blood mononuclear cells (PBMCs), white blood cells, or NK cells per microliter of the sample. In some embodiments, flow cytometric assays detecting cells expressing the receptor generally using antibodies specific for the receptors also can be performed. Cell-based assays may also be used to detect the number or percentage of functional cells, such as cells capable of binding to and / or neutralizing and / or inducing responses, e.g., cytotoxic responses, against cells of the disease or condition or expressing the antigen recognized by the receptor. In any of such embodiments, the extent or level of expression of another marker associated with the recombinant receptor (e.g. CAR- expressing cells) can be used to distinguish the administered cells from endogenous cells in a subject.

[0123] Also provided are methods and uses of the transmembrane proteins and engineered cells expressing the same, such as for use in cell therapy. Further provided are methods for engineering, preparing, and producing the transmembrane proteins and cells, compositions containing the transmembrane proteins and cells, and kits and devices containing and for using, producing, and administering the composition or cells. Also provided are methods, compounds, and compositions for producing the engineered cells. Provided are nucleic acids, such as constructs, e.g. viral vectors encoding the transmembrane proteins and / or genetically engineered recombinant receptors, and methods for introducing such nucleic acids into cells, such as by transduction. Also provided are compositions containing the transmembrane proteins and engineered cells, and methods, kits, and devices for administering the cells and compositions to subjects, such as for cell therapy. In some aspects, the cells are isolated from a subject, engineered, and administered to the same subject (e.g., autologous). In some aspects, the cells are isolated from one subject (e.g., a donor), engineered, and administered to another subject (e.g., allogeneic).I. Transmembrane Proteins

[0124] Provided in some embodiments are transmembrane proteins, such as those containing an extracellular domain (e.g., containing a cytokine or a functional portion and / or mutein thereol) and a transmembrane domain (e.g., CD8, CD25, IL15Ra, or a fragment thereof). In some embodiments, the extracellular domain contains an interleukin or a functional portion thereof. In some embodiments, the extracellular domain contains an interleukin or a mutcin thereof. In some embodiments, the interleukin is IL2 or IL15, such as full-length IL2 or full-length IL15. In some embodiments, the extracellular domain also contains a stalk region (e.g., a CD8, CD25, or IL15Rastalk). In some embodiments, the cytokine or functional portion thereof and the stalk region are joined by a linker. In some embodiments, the transmembrane domain comprises a transmembrane region and / or an intracellular region. In some embodiments, the transmembrane domain comprises a transmembrane region and an intracellular region. In some embodiments, the transmembrane protein comprises, from N-lenninus to C -terminus: the extracellular domain and the transmembrane domain. Thus, in some aspects, the transmembrane protein comprises, from N-tenninus to C-terminus: a cytokine (e.g.. an interleukin) or functional portion thereof, a linker, a stalk, a transmembrane region, and an intracellular region.

[0125] In some embodiments, the term “functional portion” may mean a sufficient portion of a cytokine (e.g.. an interleukin) or a subunit of a cytokine that is capable of binding to its cognate receptor to transduce its signal and effect immunomodulation activity. A functional portion typically contains at least or about at least 30%, 40%, 50%. 60%. 70%, 80%, 90%, 95%, or more of the full-length sequence, such as the full-length mature sequence lacking the signal peptide. Alternatively, or additionally, a functional portion of a cytokine (e.g., a “mutein”) may contain a substitution in an amino acid residue as compared to a native sequence of the cytokine. For example, a functional portion of an interleukin may contain one or more amino acid residue substitution(s) compared to the native sequence of the interleukin.

[0126] In some embodiments, the transmembrane proteins are expressed by cells. Thus, provided are cells expressing such transmembrane proteins. In some embodiments, the provided cells expressing a transmembrane protein exhibit increased or decreased activity to stimulate or suppress the activity or response of a cognate receptor compared to a reference cell, wherein the reference cell is a cell not containing the transmembrane protein, or containing an alternative transmembrane protein. In some embodiments, the provided transmembrane proteins exhibit increased or decreased binding affinity for their cognate receptor, compared to alternative transmembrane proteins. For example, in some embodiments, the provided transmembrane protein contains one or more amino acid substitutions that increase binding of the cytokine or functional portion thereof to its cognate receptor as compared to a transmembrane protein not having the one or more amino acid substitutions. In some embodiments, the provided transmembrane protein contains one or more amino acid substitutions that reduce or prevent ubiquitination and / or glycosylation of the cy tokine or functional portion thereof as compared to a transmembrane protein not having the one or more amino acid substitutions. In some embodiments, the provided transmembrane protein contains one or more amino acid substitutions that the stability of the transmembrane protein as compared to a transmembrane protein not having the one or more amino acid substitutions.A. Extracellular Domain

[0127] Provided herein are transmembrane proteins comprising (a) an extracellular domain comprising a cytokine or functional portion thereof: and (b) a transmembrane domain. In some embodiments, the extracellular domain comprises a cytokine (e.g.. interleukin) or functionalportion thereof. In some embodiments, the extracellular domain comprises a cytokine (e.g., interleukin) or functional portion thereof and a stalk. In some embodiments, the extracellular domain comprises, from N-terminus to C-tenninus: a cytokine (e.g., interleukin) or functional portion thereof and a stalk. In some aspects, the extracellular domain comprises a linker between the cytokine or functional portion thereof and the stalk. Thus, in some embodiments, the extracellular domain comprises, from N-terminus to C-terminus: a cytokine (e.g., interleukin) or functional portion thereof, a linker, and a stalk. In some embodiments, the cytokine or functional portion thereof binds to its cognate receptor on a cell (e.g., an immune cell). In some embodiments, the cytokine or functional portion thereof binds to its cognate receptor in cis and / or in trans. i. Cytokines

[0128] In some embodiments, the cytokine or functional portion thereof is a cytokine (e.g., interleukin, interferon, and / or chemokine) that binds to a receptor on an immune cell, such as a NK cell, B cell, T cell, macrophage, dendritic cell, monocyte, or other immrme cell. In some embodiments, the cytokine is an interleukin. In some embodiments, the cytokine is an interferon. In some embodiments, the cytokine is a chemokine.

[0129] In some embodiments, the cytokine is selected from IL15, IL2, ILla, ILlb, ILIRa, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL17, IL18, IL21, IL22, IL23, IL25, IL27, IL32, IL33, BNDFCD40L, CNTF, CT1, G-CSF, GM-CSF, IFNa, IFNb, IFNg, leukemia inhibitory factor, leptin, M-CSF, MIF, MIP-la, MIP-lb, MCP-1, TGFb, TNFa, VEGF, or a functional portion thereof. In some embodiments, the cytokine is a chemokine or an interleukin. In some embodiments, the cytokine or functional portion thereof is an interleukin. In some embodiments, the interleukin is selected from IL15, IL2, ILla, ILlb, IL3, IL4, IL5, IL6, IL7, IL9, IL10, IL11, IL12a, IL12b, IL13, IL17, IL18, IL21. IL22, IL23, and IL27. In some embodiments, the interleukin is selected from IL15, IL2, IL12a, IL12b, IL18, IL21. or any combination thereof. In some embodiments, the interleukin is IL 15 or a functional portion thereof. In some embodiments, the interleukin is IL 15. In some embodiments, the interleukin is IL2 or a functional portion thereof. In some embodiments, the interleukin is IL2. In some embodiments, the interleukin is IL6 or a functional portion thereof. In some embodiments, the interleukin is IL6. In some embodiments, the interleukin is IL12a or a functional portion thereof. In some embodiments, the interleukin is IL12a. In some embodiments, the interleukin is IL 12b or a functional portion thereof. In some embodiments, the interleukin is IL 12b. In some embodiments, the interleukin is IL 18 or a functional portion thereof. In some embodiments, the interleukin is IL 18. In some embodiments, the interleukin is IL21 or a functional portion thereof. In some embodiments, the interleukin is TL21. In some embodiments, the interleukin is mammalian, such as human. Thus, in some embodiments, the interleukin is a human interleukin. Non-limiting examples of cytokines of a provided transmembrane protein are set forth in Table 1. In some embodiments, the cytokine (e.g., interleukin) or functional portion thereof does not contain a signal peptide or propeptide.

[0130] In some embodiments, the interleukin or functional portion thereof comprises the amino acid sequence of any one of SEQ ID NOS:25, 44, and 62-66, or an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequency identity to any one of SEQ ID NOS: 25, 44, and 62-66.

[0131] In some embodiments, the cytokine is an interleukin or a functional portion thereof. Thus, in some aspects, the extracellular domain comprises an interleukin or a functional portion thereof. In some aspects, the extracellular domain comprises an interleukin.

[0132] In some embodiments, the interleukin is interleukin- 15 (IL15). In some embodiments, IL15 is human IL15. In some embodiments, IL15 is full-length IL15. hi some embodiments, IL 15 is full-length, human IL15. In some embodiments, IL 15 comprises the amino acid sequence set forth in SEQ ID NO:25.

[0133] In some embodiments, IL15 is a mutated (e.g., “mutein”) and / or truncated fonn of IL15. In some embodiments, IL 15 is a mutated form of IL 15 comprising an amino acid substitution. In some embodiments, IL 15 is a truncated form of IL 15. In some embodiments. IL 15 comprises an amino acid substitution. In some embodiments, IL 15 comprises a K86R substitution. In some embodiments, IL15 comprises a N112A substitution. In some embodiments. 1L15 comprises a K86R substitution and a N112A substitution. In some embodiments, IL 15 comprises the amino acid sequence of SEQ ID NO:46, or an ammo acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%. 95%. 96%. 97%. 98%. 99%. or more sequency identity to SEQ ID NO:46. In some embodiments, IL 15 comprises the amino acid sequence of SEQ ID NO:46.

[0134] In some embodiments, IL 15 is a truncated version of the native IL 15 sequence (e.g., SEQ ID NO:25). Thus, in some embodiments, IL15 comprises an amino acid sequence having at least about 50%. at least about 60%, at least about 70%. at least about 80%, or at least about 90%, at least about 91%, at least about 92%. at least about 93%, at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:25.

[0135] In some embodiments, the interleukin is interleukin-2 (IL2). In some embodiments, IL2 is human IL2. In some embodiments, IL2 is full-length IL2. In some embodiments, IL2 is full-length, human IL2. In some embodiments, IL2 comprises the amino acid sequence set forth in SEQ ID NO:44.

[0136] In some embodiments, IL2 is a mutated and / or truncated form of IL2. In some embodiments, IL2 is a mutated form of IL2 comprising an amino acid substitution. In some embodiments, IL2 is a truncated form of IL2. In some embodiments, IL2 comprises an amino acid substitution. In some embodiments, IL2 comprises a L80F substitution, a R81D substitution, a L85V substitution, a I86V substitution, a I92F substitution, or any combination thereof. In some embodiments, IL2 comprises a L80F substitution. In some embodiments, IL2 comprises a R81D substitution. In some embodiments, IL2 comprises a L85V substitution. In some embodiments, IL2 comprises a I86V substitution. In some embodiments, IL2 comprises a I92F substitution. In some embodiments. IL2 comprises a L80F substitution, a R81D substitution, a L85V substitution, a I86V substitution, and a I92F substitution. In some embodiments, IL2 comprises the amino acid sequence of SEQ ID NO:45, or an amino acid sequence that has at least 85%, 86%. 87%. 88%. 89%. 90%.91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequency identity to SEQ ID NO:45. In some embodiments, IL2 comprises the amino acid sequence of SEQ ID NO:45.

[0137] In some embodiments, IL2 is a truncated version of the native IL2 sequence (e.g., SEQ ID NO:44). Thus, in some embodiments, IL2 comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:44.

[0138] In some embodiments, the interleukin is interleukin-6 (IL6). In some embodiments, IL6 is human IL6. In some embodiments. IL6 is full-length IL6. In some embodiments, IL6 is full-length, human IL6. In some embodiments, IL6 comprises the amino acid sequence set forth in SEQ ID NO:62.

[0139] In some embodiments. 1L6 is a mutated and / or truncated form of 1L6. In some embodiments. IL6 is a mutated form of IL6 comprising an amino acid substitution. In some embodiments, IL6 is a truncated fonn of IL6. In some embodiments, IL6 comprises an amino acid substitution. In some embodiments. IL6 is a truncated version of the native IL6 sequence (e.g., SEQ ID NO:62). Thus, in some embodiments, IL6 comprises an amino acid sequence having at least about 50%, at least about 60%. at least about 70%, at least about 80%. or at least about 90%, at least about 91%, at least about 96%, at least about 93%, at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:62.

[0140] In some embodiments, the interleukin is interleukin- 10 (IL10). In some embodiments, IL10 is human IL10. In some embodiments, IL10 is full-length IL10. In some embodiments, IL 10 is full-length, human IL 10. In some embodiments, IL 10 comprises the amino acid sequence set forth in SEQ ID NO:63.

[0141] In some embodiments, IL10 is a mutated and / or truncated form of IL10. In some embodiments, IL 10 is a mutated fonn of IL 10 comprising an amino acid substitution. In some embodiments, IL10 is a truncated form of IL10. In some embodiments, IL10 comprises an amino acid substitution. In some embodiments, IL10 is a truncated version of the native IL10 sequence (e.g., SEQ ID NO:63). Thus, in some embodiments, IL10 comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, at least about 91%, at least about 96%, at least about 93%, at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:63.

[0142] In some embodiments, the interleukin is interleukin- 12-alpha (IL12a). In some embodiments, IL12a is human IL12a. In some embodiments, IL12a is full-length IL12a. In some embodiments. IL12a is full-length, human IL12a. In some embodiments, IL12a comprises the amino acid sequence set forth in SEQ ID NO:64.

[0143] In some embodiments. IL12a is a mutated and / or truncated form of IL12a. In some embodiments. IL12a is a mutated form of IL12a comprising an amino acid substitution. In someembodiments, IL12a is a truncated form of IL12a. In some embodiments, IL12a comprises an amino acid substitution. In some embodiments, IL 12a is a truncated version of the native IL 12a sequence (e.g., SEQ ID NO:64). Thus, in some embodiments, IL12a comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, at least about 91%, at least about 96%, at least about 93%, at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 64.

[0144] In some embodiments, the interleukin is interleukin- 12-beta (IL12b). In some embodiments, IL 12b is human IL 12b. In some embodiments, IL 12b is full-length IL 12b. In some embodiments, IL12b is full-length, human IL12b. In some embodiments, IL12b comprises the amino acid sequence set forth in SEQ ID NO:65.

[0145] In some embodiments. IL12b is a mutated and / or truncated form of IL12b. In some embodiments, IL12b is a mutated form of IL12b comprising an amino acid substitution. In some embodiments. IL12b is a truncated form of IL12b. In some embodiments, IL12b comprises an amino acid substitution. In some embodiments, IL12b is a truncated version of the native IL12b sequence (e.g., SEQ ID NO:65). Thus, in some embodiments, IL12b comprises an amino acid sequence having at least about 50%. at least about 60%, at least about 70%. at least about 80%, or at least about 90%, at least about 91%. at least about 96%, at least about 93%, at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 65.

[0146] In some embodiments, the interleukin is interleukin- 18 (IL18). In some embodiments, IL18 is human IL18. In some embodiments, IL18 is full-length IL18. In some embodiments, IL 18 is full-length, human IL 18. In some embodiments, IL 18 comprises the amino acid sequence set forth in SEQ ID NO:66.

[0147] In some embodiments, IL18 is a mutated and / or truncated form of IL18. In some embodiments, IL 18 is a mutated form of IL 18 comprising an amino acid substitution. In some embodiments, IL18 is a truncated form of IL18. In some embodiments, IL18 comprises an amino acid substitution. In some embodiments, IL18 is a truncated version of the native IL18 sequence (e.g., SEQ ID NO:66). Thus, in some embodiments, IL18 comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, at least about 91%, at least about 96%, at least about 93%, at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:66.

[0148] In some embodiments, the interleukin is interleukin-21 (IL21). In some embodiments, IL21 is human IL21. In some embodiments, IL21 is full-length IL21. In some embodiments, IL21 is full-length, human IL21. In some embodiments, IL21 comprises the amino acid sequence set forth in SEQ ID NO:67.

[0149] In some embodiments, IL21 is a mutated and / or truncated form of IL21. In some embodiments. IL21 is a mutated form of IL21 comprising an amino acid substitution. In some embodiments. IL21 is a truncated form of IL21. In some embodiments. IL21 comprises an amino acidsubstitution. In some embodiments, IL21 is a truncated version of the native IL21 sequence (e.g., SEQ ID NO:67). Thus, in some embodiments, IL21 comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, at least about 91%, at least about 96%, at least about 93%, at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:67. ii. Linkers

[0150] In any of the provided embodiments, the extracellular domain comprises a linker. In some aspects, the extracellular domain comprises a linker between the cytokine (e.g., interleukin) or functional portion thereof and the stalk, such that the cytokine (e.g., interleukin) and the stalk are joined by a linker. Thus, an extracellular domain may contain, from N-terminus to C-terminus: an interleukin, a linker, and a stalk.

[0151] In some embodiments, the linker is between 1 and 100 amino acids in length, such as between 2 and 50 amino acids in length or between 3 and 25 amino acids in length. In some embodiments, the linker is between 1 and 10 amino acids in length. In some embodiments, the linker is between 2 and 8 amino acids in length. In some embodiments, the linker is 2, 3, 4 ,5, 6, 7, 8. 9, or 10 amino acids in length. In some embodiments, the linker is 2 amino acids in length. In some embodiments, the linker is 3 amino acids in length. In some embodiments, the linker is 4 amino acids in length. In some embodiments, the linker is 5 amino acids in length. In some embodiments, the linker is 6 amino acids in length. In some embodiments, the linker is 7 amino acids in length. In some embodiments, the linker is 8 amino acids in length. In some embodiments, the linker is 9 amino acids in length. In some embodiments, the linker is between 10 and 25 amino acids in length. In some embodiments, the linker is 10 amino acids in length. In some embodiments, the linker is 15 amino acids in length. In some embodiments, the linker is 20 amino acids in length. In some embodiments, the linker is 25 amino acids in length.

[0152] In some embodiments, the linker is comprised of glycine and serine residues (a “Gly-Ser” linker). Thus, in some aspects, the linker is a Gly-Ser linker. In some embodiments, the linker comprises the amino acid sequence of GSG (SEQ ID NO:21). In some embodiments, the linker comprises the amino acid sequence of GGGS (SEQ ID NO:68). In some embodiments, the linker comprises the amino acid sequence of GGGGS (SEQ ID NO:69). In some embodiments, the linker comprises any repetition or combination of a linker sequence provided herein. For example, in some embodiments, the linker comprises a combination of the amino acid sequence of SEQ ID NO:69 (e.g., SEQ ID NO:1 or SEQ ID NO:2). For example, in some aspects, the linker comprises a combination of the amino acid sequences of SEQ ID NOS:68 and 69 (e.g.. SEQ ID NO:70 or SEQ ID NO:40). In some embodiments, the linker has the sequence of GGGGS(n). where n is greater than or equal to one. In some embodiments, the linker comprises the sequence of GGGGSGGGGSGGGGS (SEQ ID NO:1). In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO:2). In some embodiments, the linker comprises the amino acid sequence ofGGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO:70). In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO:40). iii. Stalk

[0153] In some embodiments, the stalk is selected from a CD8a stalk, a CD25 stalk, a IL15Ra stalk, or a functional portion thereof. In some embodiments, the stalk is a CD8a stalk or a functional portion thereof. In some embodiments, the stalk is a CD8a stalk. In some embodiments, the stalk is a CD25 stalk or a portion thereof. In some embodiments, the stalk is a CD25 stalk. In some embodiments, the stalk is a IL15Ra stalk or a portion thereof. In some embodiments, the stalk is a IL15Ra stalk. In some embodiments, the stalk does not comprise the Sushi domain of IL15Ra (e g., SEQ ID NO:61). Thus, in some aspects, the stalk does not contain, e.g., amino acid residues 1-65 of SEQ ID NO:41. Accordingly, in some embodiments, the transmembrane protein does not comprise full-length IL15Ra. In some embodiments, the stalk is mammalian, such as human. Thus, in some embodiments, the stalk is a human stalk. Non-limiting examples of stalks of a provided transmembrane protein are set forth in Table 2.

[0154] In some embodiments, the stalk or functional portion thereof comprises the amino acid sequence of any one of SEQ ID NOS:6. 29, 59, and 35, or an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequency identity to any one of SEQ ID NOS: 6, 29, 59, and 35.

[0155] In some embodiments, the stalk is a CD8a stalk. In some embodiments, the CD8a stalk is human. In some embodiments, the CD8a stalk comprises the amino acid sequence set forth in SEQ ID NO: 6.

[0156] In some embodiments, the CD8a stalk is a mutated and / or truncated form of a CD8a stalk. In some embodiments, the CD8a stalk is a mutated form of a CD8a stalk comprising anamino acid substitution. In some embodiments, the CD8a stalk is a truncated form of a CD8a stalk. In some embodiments, the CD8a stalk comprises an amino acid substitution. In some embodiments, the CD8a stalk is a truncated version of the native CD8a stalk sequence (e.g., SEQ ID NO:6). Thus, in some embodiments, the CD8a stalk comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. at least about 91%, at least about 96%. at least about 93%, at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:6.

[0157] In some embodiments, the stalk is a CD25 stalk. In some embodiments, the CD25 stalk is human. In some embodiments, the CD25 stalk comprises the amino acid sequence set forth in SEQ ID NO:29.

[0158] In some embodiments, the CD25 stalk is a mutated form of a CD25 stalk comprising an amino acid substitution. In some embodiments, the CD25 stalk comprises an amino acid substitution. In some embodiments, the CD25 stalk comprises a C27S substitution. In some embodiments, the CD25 stalk comprises the amino acid sequence of SEQ ID NO:59, or an amino acid sequence that has at least 85%, 86%, 87%, 88%. 89%. 90%, 91%, 92%, 93%. 94%. 95%, 96%, 97%, 98%, 99%, or more sequency identity7to SEQ ID NO:59. In some embodiments, the CD25 stalk comprises the amino acid sequence of SEQ ID NO:59.

[0159] In some embodiments, the CD25 stalk is a truncated version of the native CD25 stalk sequence (e.g.. SEQ ID NO:29). Thus, in some embodiments, the CD25 stalk comprises an amino acid sequence having at least about 50%. at least about 60%, at least about 70%, at least about 80%, or at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:29.

[0160] In some embodiments, the stalk is a IL15Ra stalk. In some embodiments, the IL15Ra stalk is human. In some embodiments, the IL15Ra stalk comprises the amino acid sequence set forth in SEQ ID NO:35.

[0161] In some embodiments, the IL15Ra stalk is a mutated and / or truncated form of a IL15Ra stalk. In some embodiments, the IL15Ra stalk is a mutated form of a IL15Ra stalk comprising an amino acid substitution. In some embodiments, the IL15Ra stalk is a truncated form of a IL15Ra stalk. In some embodiments, the IL15Ra stalk comprises an amino acid substitution. In some embodiments, the IL15Ra stalk is a truncated version of the native IL15Ra stalk sequence (e.g., SEQ ID NO:35). Thus, in some embodiments, the IL15Ra stalk comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, at least about 91%, at least about 96%. at least about 93%, at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:35.B. Transmembrane Domain

[0162] Provided herein are transmembrane proteins comprising (a) an extracellular domain comprising a cytokine (e.g., interleukin) or functional portion thereof; and (b) atransmembrane domain. In some embodiments, the transmembrane domain comprises a transmembrane region and an intracellular region. Thus, in some embodiments, the transmembrane domain comprises, from N-terminus to C-terminus: a transmembrane region and an intracellular region. i. Transmembrane Region

[0163] The transmembrane region in some embodiments is derived from a natural source or a synthetic source. In some embodiments, the transmembrane region is synthetic. Alternatively, when the source is natural, the transmembrane region in some aspects is derived from any membranebound or transmembrane protein.

[0164] In some embodiments, transmembrane regions include any of those derived from (e.g., comprise at least the transmembrane region of) the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16. CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane region is a transmembrane region of CD3. In some embodiments, the transmembrane region is a transmembrane region of CD4. In some embodiments, the transmembrane region is a transmembrane region of CD8 (e.g., CD8a). In some embodiments, the transmembrane region is a transmembrane region of CD28.

[0165] In some embodiments, the transmembrane region is derived from (e.g., comprises at least tire transmembrane region of) a receptor, such as an interleukin receptor. Thus, in some aspects, the transmembrane region is a transmembrane region of an interleukin receptor or a chain thereof (e g., IL15Ra or CD25). In some embodiments, the transmembrane region is a transmembrane region of IL15Ra. In some embodiments, the transmembrane region is a transmembrane region of CD25.

[0166] Non-limiting examples of transmembrane regions of a provided transmembrane protein are set forth in Table 3. In some embodiments, the transmembrane region comprises the amino acid sequence of any one of SEQ ID NOS:8, 11. 31. and 36. or an amino acid sequence that has at least 85%, 86%. 87%, 88%, 89%, 90%, 91%. 92%. 93%, 94%, 95%, 96%, 97%. 98%, 99%, or more sequency identity to any one of SEQ ID NOS: 8, 11. 31, and 36.

[0167] In some embodiments, the transmembrane region is a CD8 transmembrane region. In some embodiments, the transmembrane region is a CD8a transmembrane region. In some embodiments, the CD8a transmembrane region is a human CD8a transmembrane region. In some embodiments, the CD8a transmembrane region comprises the amino acid sequence set forth in SEQID N0:8. In some embodiments, the CD8a transmembrane region is a mutated and / or truncated form of a CD8a transmembrane region. In some embodiments, the CD8a transmembrane region is a mutated form of a CD8a transmembrane region comprising an amino acid substitution. In some embodiments, the CD8a transmembrane region is a truncated form of a CD8a transmembrane region. In some embodiments, the CD8a transmembrane region comprises an amino acid substitution. In some embodiments, the CD8a transmembrane region is a truncated version of the native CD8a transmembrane region sequence (e.g., SEQ ID NO:8). Thus, in some embodiments, the CD8a transmembrane region comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, at least about 91%. at least about 96%, at least about 93%. at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:8.

[0168] In some embodiments, the transmembrane region is a CD28 transmembrane region. In some embodiments, the CD28 transmembrane region is a human CD28 transmembrane region. In some embodiments, the CD28 transmembrane region comprises the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the CD28 transmembrane region is a mutated and / or truncated form of a CD28 transmembrane region. In some embodiments, the CD28 transmembrane region is a mutated form of a CD28 transmembrane region comprising an amino acid substitution. In some embodiments, the CD28 transmembrane region is a truncated form of a CD28 transmembrane region. In some embodiments, the CD28 transmembrane region comprises an amino acid substitution. In some embodiments, the CD28 transmembrane region is a truncated version of the native CD28 transmembrane region sequence (e.g., SEQ ID NO: 11). Thus, in some embodiments, the CD28 transmembrane region comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%. at least about 80%, or at least about 90%, at least about 91%, at least about 96%, at least about 93%, at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 11.

[0169] In some embodiments, the transmembrane region is a CD25 transmembrane region. In some embodiments, the CD25 transmembrane region is a human CD25 transmembrane region. In some embodiments, the CD25 transmembrane region comprises the amino acid sequence set forth in SEQ ID NO:31. In some embodiments, the CD25 transmembrane region is a mutated and / or truncated form of a CD25 transmembrane region. In some embodiments, the CD25 transmembrane region is a mutated form of a CD25 transmembrane region comprising an amino acid substitution. In some embodiments, the CD25 transmembrane region is a truncated form of a CD25 transmembrane region. In some embodiments, the CD25 transmembrane region comprises an amino acid substitution. In some embodiments, the CD25 transmembrane region is a truncated version of the native CD25 transmembrane region sequence (e.g., SEQ ID NO:31). Thus, in some embodiments, the CD25 transmembrane region comprises an amino acid sequence having at least about 50%. at least about 60%, at least about 70%, at least about 80%, or at least about 90%, at least about 91%. at leastabout 96%, at least about 93%, at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:31.

[0170] In some embodiments, the transmembrane region is a IL15R transmembrane region. In some embodiments, the transmembrane region is a IL15RA transmembrane region. In some embodiments, the IL15RA transmembrane region is a human IL15RA transmembrane region. In some embodiments, the IL15RA transmembrane region comprises the amino acid sequence set forth in SEQ ID NO:36. In some embodiments, the IL15RA transmembrane region is a mutated and / or truncated form of a IL15RA transmembrane region. In some embodiments, the IL15RA transmembrane region is a mutated form of a IL15RA transmembrane region comprising an amino acid substitution. In some embodiments, the IL15RA transmembrane region is a truncated form of a IL15RA transmembrane region. In some embodiments, the IL15RA transmembrane region comprises an amino acid substitution. In some embodiments, the 1L15RA transmembrane region is a truncated version of the native IL15RA transmembrane region sequence (e.g.. SEQ ID NO:36). Thus, in some embodiments, the IL15RA transmembrane region comprises an amino acid sequence having at least about 50%, at least about 60%. at least about 70%, at least about 80%. or at least about 90%, at least about 91%, at least about 96%, at least about 93%. at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:36. ii. Intracellular Region

[0171] The intracellular region in some embodiments is derived from a natural source or a synthetic source. In some embodiments, the intracellular region is synthetic. Alternatively, when the source is natural, the intracellular region in some aspects is derived from any membrane-bound or transmembrane protein. In some embodiments, the transmembrane region and the intracellular region are derived from the same protein. Thus, in some embodiments, the transmembrane domain comprises a transmembrane region and an intracellular region of the same membrane-bound or transmembrane protein.

[0172] In some embodiments, intracellular regions include any of those derived from (e.g., comprise at least an intracellular region of) the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon. CD45, CD4, CD5. CD8. CD9, CD16, CD22, CD25. CD33, CD37. CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the intracellular region is an intracellular region of CD3. In some embodiments, the transmembrane region is an intracellular region of CD4. In some embodiments, the intracellular region is an intracellular region of CD8 (e.g.. CD8a). In some embodiments, the intracellular region is an intracellular region of CD28.

[0173] In some embodiments, the intracellular region is derived from (e.g.. comprises at least an intracellular region of) a receptor, such as an interleukin receptor. Thus, in some aspects, the intracellular region is an intracellular of an interleukin receptor or a chain thereof (e.g.. IL15Ra or CD25). In some embodiments, the intracellular region is an intracellular region of IL15Ra. In some embodiments, the intracellular region is an intracellular region of CD25.

[0174] Non-limiting examples of intracellular regions of a provided transmembrane protein are set forth in Table 4. In some embodiments, the intracellular region comprises the amino acid sequence of any one of SEQ ID NOS:71, 12, 32, and 37, or an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequency identity to any one of SEQ ID NOS: 71, 12, 32, and 37.

[0175] In some embodiments, the intracellular region is a CD8 intracellular region. In some embodiments, the intracellular region is a CD8a intracellular region. In some embodiments, the CD8a intracellular region is a human CD8a intracellular region. In some embodiments, the CD8a intracellular region comprises the amino acid sequence set forth in SEQ ID NO:71. In some embodiments, the CD8a intracellular region is a mutated and / or truncated form of a CD8a intracellular region. In some embodiments, the CD8a intracellular region is a mutated form of a CD8a intracellular region comprising an amino acid substitution. In some embodiments, the CD8a intracellular region is a truncated form of a CD8a intracellular region. In some embodiments, the CD8a intracellular region comprises an amino acid substitution. In some embodiments, the CD8a intracellular region is a truncated version of the native CD8a intracellular region sequence (e.g., SEQ ID NO:71). Thus, in some embodiments, the CD8a intracellular region comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, at least about 91%, at least about 96%, at least about 93%, at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:71.

[0176] In some embodiments, the intracellular region is a CD28 intracellular region. In some embodiments, the CD28 intracellular region is a human CD28 intracellular region. In some embodiments, the CD28 intracellular region comprises the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the CD28 intracellular region is a mutated and / or truncated form of a CD28 intracellular region. In some embodiments, the CD28 intracellular region is a mutated form of a CD28 intracellular region comprising an amino acid substitution. In some embodiments, the CD28 intracellular region is a truncated form of a CD28 intracellular region. In some embodiments, the CD28 intracellular region comprises an amino acid substitution. In some embodiments, the CD28 intracellular region is a truncated version of the native CD28 intracellular region sequence (c.g., SEQ ID NO: 12). Thus, in some embodiments, the CD28 intracellular region comprises an amino acidsequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, at least about 91%, at least about 96%, at least about 93%, at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 12.

[0177] In some embodiments, the intracellular region is a CD25 intracellular region. In some embodiments, the CD25 intracellular region is a human CD25 intracellular region. In some embodiments, the CD25 intracellular region comprises the amino acid sequence set forth in SEQ ID NO:32. In some embodiments, the CD25 intracellular region is a mutated and / or truncated form of a CD25 intracellular region. In some embodiments, the CD25 intracellular region is a mutated form of a CD25 intracellular region comprising an amino acid substitution. In some embodiments, the CD25 intracellular region is a truncated form of a CD25 intracellular region. In some embodiments, the CD25 intracellular region comprises an amino acid substitution. In some embodiments, the CD25 intracellular region is a truncated version of the native CD25 intracellular region sequence (e.g., SEQ ID NO:32). Thus, in some embodiments, the CD25 intracellular region comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. at least about 91%, at least about 96%. at least about 93%, at least about 94%. or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:32.

[0178] In some embodiments, the intracellular region is a IL15R intracellular region. In some embodiments, the intracellular region is a IL15RA intracellular region. In some embodiments, the IL15RA intracellular region is a human IL15RA intracellular region. In some embodiments, the IL15RA intracellular region comprises the amino acid sequence set forth in SEQ ID NO:37. In some embodiments, the IL15RA intracellular region is a mutated and / or truncated form of a IL15RA intracellular region. In some embodiments, the IL15RA intracellular region is a mutated form of a IL15RA intracellular region comprising an amino acid substitution. In some embodiments, the IL15RA intracellular region is a truncated form of a IL15RA intracellular region. In some embodiments, the IL15RA intracellular region comprises an amino acid substitution. In some embodiments, the IL15RA intracellular region is a truncated version of the native IL15RA intracellular region sequence (e.g., SEQ ID NO:37). Thus, in some embodiments, the IL15RA intracellular region comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, at least about 91%, at least about 96%, at least about 93%, at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:37.

[0179] In some aspects, the transmembrane domain comprises any of the provided transmembrane regions in combination with, such as directly linked to, any of the provided intracellular regions. In some aspects, the transmembrane domain comprises a transmembrane region and an intracellular region from the same membrane-bound or transmembrane protein.

[0180] For example, in some aspects, the transmembrane domain is a CD8a transmembrane domain comprising a CD8a transmembrane region and a CD8a intracellular region. Insome embodiments, the CD8a transmembrane domain comprises the amino acid sequence of SEQ ID NO:27. In some embodiments, the CD8a transmembrane domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, at least about 91%, at least about 96%, at least about 93%, at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:27.

[0181] For example, in some aspects, the transmembrane domain is a CD28 transmembrane domain comprising a CD28 transmembrane region and a CD28 intracellular region. In some embodiments, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%. or at least about 90%, at least about 91%, at least about 96%, at least about 93%, at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 72.

[0182] In some aspects, the transmembrane domain is a CD25 transmembrane domain comprising a CD25 transmembrane region and a CD25 intracellular region. In some embodiments, the CD25 transmembrane domain comprises the amino acid sequence of SEQ ID NO:58. In some embodiments, the CD25 transmembrane domain comprises an amino acid sequence having at least about 50%, at least about 60%. at least about 70%, at least about 80%. or at least about 90%, at least about 91%, at least about 96%, at least about 93%. at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:58.

[0183] In some aspects, the transmembrane domain is a IL15Ra transmembrane domain comprising a IL15Ra transmembrane region and a IL15Ra intracellular region. In some embodiments, the IL15Ra transmembrane domain comprises the amino acid sequence of SEQ ID NO:38. In some embodiments, the IL15Ra transmembrane domain comprises an amino acid sequence having at least about 50%, at least about 60%. at least about 70%, at least about 80%, or at least about 90%, at least about 91%, at least about 96%, at least about 93%, at least about 94%, or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:38.C. Non-Limiting Examples of Transmembrane Proteins

[0184] In some aspects, the transmembrane protein comprises an extracellular domain comprising IL2 or a functional portion thereof and a CD8a, CD25, or IL15Ra transmembrane domain. Thus, in some aspects, the transmembrane protein is a membrane-bound interleukin-2 (mbIL2).

[0185] In some embodiments, the extracellular domain comprises IL2 (e.g., SEQ ID NO:44 or SEQ ID NO:45) and a CD8a stalk (e.g.. SEQ ID NO:6). In some embodiments, the IL2 and the CD8a stalk are connected by a linker of SEQ ID NO:21. Thus, in some embodiments, the extracellular domain comprises, from N-terminus to C-terminus: SEQ ID NO:44, SEQ ID NO:21, and SEQ ID NO:6. In some embodiments, the extracellular domain comprises, from N-terminus to C- terminus: SEQ ID NO:45. SEQ ID NO:21. and SEQ ID NO:6. In some embodiments, the transmembrane domain is a CD8a transmembrane domain comprising a transmembrane region ofCD8a (e.g., SEQ ID NO:8) and an intracellular region of CD8a (e.g., SEQ ID NO:72). Thus, in some embodiments, the transmembrane domain comprises SEQ ID NO:27. In some aspects, the transmembrane protein comprises (a) an extracellular domain comprising IL2 and a CD8a stalk; and (b) a transmembrane domain comprising a CD8a transmembrane region and a CD8a intracellular region. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%. at least about 95%, at least about 96%, at least about 97%. at least about 98%, at least about 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:52 or SEQ ID NO:55. In some embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:52 or SEQ ID NO:55. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 80%. at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:52. In some embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:52. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 80%. at least about 85%, at least about 90%, at least about 95%. at least about 96%. at least about 97%, at least about 98%, at least about 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:55. In some embodiments, the transmembrane protein comprises the amino acid sequence of ID NO:55.

[0186] In some embodiments, the extracellular domain comprises IL2 (e.g.. SEQ ID NO:44 or SEQ ID NO:45) and a CD25 stalk (e.g., SEQ ID NO:29). In some embodiments, the IL2 and the CD25 stalk are connected by a linker of SEQ ID NO:21. Thus, in some embodiments, the extracellular domain comprises, from N-terminus to C-tenninus: SEQ ID NO:44, SEQ ID NO:21, and SEQ ID NO:29. In some embodiments, the extracellular domain comprises, from N-terminus to C- terminus: SEQ ID NO:45, SEQ ID NO:21, and SEQ ID NO:29. In some embodiments, the transmembrane domain is a CD25 transmembrane domain comprising a transmembrane region of CD25 (e.g., SEQ ID NO:31) and an intracellular region of CD25 (e.g., SEQ ID NO:32). Thus, in some embodiments, the transmembrane domain comprises SEQ ID NO:58. In some aspects, the transmembrane protein comprises (a) an extracellular domain comprising IL2 and a CD25 stalk; and (b) a transmembrane domain comprising a CD25 transmembrane region and a CD25 intracellular region. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%. at least about 95%, at least about 96%, at least about 97%. at least about 98%, at least about 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:53 or SEQ ID NO:56. In some embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:53 or SEQ ID NO:56. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 80%. at least about 85%, at least about 90%, at least about 95%. at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:53. Insome embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:53. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%. at least about 95%, at least about 96%, at least about 97%. at least about 98%, at least about 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:56. In some embodiments, the transmembrane protein comprises the amino acid sequence of ID NO:56.

[0187] In some embodiments, the extracellular domain comprises IL2 (e.g., SEQ ID NO:44 or SEQ ID NO:45) and a IL15Ra stalk (e.g., SEQ ID NO:35). In some embodiments, the IL2 and the IL15Ra stalk are connected by a linker of SEQ ID NO:21. Thus, in some embodiments, the extracellular domain comprises, from N-terminus to C-terminus: SEQ ID NO:44, SEQ ID NO:21, and SEQ ID NO:35. In some embodiments, the extracellular domain comprises, from N-tenninus to C- terminus: SEQ ID NO:45, SEQ ID NO:21. and SEQ ID NO:35. In some embodiments, the transmembrane domain is an IL15Ra transmembrane domain comprising a transmembrane region of IL15Ra (e.g., SEQ ID NO:36) and an intracellular region of IL15Ra (e.g., SEQ ID NO:37). Thus, in some embodiments, the transmembrane domain comprises SEQ ID NO:38. In some aspects, the transmembrane protein comprises (a) an extracellular domain comprising IL2 and a IL15Ra stalk; and (b) a transmembrane domain comprising a IL15Ra transmembrane region and a IL15Ra intracellular region. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 80%. at least about 85%, at least about 90%, at least about 95%, at least about 96%. at least about 97%, at least about 98%, at least about 99%, or more sequence identity7to the amino acid sequence of SEQ ID NO:54 or SEQ ID NO:56. In some embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:54 or SEQ ID NO:57. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:54. In some embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:54. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%. at least about 95%, at least about 96%, at least about 97%. at least about 98%, at least about 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:57. In some embodiments, the transmembrane protein comprises the amino acid sequence of ID NO:57.

[0188] In some aspects, the transmembrane protein comprises an extracellular domain comprising IL15 or a functional portion thereof and a CD8a, CD25, or IL15Ra transmembrane domain. Thus, in some aspects, the transmembrane protein is a membrane-bound interleukin- 15 (mbIL15).

[0189] In some embodiments, the extracellular domain comprises IL15 (e.g., SEQ ID NO:25 or SEQ ID NO:46) and a CD8a stalk (e.g., SEQ ID NO:6). Thus, in some embodiments, theextracellular domain comprises, from N-terminus to C-terminus: SEQ ID NO:25 and SEQ ID NO:6. In some embodiments, the extracellular domain comprises, from N-terminus to C-terminus: SEQ ID NO:46 and SEQ ID NO:6. In some embodiments, the transmembrane domain is a CD8a transmembrane domain comprising a transmembrane region of CD8a (e.g.. SEQ ID NO:8) and an intracellular region of CD8a (e.g., SEQ ID NO:72). Thus, in some embodiments, the transmembrane domain comprises SEQ ID NO:27. In some aspects, the transmembrane protein comprises (a) an extracellular domain comprising IL 15 and a CD8a stalk; and (b) a transmembrane domain comprising a CD8a transmembrane region and a CD8a intracellular region. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:26 or SEQ ID NO:49. In some embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:26 or SEQ ID NO:49. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%. at least about 96%. at least about 97%, at least about 98%, at least about 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:26. In some embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:26. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:49. In some embodiments, the transmembrane protein comprises the amino acid sequence of ID NO:49.

[0190] In some embodiments, the extracellular domain comprises IL15 (e.g., SEQ ID NO:25 or SEQ ID NO:46) and a CD25 stalk (e.g., SEQ ID NO:29). In some embodiments, the IL15 and the CD25 stalk arc connected by a linker of SEQ ID NO:21. Thus, in some embodiments, the extracellular domain comprises, from N-terminus to C-terminus: SEQ ID NO:25, SEQ ID NO:21, and SEQ ID NO:29. In some embodiments, the extracellular domain comprises, from N-terminus to C- terminus: SEQ ID NO:46, SEQ ID NO:21, and SEQ ID NO:29. In some embodiments, the transmembrane domain is a CD25 transmembrane domain comprising a transmembrane region of CD25 (e.g., SEQ ID NO:31) and an intracellular region of CD25 (e.g., SEQ ID NO:32). Thus, in some embodiments, the transmembrane domain comprises SEQ ID NO:58. In some aspects, the transmembrane protein comprises (a) an extracellular domain comprising IL 15 and a CD25 stalk; and (b) a transmembrane domain comprising a CD25 transmembrane region and a CD25 intracellular region. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%. at least about 95%, at least about 96%, at least about 97%. at least about 98%. at least about 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:34 or SEQ ID NQ:50. In some embodiments, the transmembrane proteincomprises the amino acid sequence of SEQ ID NO:34 or SEQ ID NO:50. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:34. In some embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:34. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%. at least about 95%, at least about 96%, at least about 97%. at least about 98%, at least about 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:50. In some embodiments, the transmembrane protein comprises the amino acid sequence of ID NO:50.

[0191] In some embodiments, the extracellular domain comprises IL15 (e.g., SEQ ID NO:25 or SEQ ID NO:46) and a IL15Ra stalk (e.g.. SEQ ID NO:35). In some embodiments, the 1L15 and the IL15Ra stalk are connected by a linker of SEQ ID NO:21. Thus, in some embodiments, the extracellular domain comprises, from N-terminus to C-terminus: SEQ ID NO:25, SEQ ID NO:21, and SEQ ID NO:35. In some embodiments, the extracellular domain comprises, from N-terminus to C- terminus: SEQ ID NO:46, SEQ ID NO:21, and SEQ ID NO:35. In some embodiments, the transmembrane domain is a IL15Ra transmembrane domain comprising a transmembrane region of IL15Ra (e.g., SEQ ID NO:36) and an intracellular region of IL15Ra (e g., SEQ ID NO:37). Thus, in some embodiments, the transmembrane domain comprises SEQ ID NO:38. In some aspects, the transmembrane protein comprises (a) an extracellular domain comprising IL 15 and a IL15Ra stalk; and (b) a transmembrane domain comprising a IL15Ra transmembrane region and a IL15Ra intracellular region. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:47 or SEQ ID NO:51. In some embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:47 or SEQ ID NO:51. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:47. In some embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:47. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%. at least about 95%, at least about 96%. at least about 97%, at least about 98%. at least about 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:51. In some embodiments, the transmembrane protein comprises the amino acid sequence of ID NO:51.

[0192] In some aspects, the transmembrane protein comprises a fusion of IL 15 and IL15Ra. Thus, in some embodiments, the transmembrane protein comprises an extracellular domaincomprising IL15 and an ectodomain of IL15Ra, and a transmembrane domain of IL15Ra. In some embodiments, die extracellular domain comprises IL15 (e.g., SEQ ID NO:25 or SEQ ID NO:46) and a IL15Ra ectodomain (e.g.. SEQ ID NO:41). In some embodiments, the IL15 and the IL15Ra ectodomain are connected by a linker of SEQ ID NO:40. Thus, in some embodiments, the extracellular domain comprises, from N-terminus to C-terminus: SEQ ID NO:25, SEQ ID NO:40, and SEQ ID NO:41. In some embodiments, the extracellular domain comprises, from N-tenninus to C- terminus: SEQ ID NO:46, SEQ ID NO:40, and SEQ ID NO:41. In some embodiments, the transmembrane domain is a IL15Ra transmembrane domain comprising a transmembrane region of IL15Ra (e.g., SEQ ID NO:36) and an intracellular region of IL15Ra (e.g.. SEQ ID NO:37). Thus, in some embodiments, the transmembrane domain comprises SEQ ID NO:38. In some aspects, the transmembrane protein comprises (a) an extracellular domain comprising IL15 and a IL15Ra ectodomain; and (b) a transmembrane domain comprising a lL15Ra transmembrane region and a IL15Ra intracellular region. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%. at least about 97%, at least about 98%, at least about 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:48. In some embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID NO:48.II. Recombinant Receptors

[0193] In some embodiments, the transmembrane proteins are used in combination with and / or are expressed in or from engineered cells expressing recombinant receptors.

[0194] Thus, provided are recombinant receptors and cells expressing such receptors. In some embodiments, the recombinant receptors include chimeric receptors, including those containing antigen-binding domains or fragments thereof, such as chimeric antigen receptors (CARs) and T cell receptors (TCRs. e.g., transgenic TCRs). The chimeric receptor, such as a CAR, generally includes an extracellular antigen-binding domain linked to one or more intracellular signaling components, in some aspects via linkers and / or transmembrane domains.

[0195] In particular embodiments, the recombinant receptor, such as a chimeric receptor, contains an intracellular signaling domain, which includes an intracellular signaling region, such as an activating cytoplasmic (intracellular) domain capable of inducing a primary activation signal in a T cell, for example, a cytoplasmic signaling domain of a T cell receptor (TCR) component (e.g. a cytoplasmic signaling domain of a zeta chain of a CD3zeta (CD3z) chain or a functional variant or signaling portion thereof) and / or that comprises an immunoreceptor tyrosine-based activation motif (IT AM).

[0196] In some embodiments, the chimeric receptor further contains an extracellular antigen-binding domain that binds to an antigen. In some embodiments, the chimeric receptor is a CAR that contains an extracellular antigen-binding domain drat binds to an antigen. In someembodiments, the antigen is a protein expressed on the surface of cells. In some embodiments, the antigen is expressed by and / or associated with cells of a disease or condition.

[0197] Non-limiting examples of recombinant receptors, including CARs and recombinant TCRs, as well as methods for engineering and introducing the receptors into cells, include those described, for example, in international patent application publication numbersW02000014257, W02005044996, WO2012129514, W02013071154, W02013123061.WO2013126726, WO2013166321, WO2014031687, WO2014117121, WO2016014789W02016090312, WO2017211900, WO2018183385, W02020160050, W02020180882WO2021252804, and WO2023288185, each of which is incorporated by reference herein in its entirety. Non-limiting examples of recombinant receptors, including CARs. as well as methods for engineering and introducing the receptors into cells, include those described, for example, in international patent application publication number WO2024206439 which is incorporated by reference herein in its entirety. In some embodiments, similar methods for the construction and introduction or transfer into cells can be employed for the provided recombinant receptors.

[0198] In some embodiments, the recombinant receptor (e.g., CAR) includes an antigenbinding domain that binds to an antigen. Among the antigens targeted by the recombinant receptors are those expressed in the context of a disease, condition, or cell type to be targeted by the genetically engineered cells. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematologic cancers, cancers of the immune system, such as lymphomas, leukemias, and / or myelomas, such as B. T, and myeloid leukemias, lymphomas, and multiple myelomas. Also among the diseases and conditions are inflammatory and autoimmune diseases, such as autoimmune encephalitis, inflammatory' bowel disease (IBD), Type 1 diabetes, Guillain-Barre syndrome, chronic inflammatory' demyelinating polyneuropathy (CIDP), psoriasis, Graves’ disease, Hashimoto’s thyroiditis, systemic lupus erythematosus (SLE), lupus nephritis (LN), myasthenia gravis (MG), scleroderma (also known as systemic sclerosis; SSc), idiopathic inflammatory' myopathies (IIM; also known as myositis), vasculitis, multiple sclerosis (MS), and rheumatoid arthritis (RA). In some embodiments, the antigen is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues. In some aspect, such as in the case of an autoimmune disorder, the antigen is expressed by B cells and / or plasma cells. In other embodiments, the antigen is expressed on normal cells and / or is expressed on the engineered cells.

[0199] In some embodiments, the antigen is associated with and / or expressed by cells of a tumor or cancer. In some embodiments, the antigen is a ligand of natural killer group 2D receptor (NKG2D) (e.g.. MICA, MICB, ULBP1. ULBP2, ULBP3, ULBP4. ULBP5, and / or ULBP6), B cell maturation antigen (BCMA). CD19, CD20, CD22. CD23. CD24, CD30, CD33, CD44. CD70.CD123, CD138, CD171, claudin 6, epidermal growth factor receptor (EGFR), Fc receptor homolog 5 (FCRH5), human epidermal growth factor receptor 2 (HER2), mesothelin, mucin 1 (MUC1), receptor tyrosine kinase like orphan receptor 1 (R0R1), SLAMF7, Wilms Tumor 1 (WT1), a pathogenspecific antigen, and / or a molecule expressed by HIV, HCV, HBV. In some embodiments, the antigen is a ligand of NKG2D. In some embodiments, the antigen is BCMA. hi some embodiments, the antigen is CD19. In some embodiments, the antigen is CD20. In some embodiments, the antigen is CD38. In some embodiments, the antigen is CD70. In some embodiments, the antigen is CD138. In some embodiments, the antigen is EGFR. In some embodiments, the antigen is HER2. In some embodiments, the antigen is ROR1. In some embodiments, the antigen is SLAMF7.

[0200] In some embodiments, the antigen is associated with and / or expressed by cells of an autoimmune disease. In some embodiments, the antigen is BCMA, CD 19. CD20, CD22, CD38, CD70, CD138, GPRC5D, ROR1, SLAMF7. In some embodiments, the antigen is BCMA. In some embodiments, the antigen is CD 19. In some embodiments, the antigen is CD20. In some embodiments, the antigen is CD22. In some embodiments, the antigen is CD38. In some embodiments, the antigen is CD70. In some embodiments, the antigen is CD138. In some embodiments, the antigen is GPRC5D. In some embodiments, the antigen is ROR1. In some embodiments, the antigen is SLAMF7.

[0201] In some embodiments, the antigen is a pathogen-specific antigen, such as a viral antigen, bacterial antigen, and / or parasitic antigen.A. Antigen-Binding Domain

[0202] In some embodiments, the recombinant receptor, e.g., CAR, contains an antigenbinding domain. In some embodiments, the recombinant receptor is a CAR.

[0203] In some embodiments, the antigen-binding domain comprises an extracellular domain of a receptor. In some embodiments, the antigen-binding domain comprises an extracellular domain of NKG2D. In some such embodiments, the antigen is a cognate ligand of the receptor.

[0204] In some embodiments, the CAR is constructed with a specificity for a particular antigen, such as an antigen expressed in a particular cell type to be targeted by cell therapy. Thus, the CAR typically includes in its extracellular portion one or more antigen-binding molecules, such as one or more antigen-binding fragment, domain, or portion, or one or more antibody variable domains, and / or antibody molecules. In some embodiments, the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).

[0205] The term "antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, variable heavy chain (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variablefragments (scFv), and single domain antibodies (e.g. , sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.

[0206] In some embodiments, the antigen-binding proteins, antibodies and antigen binding fragments thereof specifically recognize an antigen of a full-length antibody. In some embodiments, the heavy and light chains of an antibody can be full-length or can be an antigenbinding portion (a Fab, F(ab')2, Fv or a single chain Fv fragment (scFv)). In other embodiments, the antibody heavy chain constant region is chosen from, e.g.. IgGl. IgG2. IgG3. IgG4. IgM, IgAl. IgA2. IgD, and IgE, particularly chosen from, e.g., IgGl, IgG2, IgG3, and IgG4, more particularly, IgGl (e.g., human IgGl). In another embodiment, the antibody light chain constant region is chosen from, e.g.. kappa or lambda, particularly kappa.

[0207] Among the provided antibodies are antibody fragments. An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; variable heavy chain (VH) regions, single-chain antibody molecules such as scFvs and single-domain VH single antibodies; and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibodies are single-chain antibody fragments comprising a variable heavy chain region and / or a variable light chain region, such as scFvs. The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy' chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. A single VH or VL domain may be sufficient to confer antigen-binding specificity'.

[0208] Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody. In some embodiments, the single-domain antibody is a came lid single-domain antibody (VHH). In some embodiments, the CAR comprises an antibody heavy chain domain that specifically binds the antigen, such as a cancer marker or cell surface antigen of a cell or disease to be targeted, such as a tumor cell or a cancer cell, such as any of the target antigens described herein or known in the art.

[0209] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In someembodiments, the antibodies are recombmantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and / or that may not be produced by enzyme digestion of a naturally-occurring intact antibody. In some embodiments, the antibody fragments are scFvs.

[0210] A “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody, refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non- human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0211] In some embodiments, the CAR contains an antibody or an antigen-binding fragment (e.g. scFv) that specifically recognizes an antigen, such as an intact antigen, expressed on the surface of a cell. In some embodiments, the antibody or an antigen-binding fragment (e g. scFv) is an anti-BCMA antibody or antigen-binding fragment. In some embodiments, the antibody or an antigen-binding fragment (e.g. scFv) is an anti-CD19 antibody or antigen-binding fragment. In some embodiments, the antibody or an antigen-binding fragment (e.g. scFv) is an anti-CD20 antibody or antigen-binding fragment. In some embodiments, the antibody or an antigen-binding fragment (e.g. scFv) is an anti-CD22 antibody or antigen-binding fragment. In some embodiments, the antibody or an antigen-binding fragment (e.g. scFv) is an anti-CD38 antibody or antigen-binding fragment. In some embodiments, the antibody or an antigen-binding fragment (e.g. scFv) is an anti-CD70 antibody or antigen-binding fragment. In some embodiments, the antibody or an antigen-binding fragment (e.g. scFv) is an anti-CD138 antibody or antigen-binding fragment. In some embodiments, the antibody or an antigen-binding fragment (e.g. scFv) is an anti-EGFR antibody or antigen-binding fragment. In some embodiments, the antibody or an antigen-binding fragment (e.g. scFv) is an anti-GPRC5D antibody or antigen-binding fragment. In some embodiments, the antibody or an antigen-binding fragment (e.g. scFv) is an anti-HER2 antibody or antigen-binding fragment. In some embodiments, the antibody or an antigen-binding fragment (e.g. scFv) is an anti-RORl antibody or antigen-binding fragment. In some embodiments, the antibody or an antigen-binding fragment (e.g. scFv) is an anti- SLAMF7 antibody or antigen-binding fragment.

[0212] In some embodiments, the CAR contains a TCR-like antibody, such as an antibody or an antigen-binding fragment (e.g. scFv) that specifically recognizes an intracellular antigen, such as a tumor-associated antigen, presented on the cell surface as a MHC -peptide complex. In some embodiments, an antibody or antigen-binding portion thereof that recognizes an MHC- peptide complex can be expressed on cells as part of a recombinant receptor, such as an antigenreceptor. Among the antigen receptors are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Generally, a CAR containing an antibody or antigen-binding fragment that exhibits TCR-like specificity directed against peptide-MHC complexes also may be referred to as a TCR-like CAR.

[0213] In some embodiments, the recombinant receptors include recombinant T cell receptors (TCRs) and / or TCRs cloned from naturally occurring T cells. Thus, in some embodiments, the recombinant receptor is a TCR.B. Transmembrane and Intracellular Regions

[0214] In any of the embodiments provided herein, the recombinant receptor includes a transmembrane region. Some embodiments include a transmembrane region from NKG2D or another transmembrane protein. For example, in some embodiments, chimeric receptors that bind to a ligand of NK.G2D comprise the extracellular and transmembrane regions of NKG2D. In several embodiments in which a transmembrane region is employed, the portion of the transmembrane protein employed retains at least a portion of its normal transmembrane region.

[0215] The antigen-binding domain generally is linked (e.g., by a transmembrane region) to an intracellular signaling region comprising one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and / or signal via another cell surface receptor. Thus, in some embodiments, the antigen-binding domain (e.g., antibody) is linked to a transmembrane region and an intracellular signaling region. In some embodiments, the transmembrane region is fused to the extracellular region. In one embodiment, a transmembrane region that naturally is associated with one of the regions in the receptor, e.g., CAR, is used. In some instances, the transmembrane region is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane regions of the same or different surface membrane proteins to minimize interactions w ith other members of the receptor complex.

[0216] The transmembrane region in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the region in some aspects is derived from any membrane -bound or transmembrane protein. Transmembrane regions include, but are not limited to, those derived from (comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and / or transmembrane regions containing functional variants thereof such as those retaining a substantial portion of the structural, e.g., transmembrane, properties thereof. In some embodiments, the transmembrane region is a transmembrane region derived from CD4, CD28. or CD8, e.g.. CD8alpha, or functional variant thereof. Alternatively, the transmembrane region in some embodiments is synthetic (e.g., de novo synthesized and / or synthesized combinations of sub-regions of two or more transmembrane regions provided for herein).

[0217] In several embodiments, however, the transmembrane region comprises at least a portion of CD8, a transmembrane glycoprotein normally expressed on both T cells and NK cells. In several embodiments, the transmembrane region comprises CD8a. In several embodiments, the transmembrane region comprises a CD8 (e.g., CD8a) hinge and a CD8 (e.g., CD8a) transmembrane protein. In several embodiments, the transmembrane region comprises a hinge, e.g. a CD8a hinge. In several embodiments, the “hinge” of CD8a comprises the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the CD8a hinge is modified or truncated. In several embodiments, the CD8a hinge comprises an amino acid sequence that has at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO:6.

[0218] In several embodiments, the transmembrane region comprises a CD8a transmembrane protein. In several embodiments, the CD8a transmembrane protein comprises the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the CD8a transmembrane protein is modified or truncated. In several embodiments, the CD8a transmembrane protein comprises an amino acid sequence that has at least 70%, at least 75%. at least 80%, at least 85%. at least 90%, at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:8.

[0219] In some embodiments, the transmembrane region comprises a CD8a hinge and a CD8a transmembrane protein. In some embodiments, the transmembrane region comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the transmembrane region comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10.

[0220] In some embodiments, the transmembrane region comprises a CD28 transmembrane protein or a fragment thereof. In several embodiments, the CD28 transmembrane protein comprises the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the CD28 transmembrane protein is modified or truncated. In several embodiments, the CD28 transmembrane protein comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11.

[0221] In any of the embodiments provided herein, the recombinant receptor includes an intracellular signaling region comprising one or more intracellular signaling components. Among the intracellular signaling regions are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and / or a signal through a costimulatory receptor alone.

[0222] The receptor, e.g., the CAR, generally includes at least one intracellular signaling component or components. In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g.. CD3zeta (CD3z) chain. Thus, in some aspects, the antigen-binding portion is linked (e.g.. by atransmembrane region) to one or more intracellular signaling components. In some embodiments, upon ligation of the CAR or other chimeric receptor, the intracellular (cytoplasmic) signaling region of the receptor activates at least one of the normal effector functions or responses of the immune cell, e.g., immune cell engineered to express the CAR. For example, in some contexts, tire CAR induces a function such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling component of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling region includes the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptors to initiate signal transduction following antigen receptor engagement.

[0223] In the context of a natural TCR. full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the receptor.

[0224] T cell activation is. in some aspects, described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary' cytoplasmic signaling sequences). In some aspects, the receptor includes one or both of such signaling components.

[0225] In some aspects, the receptor includes a primary' cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary' cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.

[0226] For example, immune cells engineered according to several embodiments disclosed herein may comprise at least one subunit of the CD3 T cell receptor complex (or a fragment thereof). In several embodiments, the intracellular signaling region comprises a CD3zeta domain. In several embodiments, the CD3zeta domain comprises the amino acid sequence set forth in SEQ ID NO: 18. In several embodiments, the CD3zeta domain is truncated or modified. In some embodiments, the CD3zeta domain comprises an amino acid sequence that has at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18.

[0227] In some embodiments, the intracellular signaling region includes a primary intracellular signaling domain (e.g., CD3 zeta) and a costimulatory signaling region, such as CD28, 4-1BB, 0X40, DAP 10, or ICOS protein or portion thereof. In several embodiments, unexpectedly enhanced signaling is achieved through the use of multiple signaling domains whose activities act synergistically.

[0228] In several embodiments, the intracellular signaling domain comprises a costimulatory signaling region of 0X40. In several embodiments, the 0X40 co-stimulatoiy signaling region comprises the amino acid sequence set forth in SEQ ID NO: 14. In several embodiments, the 0X40 co-stimulatory signaling region is truncated or modified. In several embodiments, the 0X40 co-stimulatory signaling region comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to die amino acid sequence set forth in SEQ ID NO: 14. In several embodiments, 0X40 is used as the sole co-stimulatory signaling region in the construct, however, in several embodiments. 0X40 can be used with one or more other co-stimulatory signaling regions. By way of example, combinations of CD28. 0X40, 4-1BB, and / or CD3zeta are used in some embodiments.

[0229] In several embodiments, the intracellular signaling region comprises a co- stimulatory signaling region of 4-1BB (CD137). In several embodiments, the 4-1BB co-stimulatory signaling region comprises the amino acid sequence set forth in SEQ ID NO: 16. In several embodiments, the 4-1BB co-stimulatory signaling region is truncated or modified. In several embodiments, the 4- IBB co-stimulatory signaling region comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16. In several embodiments, 4-1BB is used as the sole co-stimulatory signaling region in the construct, however, in several embodiments, 4- IBB can be used with one or more other co-stimulatory signaling regions. By way of example, combinations of CD28, 0X40, 4-1BB, and / or CD3zeta are used in some embodiments.

[0230] In several embodiments, the intracellular signaling region comprises a co- stimulatory signaling region of CD28. In several embodiments, the CD28 co-stimulatory signaling region comprises the amino acid sequence set forth in SEQ ID NO:73. In several embodiments, the CD28 co-stimulatory' signaling region is truncated or modified. In several embodiments, the CD28 co-stimulatory signaling region comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to Ore amino acid sequence set forth in SEQ ID NO:73. In several embodiments, CD28 is used as the sole co-stimulatory signaling region in the construct, however, in several embodiments. CD28 can be used with one or more other co-stimulatory signaling regions. By way of example, combinations of CD28. 0X40, 4-1BB, and / or CD3zeta are used in some embodiments.

[0231] In any of the provided embodiments, the nucleic acid encoding the recombinant receptor, or a portion thereof, is codon-optimized e.g., codon optimized for humans. In some embodiments, the polynucleotides are optimized, or contain certain features designed for optimization, such as for codon usage, to reduce RNA heterogeneity and / or to modify, e.g., increaseor render more consistent among cell product lots, expression, such as surface expression, of the encoded receptor. In some embodiments, polynucleotides, encoding chimeric receptors, are modified as compared to a reference polynucleotide, such as to remove cryptic or hidden splice sites, to reduce RNA heterogeneity. In some embodiments, polynucleotides, encoding chimeric receptors, are codon optimized, such as for expression in a mammalian, e.g., human, cell such as in a human immune cell. In some aspects, the modified polynucleotides result in improved, e.g., increased or more uniform or more consistent level of, expression, e.g., surface expression, when expressed in a cell.III. Nucleic Acids, Vectors, and Engineered Cells

[0232] Also provided are methods, uses, polynucleotides, nucleic acid sequences, compositions, and kits for producing the genetically engineered cells. In some aspects, the genetic engineering involves introduction of a nucleic acid sequence, or a polynucleotide or vector comprising die same, encoding the genetically engineering component or other component into the cell, such as a component encoding a gene-disrupting protein or nucleic acid. Among additional nucleic acids, e.g., genes for introduction, are those to improve die efficacy of therapy, such as by promoting the viability, persistence, and / or function of administered cells; to provide a genetic marker for selection and / or evaluation of the cells; and / or to improve safety.A. Cell Types

[0233] Some embodiments of the methods and compositions provided herein relate to a cell engineered to express a transmembrane protein (e.g., a membrane-bound interleukin) as described herein. In some embodiments, the cell is an immune cell. Various types of immune cells can be used, such as dendritic cells, monocytes, macrophages, T cells, Natural Killer (NK cells), NK T cells, B cells, or combinations thereof, as described in more detail below. In some embodiments, the cell is further engineered to express a recombinant receptor, e.g., CAR, that binds to an antigen. In some aspects, the cell is a stimulatory cell. In some aspects, the cell is an immortalized cell.

[0234] There are provided for herein polynucleotides and vectors that encode transmembrane proteins. In some aspects, the polynucleotide or vector further encodes (e g., bicistronically encodes) a recombinant receptor, such as a CAR. For example, some embodiments include a polynucleotide or vector that encodes, for example transmembrane protein that is a membrane -bound IL2 or a membrane-bound IL 15 transmembrane protein, to increase the survival, persistence, and / or cytotoxicity of the cell expressing the transmembrane protein. Methods of treating a disease or condition and other uses of such cells are also provided for herein. Also provided are genetically engineered cells (e.g., immune cells) expressing such transmembrane proteins.

[0235] In several embodiments, cells are genetically engineered to have enhanced surv ival, proliferation, persistence, and / or cytotoxic effects against target cells. For example, a cell of die immune sy stem may be engineered to include a transmembrane protein, and optionally a recombinant receptor, as described herein. In several embodiments, white blood cells or leukocytes,are used, since their native function is to defend the body against growth of abnormal cells and infectious disease. There are a variety of types of white bloods cells that serve specific roles in the human immune system and are therefore a preferred starting point for the engineering of cells disclosed herein. White blood cells include granulocytes and agranulocytes (presence or absence of granules in the cytoplasm, respectively). Granulocytes include basophils, eosinophils, neutrophils, and mast cells. Agranulocytes include lymphocytes and monocytes. Cells such as those that follow or are otherwise described herein may be engineered to include a transmembrane protein, or a polynucleotide or vector encoding the transmembrane protein. In several embodiments, the cells are engineered to co-express (such as bicistronically express) a recombinant receptor (e.g., CAR). In alternative embodiments, cells of a stimulatory cell line (e.g.. K562 cells or K562-derived cells) are engineered to have enhanced ability to stimulate or feed other cells. For example, in some embodiments, stimulatory cells (e.g.. K562 cells or K562-derived cells) are engineered to express a transmembrane protein as provided herein. i. Monocytes

[0236] In some embodiments, the cells comprise monocytes. Monocytes are a subtype of leukocyte. Monocytes can differentiate into macrophages and myeloid lineage dendritic cells. Monocytes arc associated with the adaptive immune system and serve the main functions of phagocytosis, antigen presentation, and cytokine production. Phagocytosis is the process of uptake of cellular material, or entire cells, followed by digestion and destruction of the engulfed cellular material.

[0237] In some embodiments, a monocyte is positive for cell surface expression of a marker selected from among the group consisting of CCR2, CCR5, CDllc, CD14, CD16, CD62L, CD68+, CX3CR1, HLA-DR. or any combination thereof. In some embodiments, a monocyte is positive for cell surface expression of CD 14. In some embodiments, a monocyte is positive for cell surface expression of CCR2. In some embodiments, a monocyte is positive for cell surface expression of CCR5. In some embodiments, a monocyte is positive for cell surface expression of CD62L.

[0238] In several embodiments, monocytes are used in connection with one or more additional engineered cells as disclosed herein. Some embodiments of the methods and compositions described herein relate to a monocyte that expresses a transmembrane protein or a polynucleotide or vector encoding the transmembrane protein. In some embodiments, the monocyte further expresses a recombinant receptor, such as a CAR. In some embodiments, the monocytes engineered to express a transmembrane protein (e.g., mbIL2 or mbIL15) are engineered to also express (e.g., bicistronically express) a recombinant receptor (e.g.. a CAR). Thus, in some embodiments, the monocytes are engineered to bicistronically express the CAR and transmembrane protein.

[0239] In some embodiments, the monocytes are autologous cells. In some embodiments, the monocytes are allogeneic cells. In some embodiments, the monocytes are obtained from a healthy donor.ii. Lymphocytes

[0240] In some embodiments, the cells comprise lymphocytes. Lymphocytes, the other primary sub-type of leukocyte include T cells (cell-mediated, cytotoxic adaptive immunity), natural killer cells (cell-mediated, cytotoxic innate immunity), and B cells (humoral, antibody -driven adaptive immunity). While B cells are engineered according to several embodiments, disclosed herein, several embodiments also relate to engineered T cells or engineered NK cells (mixtures of T cells and NK cells are used in some embodiments, either from the same donor, or different donors). Thus, in some embodiments, the cells comprise T cells. In some embodiments, the cells comprise NK cells. In some embodiments, the cells comprise T cells and NK cells. In some embodiments, the cells comprise B cells.

[0241] In several embodiments, lymphocytes are used in connection with one or more additional engineered cells as disclosed herein. Some embodiments of the methods and compositions described herein relate to a lymphocyte that expresses a transmembrane protein or a polynucleotide or vector encoding the transmembrane protein. In some embodiments, the lymphocytes further express a recombinant receptor, such as a CAR. In some embodiments, the lymphocytes engineered to express a transmembrane protein (e.g., mbIL2 or mbIL15) are engineered to also express (e.g., bicistronically express) a recombinant receptor (e.g., a CAR). Thus, in some embodiments, the lymphocytes arc engineered to bicistronically express the CAR and transmembrane protein.

[0242] In some embodiments, the lymphocytes are autologous cells. In some embodiments, the lymphocytes are allogeneic cells. In some embodiments, the lymphocytes are obtained from a healthy donor. iii. T Cells

[0243] In some embodiments, the cells comprise T cells. T cells are distinguishable from other lymphocytes sub-types (e.g., B cells or NK cells) based on the presence of a T-cell receptor on the cell surface.

[0244] T cells can be divided into various different subtypes, including effector T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cell, mucosal associated invariant T cells and gamma delta T cells. In some embodiments, a specific subtype of T cell is engineered. In some embodiments, a T cell is positive for cell surface expression of a marker selected from among the group consisting of CD3, CD4, and / or CD8. In some embodiments, a T cell is positive for cell surface expression of CD3. In some embodiments, a T cell is positive or cell surface expression of CD4. In some embodiments, a T cell is positive or cell surface expression of CD8.

[0245] In some embodiments, CD3+ T cells are engineered. In some embodiments, CD4+ T cells are engineered. In some embodiments, CD8+ T cells are engineered. In some embodiments, regulatory T cells are engineered. In some embodiments, gamma delta T cells are engineered. In some embodiments, a mixed pool of T cell subtypes is engineered. For example, in someembodiments, CD4+ and CD8+ T cells are engineered. In some embodiments, there is no specific selection of a type of T cells to be engineered to express the transmembrane proteins disclosed herein. In several embodiments, specific techniques, such as use of cytokine stimulation are used to enhance expansion / collection of T cells with a specific marker profile. For example, in several embodiments, activation of certain human T cells, e.g. CD4+ T cells, CD8+ T cells is achieved through use of CD3 and / or CD28 as stimulatory' molecules.

[0246] In several embodiments, there is provided a method of treating or preventing a disease or condition, comprising administering a therapeutically effective amount of T cells expressing the transmembrane protein (and optionally, a recombinant receptor) as described herein. In several embodiments, there is provided a method of treating or preventing a disease or condition comprising administering T cells expressing a transmembrane protein as described herein.

[0247] In several embodiments, T cells are used in connection with one or more additional engineered cells as disclosed herein. Some embodiments of the methods and compositions described herein relate to a T cell that expresses a transmembrane protein or a polynucleotide or vector encoding the transmembrane protein. In some embodiments, the T cell further expresses a recombinant receptor, such as a CAR. In some embodiments, the T cells engineered to express a transmembrane protein (e.g., mbIL2 or mbIL15) are engineered to also express (e.g., bicistronically express) a recombinant receptor (e.g., a CAR). Thus, in some embodiments, the T cells are engineered to bicistronically' express the CAR and transmembrane protein.

[0248] In some embodiments, the T cells are autologous cells. In some embodiments, the T cells are allogeneic cells. In some embodiments, the T cells are obtained from a healthy donor. iv. NK Cells

[0249] In some embodiments, the cells comprise natural killer (NK) cells. In several embodiments, there is provided a method of treating or preventing a disease or condition, comprising administering natural killer (NK) cells expressing a transmembrane protein as described herein. In some embodiments, the NK cells further express a recombinant receptor. In several embodiments, NK cells are preferred because the natural cytotoxic potential of NK cells is relatively high. In several embodiments, it is unexpectedly beneficial that the engineered cells disclosed herein exhibit increased survival, proliferation, persistence, and / or cytotoxicity against target cells.

[0250] In some embodiments, a NK cell is positive for cell surface expression of a marker selected from among the group consisting of CCR7. CD16, CD56. CD57, CD11, CX3CR1, a Killer Ig-like receptor (KIR), NKp30, NKp44. NKp46. or any combination thereof. In some embodiments, a NK cell is positive for cell surface expression of CD 16. In some embodiments, a NK cell is positive for cell surface expression of CD56. In some embodiments, a NK cell is positive for cell surface expression of a Killer Ig-like receptor.

[0251] Some embodiments of the methods and compositions described herein relate to NK cells engineered to express the transmembrane protein (and optionally, a recombinant receptor) asdescribed herein. In several embodiments, there is provided a method of treating or preventing a disease or condition comprising administering NK cells expressing a transmembrane protein as described herein. In several embodiments, NK cells are used in connection with one or more additional engineered cells as disclosed herein. Some embodiments of the methods and compositions described herein relate to a NK cell that expresses a transmembrane protein or a polynucleotide or vector encoding the transmembrane protein. In some embodiments, the NK cell further expresses a recombinant receptor, such as a CAR. In some embodiments, the NK cells engineered to express a transmembrane protein (e.g.. mbIL2 or mbIL15) are engineered to also express (e.g., bicistronically express) a recombinant receptor (e.g., a CAR). Thus, in some embodiments, the NK cells are engineered to bicistronically express the CAR and transmembrane protein.

[0252] In some embodiments, the NK cells are autologous cells. In some embodiments, the NK cells are allogeneic cells. In some embodiments, the NK cells are obtained from a healthy donor.

[0253] In some embodiments, the NK cells are derived from cell line NK-92. NK-92 cells are derived from NK cells, but lack major inhibitory receptors displayed by normal NK cells, while retaining the majority of activating receptors. Some embodiments of NK-92 cells described herein related to NK-92 cell engineered to silence certain additional inhibitory receptors, for example, SMAD3, allowing for upregulation of interferon-y (IFNy), granzyme B. and / or perforin production. Additional information relating to the NK-92 cell line is disclosed in WO 1998 / 49268 and U.S. Patent Application Publication No. 2002-0068044 and incorporated in their entireties herein by reference.

[0254] In some embodiments, the NK cells are used in combination with T cells. Thus, in some embodiments, the cells comprise T cells and NK cells (either from the same donor or from different donors). For example, in one embodiment, primary NK cells are used in combination with primary T cells. v. Hematopoietic Stem Cells

[0255] In some embodiments, the cells comprise hematopoietic stem cells (HSCs). In some embodiments. HSCs are used in the methods disclosed herein. In some embodiments, a HSC is positive for cell surface expression of a marker selected from among the group consisting of CD34, CD59, and CD90. In some embodiments, a HSC is positive for cell surface expression of CD34. In some embodiments, a HSC is positive for cell surface expression of CD59. In some embodiments, a HSC is positive for cell surface expression of CD90.

[0256] In several embodiments, HSCs are used in connection with one or more additional engineered cells as disclosed herein. Some embodiments of the methods and compositions described herein relate to a HSC that expresses a transmembrane protein (e.g., mbIL2 or mbIL15) or a polynucleotide or vector encoding the transmembrane protein. In some embodiments, the HSC further expresses a recombinant receptor, such as a CAR. In some embodiments, the HSCs engineered to express a transmembrane protein (e.g., mbIL2 or mbIL15) are engineered to also express (e.g.,bicistronically express) a recombinant receptor (e.g., a CAR). Thus, in some embodiments, the HSCs are engineered to bicistronically express the CAR and transmembrane protein.

[0257] In some embodiments, the HSCs are autologous cells. In some embodiments, the HSCs are allogeneic cells. In some embodiments, the HSCs are obtained from a healthy donor. vi. Induced Pluripotent Stem Cells

[0258] In some embodiments, the cells are derived (differentiated) from pluripotent stem cells (PSCs). In some embodiments, the cells are immune cells (e.g., NK and / or T cells) derived from induced pluripotent stem cells (iPSCs) are used. For example, in some embodiments, NK cells are derived from iPSCs. In some embodiments, induced pluripotent stem cells (iPSCs) are used in a method disclosed herein. iPSCs are used, in several embodiments, to leverage their ability to differentiate and derive into non-pluripotent cells, including, but not limited to, CD34 cells, hemogenic endothelium cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, and B cells comprising one or several genetic modifications at selected sites through differentiating iPSCs or less differentiated cells comprising die same genetic modifications at the same selected sites. In several embodiments, the iPSCs are used to generate iPSC-derived NK or T cells. In several embodiments, the iPSCs are used to generate iPSC-derived NK cells. In several embodiments, the iPSCs are used to generate iPSC-derived T cells.

[0259] Several embodiments of the methods and compositions disclosed herein relate to induced pluripotent stem cells engineered to express a transmembrane protein. In some embodiments, the iPSCs engineered to express a transmembrane protein (e.g., mbIL2 or mbIL15) are engineered to also express (e.g., bicistronically express) a recombinant receptor (e.g., CAR).

[0260] In several embodiments, the engineered iPSCs are differentiated into NK. T, or other immune cells, such as for use in a composition or method provided herein. In several embodiments, the engineered iPSCs are differentiated into NK cells. In several embodiments, the engineered iPSCs are differentiated into T cells. In several embodiments, the engineered iPSCs are differentiated into NK and T cells. vii. Stimulatory Cells

[0261] In some embodiments, the cells are stimulatory cells or are derived from a stimulatory cell line. In some embodiments, the cells are stimulatory cells. In some embodiments, the cells are used to stimulate one or more other cell types (e.g., immune cells). Thus, in some embodiments, the cells are a stimulatory cell line used to stimulate immune cells (e.g.. NK cells).

[0262] For example, in several embodiments cell lines are used in a co-culture with a population of immune cells that are to be expanded. Such cell lines are referred to herein as "stimulatory cells”. In several embodiments, the stimulatory cells are themselves genetically modified. In some embodiments, the stimulatory cells do not express MHC I molecules, which havean inhibitor}' effect on NK cells. In some embodiments, the stimulator}' cells need not entirely lack MHC I expression, however they may express MHC I molecules at a lower level than a wildtype cell. In additional embodiments, the stimulatory cells also have reduced (or lack) MHC II expression, as well as having reduced (or lacking) MHC I expression. In some embodiments, other cell lines that may initially express MHC class I molecules can be used, in conjunction with genetic modification of those cells to reduce or knock out MHC I expression.

[0263] In several embodiments, the stimulatory cells are immortalized, e.g., a cancer cell line. However, in several embodiments, the stimulatory cells are primary cells. Various cell ty pes can be used as stimulator ’ cells, depending on the embodiment. These include, but are not limited to, K562 cells, certain Wilm’s Tumor cell lines (for example Wilms tumor cell line HFWT), endometrial tumor cells (for example, HHUA). melanoma cells (e.g., HMV-II), hepatoblastoma cells (e.g., HuH- 6), lung small cell carcinoma cells (e.g., Lu-130 and Lu-134-A). neuroblastoma cells (e.g., NB19 and NB69). embryonal carcinoma testis cells (e.g.. NEC14). cervical carcinoma cells (TCO-2). neuroblastoma cells (e.g., TNB1), 721.221 EBV transformed B cell line, among others. In some embodiments, the stimulatory cells are immortalized cells. In some embodiments, the stimulatory cells comprise K562 cells. Thus, in some embodiments, the cells are K562 cells or are derived from the K562 cell line. In some embodiments, the stimulatory cells are K562 cells. In some embodiments, the stimulatory cells are derived from the K562 cell line.

[0264] In some aspects, the stimulatory cells are engineered to express certain stimulatory molecules (e.g. interleukins, CD3, 4-1BBL, etc.) to promote immune cell expansion and activation. Several embodiments of the methods and compositions disclosed herein relate to stimulator ' cells engineered to express a transmembrane protein (e.g., a membrane-bound interleukin). In some embodiments, the stimulatory cells are engineered to express a mbIL2 transmembrane protein. In some embodiments, the stimulator}' cells are engineered to express any of the mbIL2 transmembrane proteins as provided herein. In some embodiments, the stimulator}' cells arc engineered to express a mbIL15 transmembrane protein. In some embodiments, the stimulatory cells are engineered to express any of the mbIL15 transmembrane proteins as provided herein. In some embodiments, the stimulatory cells also express 4-1BBL. In some embodiments, the stimulatory cells express 4-1BBL and a transmembrane protein (e.g., mbIL15). In some embodiments, tire stimulatory cells express 4- 1BBL and a transmembrane protein as provided herein (e.g., mbIL15).

[0265] Thus, provided herein are stimulatory cells, such as K562 cells, expressing a mbIL15 transmembrane protein as described herein. Also provided herein are stimulatory cells, such as K562 cells, expressing a mbIL2 transmembrane protein as described herein. In some embodiments, the stimulatory cells express a mbIL15 transmembrane protein and a mbIL2 transmembrane protein.B. Preparation of Cells for Genetic Engineering

[0266] In some embodiments, preparation of the engineered cells includes one or more culture and / or preparation steps. The cells for introduction of the transmembrane protein (e.g., mbIL2or mbIL15) may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the sample is an apheresis (e.g., leukapheresis) sample.

[0267] In some embodiments, the subject from which the cells are isolated is one not having the disease or condition in need of a cell therapy or not to which a cell therapy will be administered. In some embodiments, the cells are isolated from a subject that is different than the subject in need of a cell therapy or to which a cell therapy will be administered. Thus, in some embodiments, the cells are allogeneic to the subject to whom they are administered.

[0268] In some embodiments, the subject from which the cells are isolated is one having the disease or condition or in need of a cell therapy or to which a cell therapy will be administered. In some embodiments, the cells are isolated from the subject to which a cell therapy will be administered. Thus, in some embodiments, the cells are autologous to the subject to whom they are administered.

[0269] The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g.. transduction with viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0270] In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample or is or is derived from an apheresis (e.g., a leukapheresis) product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0271] The cells in some embodiments are primary cells, e.g., primary human cells. In some embodiments, the cells are immune cells, e.g. primary NK cells or primary T cells. In some embodiments, the cells are NK cells (e.g., primary NK cells).

[0272] In some embodiments, isolation of the cells includes one or more preparation and / or non-affinity -based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components.

[0273] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis (e.g., leukapheresis). The samples, in some aspects, contain lymphocytes, including NK cells, T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects contain cells other than red blood cells and platelets.

[0274] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity -based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

[0275] Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use. and / or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

[0276] The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular ty pe, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0277] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality' of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

[0278] For example, in some aspects, NK cells or specific subpopulations thereof, such as cells positive or expressing high levels of one or more surface markers, e.g., CD56+, CCR7+, CD16+. CD57+. CD11+, CX3CR1+. a Killer Ig-like receptor (KIR) +. NKp30+. NKp44+, or NKp46+ NKcells, are isolated by positive or negative selection techniques. For example, CD56+ NK cells can be positively selected using anti-CD56 conjugated magnetic beads. Additionally, T cells can be removed from cell populations by negative selection for CD3 expression. For example, NK cells or subpopulations thereof can be substantially depleted of T cells by negative selection for CD3- expressing cells.

[0279] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD 14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations.

[0280] In some embodiments, the cells (e.g., NK cells) are expanded in culture prior to, during, and / or following genetic engineering. In some embodiments, the cells are expanded in culture prior to genetic engineering. In some embodiments, the cells are expanded in culture following genetic engineering. In some embodiments, the cells are expanded in culture prior to and following genetic engineering. Methods for expanding cells are known in the art and include any of those described in US Patent Nos. 7,435,596 and 8.026,097; and International Patent Application Nos. PCT / SG2018 / 050138; PCT / US2020 / 044033; PCT / US2021 / 071330; PCT / US2022 / 074164, and PCT / US2024 / 013779.

[0281] In some embodiments, expanding the cells in culture comprises co-culturing the cells with stimulatory cells. In some embodiments, the stimulatory cells express IL15 (e.g., membranebound IL15) and / or 4-1BBL. In some embodiments, the stimulatory cells express IL15 (e.g., membrane -bound IL 15.) In some embodiments, the stimulatory cells express 4-1 BBL. In some embodiments, the stimulatory cells express membrane-bound interleukin 15 (mbIL15) and 4-1BBL. In some embodiments, the IL 15 transmembrane protein is any of the transmembrane proteins as provided herein. Thus, in some embodiments, the stimulator}' cells express an IL 15 transmembrane protein (mbIL15) as provided herein. In some embodiments, the stimulatory' cells do not express MHCI molecules. In some embodiments, the stimulatory cells do not express MHCII molecules. In some embodiments, the stimulatory cells are immune cells. In some embodiments, the stimulatory cells are K562 cells. Engineered stimulatory cells are disclosed in, for example, International Patent Application PCT / SG2018 / 050138.

[0282] In some embodiments, expanding the cells in culture comprises culturing the cells in the presence of IL2, IL12, and / or IL18. In some embodiments, the cells are cultured in the presence of IL2. In some embodiments, the cells are cultured in the presence of IL 12. In some embodiments, the cells are cultured in the presence of IL 18. In some embodiments, the cells are cultured in the presence of IL12 and IL18. In some embodiments, the cells are cultured in the presence of IL2. IL12. and IL18. In some embodiments, the preparation methods include steps for freezing, e.g.. cryopreserving, thecells, either before or after isolation, engineering, and / or expansion. In some embodiments, the cells are suspended in a freezing solution. Thus, provided are compositions comprising cells produced by any of the embodiments described herein and a cryopreservation solution. Any of a variety of known freezing solutions and parameters in some aspects may be used.C. Genetic Editing

[0283] Provided herein are cells (e.g., immune cells) expressing a transmembrane protein as described herein and methods of producing and using the same. In some aspects, the cells are also engineered to express a recombinant receptor. In some aspects, in connection with the provided methods, the cells are genetically edited to reduce expression of a target protein. In some aspects, the methods comprise genetically editing the cells, such as to reduce expression of a target protein. Expression of a target protein can be reduced by disrupting a gene (a target gene) encoding the target protein or a portion thereof. Alternatively, in some embodiments, expression of a target protein is reduced by an inhibitory nucleic acid molecule, such as one that is complementary to, targets, inhibits and / or binds a gene (a target gene) encoding the target protein or a portion thereof. In some of any such embodiments, the inhibitory nucleic acid molecule includes an RNA interfering agent. In some of any such embodiments, the inhibitor} nucleic acid is or contains or encodes a small interfering RNA (siRNA), a microRNA-adapted shRNA, a short hairpin RNA (shRNA), a hairpin siRNA, a precursor microRNA (pre-miRNA) or a microRNA (miRNA). In some of any such embodiments, the inhibitory nucleic acid molecule contains a sequence complementary to a target protein-encoding nucleic acid. In some of any such embodiments, the inhibitory nucleic acid molecule contains an antisense oligonucleotide complementary to a target protein-encoding nucleic acid.

[0284] It is contemplated that the cells can be genetically edited at any point prior to, during, and / or after genetic engineering of the cells to express a transmembrane protein and / or a recombinant receptor. For example, in some embodiments, the cells are genetically edited prior to genetic engineering of the cells to express a transmembrane protein. In some embodiments, the cells arc genetically edited concurrent with genetic engineering of the cells to express a transmembrane protein. In some embodiments, the cells are genetically edited after genetic engineering of the cells to express a transmembrane protein. In some embodiments, the cells are genetically edited prior to genetic engineering of the cells to express a recombinant receptor. In some embodiments, the cells are genetically edited concurrent with genetic engineering of the cells to express a recombinant receptor. In some embodiments, the cells are genetically edited after genetic engineering of the cells to express a recombinant receptor. In several embodiments, genetic engineering and gene editing are substantially contemporaneous.

[0285] As discussed below, in several embodiments, gene editing is employed to reduce or eliminate expression of a target protein, for example by disrupting a gene encoding the protein.

[0286] In several embodiments, gene editing can reduce transcription of a target gene by about 30%, about 40%, about 50%. about 60%. about 70%, about 75%, about 80%. about 85%. about90%, about 95%, about 97%, about 98%, about 99%, or more (including any amount between those listed). In several embodiments, gene editing reduces transcription of a target gene by at least about 30%. In several embodiments, gene editing reduces transcription of a target gene by at least about40%. In several embodiments, gene editing reduces transcription of a target gene by at least about50%. In several embodiments, gene editing reduces transcription of a target gene by at least about60%. In several embodiments, gene editing reduces transcription of a target gene by at least about70%. In several embodiments, gene editing reduces transcription of a target gene by at least about80%. In several embodiments, gene editing reduces transcription of a target gene by at least about90%. In several embodiments, the gene is completely knocked out, such that transcription of the target gene is eliminated (undetectable).

[0287] In several embodiments, gene editing can reduce expression of a target protein by about 30%, about 40%, about 50%. about 60%. about 70%, about 75%, about 80%. about 85%. about 90%, about 95%, about 97%, about 98%, about 99%. or more (including any amount between those listed). In several embodiments, gene editing reduces expression of a target protein by at least about 30%. In several embodiments, gene editing reduces expression of a target protein by at least about40%. In several embodiments, gene editing reduces expression of a target protein by at least about50%. In several embodiments, gene editing reduces expression of a target protein by at least about60%. In several embodiments, gene editing reduces expression of a target protein by at least about70%. In several embodiments, gene editing reduces expression of a target protein by at least about80%. In several embodiments, gene editing reduces expression of a target protein by at least about90%. In several embodiments, the gene is completely knocked out, such that expression of the target protein is eliminated (undetectable).

[0288] In several embodiments, an inhibitory nucleic acid molecule (e.g., an RNA interfering agent) can reduce expression of a target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amount between those listed). In several embodiments, the inhibitory nucleic acid molecule reduces expression of a target protein by at least about 30%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of a target protein by at least about 40%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of a target protein by at least about 50%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of a target protein by at least about 60%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of a target protein by at least about 70%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of a target protein by at least about 80%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of a target protein by at least about 90%. In several embodiments, the inhibitory nucleic acid molecule eliminates expression of the target protein, such that it is undetectable.

[0289] In several embodiments, gene editing is used to “knock in” or otherwise increase transcription of a target gene. In several embodiments, transcription of a target gene is increased by about 30%, about 40%, about 50%. about 60%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, about 97%, about 98%, about 99%. or more (including any amount between those listed). In several embodiments, transcription of a target gene is increased by at least about 30%. In several embodiments, transcription of a target gene is increased by at least about 40%. In several embodiments, transcription of a target gene is increased by at least about 50%. In several embodiments, transcription of a target gene is increased by at least about 60%. In several embodiments, transcription of a target gene is increased by at least about 70%. In several embodiments, transcription of a target gene is increased by at least about 80%. In several embodiments, transcription of a target gene is increased by at least about 90%. In several embodiments, transcription of a target gene is increased by at least about 100%.

[0290] In several embodiments, gene editing is used to “knock in” or otherwise enhance expression of a target protein. In several embodiments, expression of a target protein can be enhanced by about 30%. about 40%. about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%. about 98%. about 99%, or more (including any amount between those listed). In several embodiments, expression of a target protein is increased by at least about30%. In several embodiments, expression of a target protein is increased by at least about 40%. In several embodiments, expression of a target protein is increased by at least about 50%. In several embodiments, expression of a target protein is increased by at least about 60%. In several embodiments, expression of a target protein is increased by at least about 70%. In several embodiments, expression of a target protein is increased by at least about 80%. In several embodiments, expression of a target protein is increased by at least about 90%. In several embodiments, expression of a target protein is increased by at least about 100%.

[0291] As discussed in more detail below, a variety of approaches can be employed in a given embodiment to improve or alter one or more characteristics of cells for immunotherapy. Genetic editing can be used to reduce, eliminate (e.g., knockout), or increase expression of a target gene. For example, the transcription of the target gene and / or the translation of a protein encoded by the target gene (e.g., a target protein) can be reduced, eliminated (e.g., knocked out), or increased. The target gene can be implicated in the immune functionality' of the cell or be a part of a signaling pathway for which an increase or decrease in function is desired. Further detailed below are various gene targets. Disruption of certain genes in (e.g., NK cells) can increase persistence of those cells.

[0292] In several embodiments, genetic editing (whether knock out or knock in) of a target gene is accomplished through targeted introduction of DNA breakage, and a subsequent DNA repair mechanism. In several embodiments, double strand breaks of DNA are repaired by non-homologous end joining (NHEJ). wherein enzymes are used to directly join the DNA ends to one another to repair the break. NHEJ is an error-prone process. In general, in the absence of a repair template, the NHEJprocess re-ligates the ends of the cleaved DNA strands, which frequently results in nucleotide deletions and insertions at die cleavage site. In several embodiments, however, double strand breaks are repaired by homology directed repair (HDR), which is advantageously more accurate, thereby allowing sequence specific breaks and repair. HDR uses a homologous sequence as a template for regeneration of missing DNA sequences at the break point, such as a vector with the desired genetic elements (e.g., an insertion element to disrupt the coding sequence of a TCR subunit) within a sequence that is homologous to the flanking sequences of a double strand break. This will result in the desired change (e.g.. insertion) being inserted at the site of the DSB. The HDR pathway can occur by way of the canonical HDR pathway or the alternative HDR pathway. Unless otherwise indicated, the term "HDR" or ‘"homology -directed repair” as used herein encompasses both canonical HDR and alternative HDR.

[0293] Canonical HDR or ""canonical homology-directed repair” or “cHDR,” are used interchangeably, and refers to the process of repairing DNA damage using a homologous nucleic acid (e.g., an endogenous homologous sequence, such as a sister chromatid; or an exogenous nucleic acid, such as a donor template). Canonical HDR typically acts when there has been a significant resection at the DSB, forming at least one single-stranded portion of DNA. In a normal cell, canonical HDR typically involves a series of steps such as recognition of the break, stabilization of the break, resection, stabilization of single-stranded DNA, formation of a DNA crossover intermediate, resolution of the crossover intermediate, and ligation. The canonical HDR process requires RAD51 and BRCA2, and the homologous nucleic acid, e.g., repair template, is typically double-stranded. In canonical HDR, a double-stranded polynucleotide, e.g., a double-stranded repair template, is introduced, which comprises a sequence that is homologous to the targeting sequence, and which will either be directly integrated into the targeting sequence or will be used as a template to insert the sequence, or a portion the sequence, of the repair template into the target gene. After resection at the break, repair can progress by different pathways, e.g., by the double Holliday junction model (also referred to as the double strand break repair, or DSBR, pathway), or by the synthesis-dependent strand annealing (SDSA) pathway.

[0294] In the double Holliday junction model, strand invasion occurs by the two single stranded overhangs of the targeting sequence to the homologous sequences in the double-stranded polynucleotide, e.g., double stranded donor template, which results in the formation of an intermediate with two Holliday junctions. The junctions migrate as new DNA is synthesized from the ends of the invading strand to fill the gap resulting from the resection. The end of the newly synthesized DNA is ligated to the resected end, and the junctions are resolved, resulting in the insertion at the targeting sequence, or a portion of the targeting sequence that includes the gene variant. Crossover with the polynucleotide, e.g., repair template, may occur upon resolution of the junctions.

[0295] In the SDSA pathway, only one single stranded overhang invades the polynucleotide, e.g., donor template, and new DNA is synthesized from the end of the invading strand to fill the gap resulting from resection. The newly synthesized DNA then anneals to the remaining single stranded overhang, new DNA is synthesized to fill in the gap, and the strands are ligated to produce the modified DNA duplex.

[0296] Alternative HDR, or “alternative homology -directed repair,” or “alternative HDR,” are used interchangeably, and refers, in some embodiments, to the process of repairing DNA damage using a homologous nucleic acid (e.g., an endogenous homologous sequence, such as a sister chromatid; or an exogenous nucleic acid, such as a repair template). Alternative HDR is distinct from canonical HDR in that the process utilizes different pathways from canonical HDR, and can be inhibited by the canonical HDR mediators. RAD51 and BRCA2. Moreover, alternative HDR is also distinguished by the involvement of a single-stranded or nicked homologous nucleic acid template, e.g., repair template, whereas canonical HDR generally involves a double-stranded homologous template. In the alternative HDR pathway, a single strand template polynucleotide, e.g., repair template, is introduced. A nick, single strand break, or DSB at the cleavage site, for altering a desired target site, e.g., a gene variant in a target gene, is mediated by a nuclease molecule, and resection at the break occurs to reveal single stranded overhangs. Incorporation of the sequence of the template polynucleotide, e.g., repair template, to alter the target site of the DNA typically occurs by the SDSA pathway, as described herein. In some embodiments, HDR is carried out by introducing, into a cell, one or more agent(s) capable of inducing a DSB, and a repair template, e.g., a single-stranded oligonucleotide. The introducing can be carried out by any suitable delivery. The conditions under which HDR is allowed to occur can be any conditions suitable for carrying out HDR in a cell.

[0297] In several embodiments, gene editing is accomplished by one or more of a variety of engineered nucleases. In several embodiments, restriction enzymes are used, particularly when double strand breaks arc desired at multiple regions. In several embodiments, a bioengineered nuclease is used. Depending on the embodiment, one or more of a Zinc Finger Nuclease (ZFN), transcription-activator like effector nuclease (TALEN), meganuclease and / or clustered regularly interspaced short palindromic repeats (CRISPR / Cas9) system are used to specifically edit the genes encoding one or more of the TCR subunits.

[0298] Meganucleases are characterized by their capacity to recognize and cut large DNA sequences (from 14 to 40 base pairs). In several embodiments, a meganuclease from the LAGLID ADG family is used, and is subjected to mutagenesis and screening to generate a meganuclease variant that recognizes a unique sequence(s), such as a specific site in a target gene, or any other target gene disclosed herein. In several embodiments, two or more meganucleases, or functional fragments thereof, are fused to create a hybrid enzyme that recognizes a desired target sequence within the target gene.

[0299] In contrast to meganucleases, ZFNs and TALEN function based on a non-specific DNA cutting catalytic domain which is linked to specific DNA sequence recognizing peptides such as zinc fingers or transcription activator-like effectors (TALEs). Advantageously, the ZFNs and TALENs thus allow sequence-independent cleavage of DNA, with a high degree of sequencespecificity in target recognition. Zinc finger motifs naturally function in transcription factors to recognize specific DNA sequences for transcription. The C-terminal part of each finger is responsible for the specific recognition of the DNA sequence. While the sequences recognized by ZFNs are relatively short, (e.g., ~3 base pairs), in several embodiments, combinations of 2, 3. 4, 5, 6, 7, 8. 9, 10 or more zinc fingers whose recognition sites have been characterized are used, thereby allowing targeting of specific sequences. The combined ZFNs are then fused with the catalytic domain(s) of an endonuclease, such as FokI (optionally a FokI heterodimer), to induce a targeted DNA break.

[0300] Transcription activator-like effector nucleases (TALENs) are specific DNA-binding proteins that feature an array of 33 or 34-amino acid repeats. Like ZFNs. TALENs are a fusion of a DNA cutting domain of a nuclease to TALE domains, which allow for sequence-independent introduction of double stranded DNA breaks with highly precise target site recognition. TALENs can create double strand breaks at the target site that can be repaired by error -prone non-homologous endjoining (NHEJ), resulting in gene disruptions through the introduction of small insertions or deletions. Advantageously. TALENs are used in several embodiments, at least in part due to their higher specificity in DNA binding, reduced off-target effects, and ease in construction of the DNA-binding domain.

[0301] CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are genetic elements that bacteria use as protection against viruses. The repeats are short sequences that originate from viral genomes and have been incorporated into the bacterial genome. Cas (CRISPR associated proteins) process these sequences and cut matching viral DNA sequences. By introducing plasmids containing Cas genes and specifically constructed CRISPRs into eukary otic cells, the eukaryotic genome can be cut at any desired position. Additional information on CRISPR can be found in US Patent Publication No. 2014 / 0068797, which is incorporated by reference herein.

[0302] In several embodiments, CRISPR is used to disrupt a target gene. Depending on the embodiment and which target gene is to be edited, a Class 1 or Class 2 Cas is used. In several embodiments, a Class 1 Cas is used, and the Cas type is selected from the following types: I. IA, IB, IC, ID, IE, IF, IU, III, IIIA. IIIB, IIIC, IIID, IV IVA, IVB, and combinations thereof. In several embodiments, the Cas is selected from the group consisting of Cas3, Cas8a, Cas5, Cas8b. Cas8c, CaslOd. Csel. Cse2. Csyl. Csy2, Csy3, GSU0054, CaslO, Csm2, Cmr5. CaslO, Csxl l, CsxlO, Csfl, and combinations thereof. In several embodiments, the Cas is Cas3. In several embodiments, a Class 2 Cas is used, and the Cas type is selected from the following types: II. IIA, IIB, IIC. V, VI, and combinations thereof. In several embodiments, the Cas is selected from the group consisting of Cas9. Csn2. Cas4. Casl2a (previously known as Cpfl). C2cl, C2c3, Casl3a (previously known as C2c2).Casl3b, Casl3c, CasX, CasY and combinations thereof. In some embodiments, the Cas is Cas9. In some embodiments, class 2 CasX is used, wherein CasX can form a complex with a guide nucleic acid and wherein the complex can bind to a target DNA, and wherein the target DNA comprises a non-target strand and a target strand. In some embodiments, class 2 CasY is used, wherein CasY is capable of binding and modifying a target nucleic acid and / or a polypeptide associated with target nucleic acid.

[0303] Unless indicated otherwise to the contrary, the sequences provided for guide RNAs that are recited using deoxyribonucleotides refer to the target DNA and shall be considered as also referencing those guides used in practice (e.g., employing ribonucleotides, where the ribonucleotide uracil is used in lieu of deoxyribonucleotide thymine or vice-versa where thymine is used in lieu of uracil, wherein both are complementary base pairs to adenine when reciting either an RNA or DNA sequence).

[0304] In some embodiments, editing of a target gene advantageously imparts to the edited cells enhanced expansion, cytotoxicity and / or persistence. For example, in some embodiments, the cells are genetically edited to increase IL 15 levels and / or signaling, such as by reducing or eliminating expression of the cytokine-inducible SH2-containing protein (Cis) (e g., by disrupting the CISH gene encoding Cis). By w ay of non-limiting example, IL 15 is a positive regulator of NK cells, which can enhance one or more of NK cell homing, NK cell migration, NK cell expansion / proliferation, NK cell cytotoxicity7, and / or NK cell persistence. In CD8+ T cells, CISH actively7silences TCR signaling to maintain tumor tolerance, and CISH has been shown to be a downstream negative regulator of IL-15 receptor signaling (Palmer et al., J. Exp. Med. (2015) 212(12):2095-2113). In NK and T cells, CISH plays a role in checkpoint maturation and proliferation (Delconte et al., Nature Immunol (2016) 17:816-24). Thus, according to several embodiments, genetically editing CISH increases the persistence, proliferation, and / or cytotoxicity’, or otherwise enhances the efficacy, of cells as disclosed herein.

[0305] In several embodiments, CISH genetic editing activates or inhibits a wide variety’ of pathways. The CIS protein is a negative regulator of IL15 signaling by way of, for example, inhibiting JAK-STAT signaling pathways. These pathways would typically lead to transcription of IL15- responsive genes (including CISH). In several embodiments, disruption of CISH disinhibits JAK- STAT (e.g., JAK1-STAT5) signaling and there is enhanced transcription of IL 15 -responsive genes. In several embodiments, disruption of CISH yields enhanced signaling through mammalian target of rapamycin (mTOR). with corresponding increases in expression of genes related to cell metabolism and respiration. In several embodiments, disruption of CISH yields IL 15 induced increased expression of IL-2Ra (CD25), but not IL-15Ra or IL-2 / 15R , enhanced NK cell membrane binding of IL15 and / or IL2, increased phosphory lation of STAT-3 and / or STAT-5, and elevated expression of the antiapoptotic proteins, such as Bcl-2. In several embodiments, CISH disruption results in IL15- induced upregulation of selected genes related to mitochondrial functions (e.g.. electron transportchain and cellular respiration) and cell cycle. Thus, in several embodiments, CISH disruption by genetic editing enhances the NK cell cytotoxicity and / or persistence, at least in part via metabolic reprogramming. In several embodiments, negative regulators of cellular metabolism, such as TXNIP, are downregulated in response to CISH disruption. In several embodiments, promotors for cell survival and proliferation including BIRC5 (Survivin), TOP2A, CKS2, and RACGAP1 are upregulated after CISH disruption, whereas antiproliferative or proapoptotic proteins such as TGFB1, ATM, and PTCHI are downregulated. In several embodiments, CISH disruption alters the state (e.g., activates or inactivates) signaling via or through one or more of CXCL-10, IL2, TNF, IFNg. IL13. IL4, Jnk, PRF1, STAT5. PRKCQ, IL2 receptor Beta, SOCS2, MYD88, STAT3, STAT1, TBX21. LCK. JAK3, IL& receptor, ABL1, IL9. STAT5A. STAT5B. Tcf7, PRDM1, and / or EOMES.

[0306] In several embodiments, CISH editing endows a cell with enhanced ability to home to a target site. In several embodiments, CISH editing endows a cell with enhanced ability to migrate, e.g., within a tissue in response to. for example chemoattractants or away from repellants. In several embodiments, CISH editing endows a cell with enhanced ability to be activated, and thus exert, for example, anti-tumor effects. In several embodiments, CISH editing endows a cell with enhanced proliferative ability, which in several embodiments, allows for generation of robust cell numbers from a donor blood sample. In addition, in such embodiments, cells edited for CISH and engineered to express a CAR are more readily, robustly, and consistently expanded in culture. In several embodiments, CISH genetic editing endows a cell with enhanced cytotoxicity. In several embodiments, the editing of CISH synergistically enhances the cytotoxic effects of cells that express a transmembrane protein as provided herein, a CAR, or both.D. Vectors and Methods for Genetic Engineering

[0307] Various methods for the introduction of genetically engineered components, e.g., transmembrane proteins and recombinant receptors, e.g., CARs or TCRs. are well known and may be used with the provided methods and compositions. Exemplary' methods include those for transfer of nucleic acids encoding the transmembrane proteins or receptors, including via viral vectors, e.g., retroviral or lentiviral, non-viral vectors, or transposons. Methods of gene transfer can include transduction, electroporation or any other method that results in gene transfer into the cell.

[0308] In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV); recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors: via electroporation; via transposition; calcium phosphate transfection; protoplast fusion: cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment; or strontium phosphate DNA co-precipitation. In some embodiments, cells (e.g.. immune cells, such as NK cells) can be genetically engineered before, during, and / or after expansion, e.g., with a transmembrane protein, a recombinant receptor (e.g., CAR), or both.

[0309] Also provided are methods, polynucleotides, compositions, and kits for expressing die transmembrane proteins and / or recombinant receptors, and for producing die genetically engineered cells expressing such molecules. In some embodiments, one or more transmembrane proteins can be genetically engineered into cells or a plurality of cells. In some embodiments, one or more binding molecules, including recombinant receptors (e.g., CARs) can also be genetically engineered into cells or a plurality of cells, including cells engineered to express a transmembrane protein. In some embodiments, a transmembrane protein and a recombinant receptor are encoded by the same or separate nucleic acid molecules. In some embodiments, a transmembrane protein and a recombinant receptor are encoded by the same nucleic acid molecule. In some embodiments, a transmembrane protein and a recombinant receptor are encoded by separate nucleic acid molecules. In some cases, the polynucleotide containing nucleic acid sequence(s) encoding a transmembrane protein and / or a recombinant receptor (e.g., CAR) contains a signal sequence that encodes a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a native polypeptide. In other aspects, the signal sequence may encode a heterologous or non-native signal peptide. In some aspects, a non-limiting exemplary signal peptide comprises a CD8 alpha (CD8a) signal peptide set forth in SEQ ID NO:4.

[0310] In some embodiments the vector or construct can contain promoter and / or enhancer or regulatory elements to regulate expression of the encoded transmembrane protein and / or recombinant receptor. In some examples the promoter and / or enhancer or regulatory elements can be condition-dependent promoters, enhancers, and / or regulatory elements. In some examples these elements drive expression of the transgene.

[0311] In some embodiments, the vector or construct can contain a single promoter that drives the expression of one or more nucleic acid molecules. In some embodiments, such nucleic acid molecules, e.g., transcripts, can be multicistronic (bicistronic or tricistronic). For example, in some embodiments, transcription units can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), which allows co-expression of gene products (e.g. encoding a transmembrane protein and a recombinant receptor) by a message from a single promoter. Alternatively, in some cases, a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF), two or three genes (e.g. encoding a transmembrane protein and a recombinant receptor) separated from one another by sequences encoding a self-cleavage peptide (e.g., 2A cleavage sequences) or a protease recognition site. The ORF thus encodes a single polypeptide, which, either during (in the case of T2A peptide) or after translation, is cleaved into the individual proteins. In some cases, the peptide, such as T2A peptide, can cause the ribosome to skip (ribosome skipping) synthesis of a peptide bond at the C-terminus of a 2A peptide, leading to separation between the end of the 2A peptide sequence and the next peptide downstream. Many 2A peptides are known. Examples of 2A peptide sequences that can be used in the methods and polynucleotides disclosed herein, without limitation. 2A sequences from the foot-and-mouth disease virus (F2A, e.g., SEQ ID NO:77), equinerliinitis A virus (E2A, e.g., SEQ ID NO:76), Thosea asigna virus (T2A, e.g. SEQ ID NO:20, encoded by SEQ ID NO:19; SEQ ID NO: 23, encoded by SEQ ID NO:22; or SEQ ID NO:74), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO:75). In some embodiments, the 2A peptide is E2A (e.g.. SEQ ID NO:76). In some embodiments, the 2A peptide is F2A (e.g., SEQ ID NO:77). In some embodiments, the 2A peptide is P2A (e.g., SEQ ID NO:75). In some embodiments, the 2A peptide is T2A (e.g., SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:74). In some embodiments, the one or more different or separate promoters drive the expression of a nucleic acid molecule encoding a recombinant receptor and a nucleic acid encoding a transmembrane protein.

[0312] Any of the transmembrane proteins and / or recombinant receptors, can be encoded by polynucleotides containing one or more nucleic acid molecules encoding the molecules, in any combinations or arrangements. For example, one. two, three or more polynucleotides can encode one, two, three or more different molecules. In some embodiments, one vector or construct contains nucleic acid molecules encoding one or more transmembrane proteins, and a separate vector or construct contains nucleic acid molecules encoding a recombinant receptor.

[0313] Also provided are compositions containing one or more of the polynucleotides, vectors or constructs, such as any described above. In some embodiments, the polynucleotides, vectors, constructs or compositions can be used to engineer immune cells, such as NK or T cells, to express any of the transmembrane proteins, and optionally a recombinant receptor. The genetic engineering generally involves introduction of a nucleic acid encoding the transmembrane protein and / or recombinant receptor into the cell, such as by retroviral transductions, transfection, or transformation. In some embodiments, provided herein are polynucleotides containing a nucleic acid sequence encoding a transmembrane protein and a nucleic acid sequence encoding a recombinant receptor. The polynucleotides may include those encompassing natural and / or non-naturally occurring nucleotides and bases, e.g., including those with backbone modifications. The terms “nucleic acid molecule’’, “nucleic acid” and “polynucleotide” may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. “Nucleic acid sequence” refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide. Also provided are polynucleotides that have been optimized for codon usage.

[0314] Also provided are vectors containing the polynucleotides, such as any of the polynucleotides described herein, and cells containing the vectors, e.g., for producing the transmembrane proteins. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is a lentiviral vector. Also provided are methods for producing the transmembrane proteins or cells expressing the same. In a further embodiment, one or more vectors (e.g., expression vectors) comprising such polynucleotides are provided. In a further embodiment, a host cell comprising such polynucleotides is provided. In another such embodiment, a host cell comprises (e.g.. has been transformed with) a vector comprisinga nucleic acid sequence that encodes a transmembrane protein and / or a nucleic acid sequence that encodes a recombinant receptor. In some embodiments, one or more such host cells are provided. In some embodiments, a composition containing one or more such host cells are provided.

[0315] Also provided are methods of making the transmembrane proteins. For recombinant production of the transmembrane proteins, a nucleic acid sequence encoding a transmembrane protein, e.g., as described herein, may be isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid sequences may be readily isolated and sequenced using conventional procedures.IV. Compositions and Formulations

[0316] Also provided arc compositions including the transmembrane proteins, polynucleotides encoding the transmembrane proteins, vectors comprising the poly nucleotides, and cells expressing the transmembrane proteins, including pharmaceutical compositions and formulations. Also provided are compositions comprising engineered cells that express the transmembrane proteins provided herein, including pharmaceutical compositions and formulations. In some embodiments, the engineered cells also express a recombinant receptor (e.g., a CAR).

[0317] Provided are pharmaceutical formulations comprising a transmembrane protein (e.g., mbIL2 or mbIL15), engineered cells expressing said transmembrane proteins (e.g., mbIL2 or mbIL15), a plurality of engineered cells expressing said transmembrane proteins (e.g., mbIL2 or mbIL15) and / or additional agents for combination treatment or therapy. In some embodiments, any of the engineered cells also express a recombinant receptor (e.g.. a CAR). The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.

[0318] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0319] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0320] In some aspects, the choice of carrier is determined in part by the particular cell, recombinant receptor, transmembrane protein, and / or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, mcthylparabcn, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% toabout 2% by weight of the total composition. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immuno globulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA: sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0321] In some aspects, a buffer is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffers is used. The buffering agent or mixtures thereof are typically present in an amount of from about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known.

[0322] Formulations of the transmembrane proteins and / or engineered cells described herein can include lyophilized formulations and aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts drat are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, the cells or antibodies are administered in the form of a salt, e.g., a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, p- toluenesulphonic acid.

[0323] The pharmaceutical composition in some embodiments contains the transmembrane proteins and / or cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeatedadministrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

[0324] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdennal, intramuscular, intranasal, buccal, sublingual, or suppository' administration. In some embodiments, the agent or cell population is administered to the subject by intravenous, intraperitoneal, or subcutaneous injection using peripheral systemic delivery.

[0325] In some embodiments, the compositions are provided as sterile liquid formulations (e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions), which in some aspects may be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, particularly by injection. The liquid composition can comprise a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyols (e g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0326] Sterile injectable solutions can be prepared by incorporating the agent or cell into a solvent, such as an admixture with a suitable carrier, diluent, or excipient (e.g., sterile water, saline, glucose, dextrose, and the like). Formulations for in vivo administration are typically sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes. In some embodiments, the dose of engineered cells administered is in a cryopreserved composition. In some aspects, the composition is administered after thawing the crvopreserved composition.

[0327] Also provided are pharmaceutical compositions for combination therapy . Any of the additional agents for combination therapy described herein, such as agents described in Section V.B., can be prepared and administered as one or more pharmaceutical compositions, with the engineered cells described herein. The combination therapy can be administered in one or more pharmaceutical compositions, e.g., where the cells are in the same pharmaceutical composition as the additional agent, or in separate pharmaceutical compositions. For example, in some embodiments, the additional agent is an additional engineered cell, e.g., cell engineered to express a different recombinant receptor that targets a different antigen or a different epitope on the antigen, and is administered in the same composition or in a separate composition. In some embodiments, each of the pharmaceutical compositions is formulated in a suitable formulation according to the recombinant receptor, cell, e.g., engineered cell, and / or additional agent, and the particular dosage regimen and / or method of delivery.V. Methods and Uses

[0328] Provided herein are methods of treatment, e.g., comprising administering any of the transmembrane proteins, engineered cells, populations of engineered cells, and compositions, and uses of such transmembrane proteins, engineered cells, populations of engineered cells, and compositions to treat or prevent diseases, conditions, and disorders, including cancers, infectious diseases, and / or autoimmune diseases. In some aspects, also provide are methods of administering any of the populations of cells described herein or a composition containing a population of cells to a subject (e.g., a subject having a disease or disorder). In some aspects, there is also provided use of any of the populations of cells described herein or a composition comprising the population of cells for treating a disease or disorder. In some aspects, there is also provided a use of any of the populations of cells described herein or a composition comprising the population of cells for the manufacture of a medicament to treat a disease or condition. In some aspects, also provided is any of the populations of cells described herein or a composition comprising the population of cells for use in treating a disease or disorder, or for administration to a subject having a disease or disorder.A. Therapeutic and Prophylactic Methods and Uses

[0329] Also provided are methods of administering and uses, such as therapeutic and prophylactic uses, of the transmembrane proteins, engineered cells expressing the transmembrane proteins, plurality of engineered cells expressing the transmembrane proteins, and / or compositions comprising the same. Such methods and uses include therapeutic methods and uses, for example, involving administration of the molecules (e.g.. transmembrane proteins), cells (e.g., engineered cells), or compositions containing the same, to a subject having or suspected of having a disease, condition, or disorder. In some embodiments, the transmembrane protein, cell, and / or composition is / are administered in an effective amount to effect treatment of the disease or disorder.

[0330] Provided herein are uses of the transmembrane proteins (mbILs) and cells (e.g., engineered cells expressing a mbIL and optionally a recombinant receptor) in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, the methods are carried out by administering the transmembrane proteins or cells, or compositions comprising the same, to the subject having, having had, or suspected of having the disease or condition. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject. Also provided herein are of use of any of tire compositions, such as pharmaceutical compositions provided herein, for the treatment of a disease or disorder, such as use in a treatment regimen.

[0331] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate ofdisease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.

[0332] As used herein, “delaying development of a disease’’ means to defer, hinder, slow, retard, stabilize, suppress and / or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or subject being treated. In some embodiments, the provided molecules and compositions are used to delay development of a disease or to slow the progression of a disease.

[0333] “Preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease.

[0334] As used herein, to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, a composition or cell which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the composition or cell.

[0335] An “effective amount” of an agent, e.g., a pharmaceutical formulation, binding molecule, antibody, cells, or composition, in the context of administration, refers to an amount effective, at dosages / amounts and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.

[0336] A “therapeutically effective amount” of an agent, e.g., a pharmaceutical formulation, transmembrane protein, cells, or composition refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and / or pharmacokinetic or pharmacody namic effect of the treatment. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered. In some embodiments, the provided methods involve administering the transmembrane proteins, cells, and / or compositions at effective amounts, e.g., therapeutically effective amounts.

[0337] A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0338] As used herein, a "subject” or an “individual” is a mammal. In some embodiments, a “mammal” includes humans, non-human primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, monkeys, etc. In some embodiments, the subject is human.

[0339] Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions.

[0340] In some embodiments, the disease or condition can be any in which expression of an antigen is associated with and / or involved in the etiology' of a disease, condition, or disorder, e.g., causes, exacerbates, or otherwise is involved in such disease, condition, or disorder. Exemplary diseases and conditions can include diseases or conditions associated with malignancy or transformation of cells (e.g., cancer), autoimmune or inflammatory disease, or an infectious disease, e.g., caused by a bacterial, viral, or other pathogen. Exemplary antigens, which include antigens associated with various diseases and conditions that can be treated, are described herein. In some aspects, cells further express a recombinant receptor, such as a CAR. In particular embodiments, the recombinant receptor, e.g.. a CAR or transgenic TCR. binds to an antigen associated with the disease or condition.

[0341] In some embodiments, the disease or condition is a cancer and / or a tumor, such as a solid tumor, lymphoma, leukemia, blood tumor, metastatic tumor, or other cancer or tumor type.

[0342] In some embodiments, the disease or condition is an infectious disease or condition, such as, but not limited to, viral, retroviral, bacterial, and protozoal infections, immunodeficiency, Cytomegalovirus (CMV), Epstein-Barr virus (EBV). adenovirus, BK polyomavirus.

[0343] In some embodiments, the disease or condition is an autoimmune or inflammatory disease or condition. In some embodiments, the disease or condition is an autoimmune disease. In some embodiments, the disease or condition is an inflammatory disease. Autoimmune or inflammatory diseases or conditions include but are not limited to autoimmune encephalitis, arthritis, e.g.. rheumatoid arthritis (RA), Type 1 diabetes, systemic lupus erythematosus (SLE), lupus nephritis (LN), inflammatory' bowel disease (IBD), Guillain-Barre syndrome, chronic inflammatory' demyelinating polyneuropathy (CIDP), psoriasis, scleroderma, autoimmune thyroid disease, Grave's disease, Crohn's disease, multiple sclerosis (MS), myasthenia gravis (MG), idiopathic inflammatory myopathies (also known as myositis), vasculitis (e.g., ANCA-associated vasculitis; AAV), asthma, and / or a disease or condition associated with transplant. Other autoimmune diseases include but are not limited to IgA nephropathy (IgAN), ankylosing spondylitis (AS), antiphospholipid syndrome (APS), autoimmune hepatitis (AIH), bullous pemphigoid (BP), chronic graft-versus-host-disease (cGvHD), cold agglutinin disease (CAD), IgG4-related disease (IgG4-RD). neuromyelitis optica spectrum disorder (NMOSD), pemphigus vulgaris (PV), primary biliary' cholangitis (PBC), primary membranous nephropathy (pMN), primary progressive multiple sclerosis (PPMS), primary sclerosing cholangitis (PSC), Sjogren’s syndrome, and warm autoimmune hemolytic anemia (wAIHA).

[0344] In some embodiments, the antigen associated with the disease or disorder is selected from the group consisting of BCMA. CD19, CD20, CD22, CD23, CD24. CD30, CD33. CD38, CD44, CD70, CD123, CD138, CEA, CD7, CS1. c-Met, a cyclin. EGFR, EGP-2, EGP-4, ephrinB2, estrogen receptor, ErbB2, 3, or 4, FBP, fetal acetylcholine receptor, GD2, GD3. GPRC5D. HER2. HMW-MAA, IL-22R-alplia, IL-13R-alpha2, kappa light chain, Lewis Y, MAGE-A1, MAGE-A3, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, oncofetal antigen, progesterone receptor, prostate specific antigen, PSMA, R0R1, SLAMF7, TAG72, VEGF-R2, Wilms Tumor 1 (WT-1), biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens.

[0345] The provided methods and uses include methods and uses for cell therapy. In some embodiments, the methods include administration of the engineered cells or a composition containing the cells to a subject, tissue, or cell, such as one having, at risk for, or suspected of having the disease, condition or disorder. In some embodiments, the cells, populations, and compositions are administered to a subject having the particular disease or condition to be treated, e.g., via cell therapy, such as NK or T cell therapy. In some embodiments, the cells or compositions are administered to the subject, such as a subject having or at risk for the disease or condition, ameliorate one or more symptom of the disease or condition.

[0346] In some embodiments, the cell therapy, e.g.. T cell therapy, is carried out by autologous transfer, in which the cells are isolated and / or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.

[0347] In some embodiments, the cell therapy, e.g., NK cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and / or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or super type as the first subject.

[0348] In some embodiments, the subject, to whom the cells, cell populations, or compositions are administered, is a primate, such as a human. In some embodiments, the subject is a human. In some embodiments, the subject is at least about 18 years of age. In some embodiments, the subject, to whom the cells, cell populations, or compositions are administered, is a non-human primate. In some embodiments, the non-human primate is a monkey (e.g., cynomolgus monkey) or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent (e.g., mouse, rat. etc.).

[0349] In some embodiments, prior to the initiation of administration of the engineered cells, the subject has received one or more prior therapies (e.g., for the disease or condition). In some embodiments, the subject has received at least 1, 2, 3, 4. 5. 6, 7, 8, 9, 10, 11, 12. 13. 14, 15, 16, 17. 18, 19 or 20 or more prior therapies. In some embodiments, the subject has received at least 3. 4. 5, 6, 7. 8. 9, 10 or more prior therapies. In some embodiments, the subject has received at least 1 priordierapy. In some embodiments, the subject has received at least 2 prior therapies. In some embodiments, tire subject has received at least 3 prior therapies. In some embodiments, the subject has received at least 4 prior therapies. In some aspects, the subject has relapsed or has been refractory to the one or more prior therapies (e.g., for the disease or condition).

[0350] For the prevention or treatment of disease, the appropriate dosage of the engineered cells may depend on the type of disease to be treated, the type of transmembrane protein and / or recombinant receptor, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the cells, and the discretion of the attending physician. The compositions and molecules and cells are in some embodiments suitably administered to the patient at one time or over a series of treatments.

[0351] In some embodiments, the dose and / or frequency of administration is determined based on efficacy and / or response. In some examples, dose and / or frequency of administration is determined by the expansion and persistence of the cells in the blood and / or bone marrow. In some embodiments, dose and / or frequency of administration is determined based on the activity (e.g., cytotoxic activity) of the cells.

[0352] In certain embodiments, treatment of a subject with a genetically engineered cell(s) described herein achieves one, two, three, four, or more of the following effects, including, for example: (i) reduction or amelioration the severity of disease or symptom associated therewith; (ii) reduction in the duration of a symptom associated with a disease; (iii) protection against the progression of a disease or symptom associated therewith; (iv) regression of a disease or symptom associated therewith; (v) protection against the development or onset of a symptom associated with a disease; (vi) protection against the recurrence of a symptom associated with a disease; (vii) reduction in the hospitalization of a subject; (viii) reduction in the hospitalization length; (ix) an increase in the survival of a subject with a disease; (x) a reduction in the number of symptoms associated with a disease; and / or (xi) an enhancement, improvement, supplementation, complementation, or augmentation of the prophylactic or therapeutic effect(s) of another therapy.

[0353] In some embodiments, the methods comprise administering a dose of the engineered cells or a composition comprising a dose of the engineered cells. In some embodiments, the engineered cells or compositions containing engineered cells can be used in a treatment regimen, wherein the treatment regimen comprises administering a dose of the engineered cells or a composition comprising a dose of the engineered cells. In some embodiments, the dose can contain, for example, a particular number or range of transmembrane protein and / or recombinant receptorexpressing immune cells (e.g., NK cells), such as any number of such cells described herein. In some embodiments, a composition containing a dose of the cells can be administered. In some aspects, the number, amount or proportion of transmembrane protein- and / or CAR-expressing cells in a cell population or a cell composition can be assessed by detection of a surrogate marker, e.g., by flowcytomctry or other means, or by detecting binding of a labelled molecule, such as a labelled antigen, that can specifically bind to the transmembrane protein or recombinant receptors provided herein.

[0354] Doses of immune cells such as NK and / or T cells may range, depending on the embodiments, from about 105 cells per kg to about 1012 cells per kg (e.g., 105-107, 107-1010, 1010- 1012 and overlapping ranges therein). In one embodiment, a dose escalation regimen is used. In several embodiments, a range of immune cells such as NK and / or T cells is administered, for example between about 1 x 106 cells / kg to about 1 x 108 cells / kg. In several embodiments, a range of NK cells is administered, for example between about 1 x 106 cells / kg to about 1 x 108 cells / kg. In several embodiments, a range of T cells is administered, for example between about 1 x 106 cells / kg to about 1 x 108 cells / kg. In some embodiments, the cells are CAR-expressing cells.

[0355] In some embodiments, a dose of engineered cells comprises between about 300 x 106 and 1 x 1010 NK cells or between 1 x 109 and 3 x 109 NK cells. In some embodiments, a dose of engineered cells comprises about 300 x 106 NK cells. In some embodiments, a dose of engineered cells comprises about 1 x 109 NK cells. In some embodiments, a dose of engineered cells comprises about 1.5 x 109 NK cells. In some embodiments, a dose of engineered cells comprises about 2 x 109 NK cells. In some embodiments, a dose of engineered cells comprises about 2.5 x 109 NK cells. In some embodiments, a dose of engineered cells comprises about 3 x 109 NK cells. In some embodiments, the NK cells are CAR-expressing NK cells.

[0356] In several embodiments, multiple doses are used, for example, two, three, four, or more doses within a dosing cycle. Such multi-dose cycles can be repeated one or more times, as needed to treat and / or prevent disease progression. In several embodiments, dosing is, for example, 3 doses of about 1.5 x 109 NK cells or about 1.5 x 109 NK cells administered over about 21 to 28 days. In several embodiments, dosing is, for example, 3 doses of about 1.0 x 109 NK cells or about 1.5 x 109 NK cells administered over about 8 to 15 days. In several embodiments, dosing is, for example, 3 doses of about 1.0 x 109 NK cells or about 1.5 x 109 NK cells administered over about 8 days. In some embodiments, each dose is separated by about 3-5 days. In several embodiments, dosing is, for example, 3 doses of about 1.0 x 109 NK cells or about 1.5 x 109 NK cells administered over about 15 days. In some embodiments, each dose is separated by about 7 days. In several embodiments, a dosing cycle comprises 3 doses of about 1.5 x 109 NK cells administered over about 21 to 28 days. In several embodiments, a dosing cycle comprises 3 doses of about 2.0 x 109 NK cells administered over about 21 to 28 days. In several embodiments, a dosing cycle comprises 3 doses of about 2.0 x 109 NK cells administered over about 8 to 15 days. In several embodiments, a dosing cycle comprises 3 doses of about 2.0 x 109 NK cells administered over about 8 days. In some embodiments, each dose is separated by about 3-5 days. In several embodiments, a dosing cycle comprises 3 doses of about 2.0 x 109 NK cells administered over about 15 days. In some embodiments, each dose is separated by about 7 days. In some embodiments, the NK cells are CAR-expressing NK cells.

[0357] In several embodiments, the administration of engineered cells is preceded by a lymphodepleting therapy (also referred to as “lymphodepletion”). In several embodiments, prior to administration of the engineered cells or a composition comprising the same, the subject is administered a lymphodepleting therapy. In several embodiments, a combination of chemotherapeutic agents is used for lymphodepletion. In several embodiments, a single chemotherapeutic agent is used for lymphodepletion. In several embodiments, wherein a combination of chemotherapeutic agents is used, agents with different mechanisms of actions are optionally used. In several embodiments, different classes of agents are optionally used. In several embodiments, an antimetabolic agent is used. In several embodiments, the antimetabolic agent inhibits and / or prevents cell replication. In several embodiments, the antimetabolic agent is an altered nucleotide that disrupts DNA replication, making it effective in targeting rapidly dividing tumor cells.

[0358] In several embodiments, the lymphodepleting therapy comprises cytosine arabinoside (Ara-C). In several embodiments, a dose of Ara-C is between about 0.2 and about 10 g / m2. including doses of about 0.2 g / m2, about 0.5 g / m2. about 1.0 g / m2, about 1.5 g / m2, about 2.0 g / m2, about 2.5 g / m2, about 3.0 g / m2, about 3.5 g / m2. about 4.0 g / m2. about 5.0 g / m2, about 6.0 g / m2, about 7.0 g / m2, about 8.0 g / m2, about 9.0 g / m2. about 10.0 about 1.5 g / m2, or any dose between those listed. In several embodiments, a dose of Ara-C is about 0.5 g / m2 of Ara-C. In several embodiments, a dose of Ara-C is about 1 g / m2 of Ara-C. In several embodiments, a dose of Ara-C is about 2 g / m2 of Ara-C. In several embodiments, the dose of Ara-C is given daily for at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In several embodiments, the dose of Ara-C is given daily for about 5 days. In several embodiments, if necessary, the dose can be split and given, for example, twice daily. In several embodiments, an additional agent is used in combination with the Ara-C. In several embodiments, the additional agent is also an antimetabolite. In several embodiments, the additional agent inhibits one or more of DNA polymerase alpha, ribonucleotide reductase and / or DNA primasc, thus inhibiting DNA synthesis.

[0359] In several embodiments, the lymphodepleting therapy comprises fludarabine. In several embodiments, a dose of fludarabine is between about 5.0 mg / m2 and about 200 mg / m2, including doses of about 5.0 mg / m2, about 10.0 mg / m2. about 15.0 mg / m2, about 20.0 mg / m2, about 25.0 mg / m2, about 30.0 mg / m2, about 35.0 mg / m2, about 40.0 mg / m2, about 45.0 mg / m2, about 50.0 mg / m2, about 60.0 mg / m2, about 70.0 mg / m2, about 80.0 mg / m2, about 90.0 mg / m2, about 100.0 mg / m2, about 125.0 mg / m2, about 150.0 mg / m2, about 175.0 mg / m2, about 200.0 mg / m2, or any dose between those listed. In several embodiments, a dose of fludarabine is about 20 mg / m2. In several embodiments, a dose of fludarabine is about 25 mg / m2. In several embodiments, a dose of fludarabine is about 30 mg / m2. In several embodiments, a dose of fludarabine is about 35 mg / m2. In several embodiments, a dose of fludarabine is about 40 mg / m2. In several embodiments, the dose of fludarabine is given daily for at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In several embodiments, the dose of fludarabine is given daily for about 3 days.In several embodiments, the dose of fludarabine is given daily for about 5 days. In several embodiments, if necessary, the dose can be split and given, for example, twice daily.

[0360] In several embodiments, the lymphodepleting therapy comprises fludarabine and Ara- C. In some embodiments, a daily dose of fludarabine is between about 20 mg / m2 and 40 mg / m2 and a daily dose of Ara-C is between about 1.5 g / m2 and 2.5 g / m2. In several embodiments, a daily dose of fludarabine is about 30 mg / m2 and a daily dose of Ara-C is about 2 g / m2. In several embodiments, the combination of fludarabine and Ara-C (or any other agent or agents as disclosed herein) is administered for about 5 days, with the administration started about 7 days prior to the first administration of the engineered cells (for example day -7 to day -3). In several embodiments, lymphodepletion is started at day -5 prior to administration of engineered cells, n several embodiments, this combination advantageously functions not only as a lymphodepletion regimen, but as an anti-cancer agent as well (in addition to the engineered cells). In several embodiments, the lymphodepletion regimen works synergistically with the engineered cells to provide effect reduction and / or elimination of cancerous cells.

[0361] In several embodiments, the lymphodepleting therapy comprises cyclophosphamide. In several embodiments, a dose of cyclophosphamide is between about 100 mg / m2 and about 1000 mg / m2, including doses of about 100.0 mg / m2, about 200 mg / m2, about 300 mg / m2. about 400 mg / m2, about 500 mg / m2, about 600 mg / m2, about 700 mg / m2, about 800 mg / m2, about 900 mg / m2, about 1000 mg / m2, or any dose between those listed. In several embodiments, a dose of cyclophosphamide is about 300 mg / m2. In several embodiments, a dose of cyclophosphamide is about 500 mg / m2. In several embodiments, the dose of cyclophosphamide is given daily for at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In several embodiments, the dose of cyclophosphamide is given daily for at least about 3 days. In several embodiments, if necessary, the dose can be split and given, for example, twice daily.

[0362] In several embodiments, the lymphodepleting therapy comprises fludarabine and cyclophosphamide. In some embodiments, a daily dose of fludarabine is between about 20 mg / m2 and 40 mg / m2 and a daily dose of cyclophosphamide is between about 300 mg / m2 and 500 mg / m2. In several embodiments, a daily dose of fludarabine is about 25 mg / m2 and a daily dose of cyclophosphamide is about 300 mg / m2. In several embodiments, a daily dose of fludarabine is about 25 mg / m2 and a daily dose of cyclophosphamide is about 500 mg / m2. In several embodiments, a daily dose of fludarabine is about 30 mg / m2 and a daily dose of cyclophosphamide is about 300 mg / m2. In several embodiments, a daily dose of fludarabine is about 30 mg / m2 and a daily dose of cyclophosphamide is about 500 mg / m2. In several embodiments, the combination of fludarabine and cyclophosphamide is administered for about 5 days, with the administration started about 7 days prior to the first administration of the engineered cells (for example day -7 to day -3). In several embodiments, lymphodepletion is started at day -5 prior to administration of engineered cells.

[0363] In several embodiments, the lymphodepleting therapy comprises cyclophosphamide. In several embodiments, a dose of cyclophosphamide is betw een about 500 mg / m2 and about 2000 mg / m2, including doses of about 500 mg / m2, about 600 mg / m2, about 700 mg / m2, about 800 mg / m2, about 900 mg / m2. about 1000 mg / m2, about 1100 mg / m2, about 1200 mg / m2, about 1300 mg / m2, about 1400 mg / m2, about 1500 mg / m2, about 1600 mg / m2, about 1700 mg / m2, about 1800 mg / m2, about 2000 mg / m2, or any dose between those listed. In several embodiments, a dose of cyclophosphamide is about 500 mg / m2. In several embodiments, a dose of cyclophosphamide is about 1000 mg / m2. In several embodiments, a dose of cyclophosphamide is about 1500 mg / m2. In several embodiments, a single dose of cyclophosphamide is given on one day. In several embodiments, the dose of cyclophosphamide is given daily for at least about 3 days. In several embodiments, the dose of cyclophosphamide is given daily for at least about 5 days. In several embodiments, if necessary, the dose can be split and given, for example, twice daily.

[0364] In several embodiments, the lymphodepleting therapy comprises fludarabine and cyclophosphamide. In some embodiments, a daily dose of fludarabine is between about 20 mg / m2 and 40 mg / m2 and a single dose of cyclophosphamide is between about 1000 mg / m2 and 2000 mg / m2. In several embodiments, a daily dose of fludarabine is about 25 mg / m2 and a single dose of cyclophosphamide is about 1000 mg / m2. In several embodiments, a daily dose of fludarabine is about 25 mg / m2 and a single dose of cyclophosphamide is about 1500 mg / m2. In several embodiments, a daily dose of fludarabine is about 25 mg / m2 and a single dose of cyclophosphamide is about 2000 mg / m2. In several embodiments, a daily dose of fludarabine is about 30 mg / m2 and a single dose of cyclophosphamide is about 1000 mg / m2. In several embodiments, a daily dose of fludarabine is about 30 mg / m2 and a single dose of cyclophosphamide is about 1500 mg / m2. In several embodiments, a daily dose of fludarabine is about 30 mg / m2 and a single dose of cyclophosphamide is about 2000 mg / m2. In several embodiments, the daily doses of fludarabine are administered for about 3 days, with the fludarabine administration started about 5 days prior to the first administration of tire engineered cells (for example day -5 to day -3) and the single dose of cyclophosphamide administered about 3 days prior to the first administration of the engineered cells (for example day -3). In several embodiments, a daily dose of about 25 mg / m2 of fludarabine is administered on each of 5 days, 4 days, and 3 days prior to the first administration of the engineered cells. In several embodiments, a single dose of about 1000 mg / m2 of cyclophosphamide is administered 3 days prior to the first administration of the engineered cells. In several embodiments, a daily dose of about 25 mg / m2 of fludarabine is administered on each of 5 days, 4 days, and 3 days prior to the first administration of the engineered cells, and a single dose of about 1000 mg / m2 of cyclophosphamide is administered 3 days prior to the first administration of the engineered cells.

[0365] In certain embodiments, a dose of a genetically engineered cell(s) described herein or composition thereof is administered to a subject every day, every other day, every couple of days, every third day. once a week, twice a week, three times a week, or once every two weeks. In otherembodiments, two, three or four doses of a genetically engineered cell(s) described herein or composition thereof is administered to a subject every day, every' couple of days, every drird day, once a week or once every' two weeks. In some embodiments, a dose(s) of a genetically engineered cell(s) described herein or composition thereof is administered for 2 days, 3 days, 5 days, 7 days, 14 days, or 21 days. In certain embodiments, a dose of a genetically engineered cell(s) described herein or composition thereof is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months. 5 months, 6 months or more.

[0366] In several embodiments, a subject is subject to lymphodepletion at least one time prior to administration of genetically engineered cells as disclosed herein. In several embodiments, lymphodepletion is performed before one or more additional doses of engineered cells are administered. In several embodiments, a dosing cycle is used that comprises lymphodepletion followed by at least two doses of engineered cells as disclosed herein, with the two doses separated by a time interval. In several embodiments, the time interval is 2. 3. 4, 5, 6, 7. 8. 9. 10. 11, 12, 13, 14. 15. 16, 17, 18, 19, 20, 21, or more days (including intervals falling between the time marking a price interval since the last administration, e.g.. 84 horns, or 3.5 days). In several embodiments, the dosing cycle itself is approximately 14, 21. 28, 35, 42 or more days. In several embodiments, three doses are each administered about 7 days apart from each other. In several embodiments, three doses are each administered between about 3 days and about 7 days apart from each other. In several embodiments, a subject receives a first dose on day 0 of the cycle, a second dose on day 7 of the cycle and a third dose on day 14 of the cycle. In several embodiments, a subject receives a first dose on day 0 of the cycle, a second dose on day 3 of the cycle and a third dose on day 7 of the cycle. In several embodiments, lymphodepletion is performed prior to the initiation of each dosing cycle, if subsequent dosing cycles are provided. For example, in several embodiments, a subject undergoes lymphodepletion, receives a plurality of doses of engineered cells according to a cycle, is evaluated at the end of the cycle time and, if deemed necessary' undergoes a second lymphodepletion followed by a second dosing cycle. Depending on the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more dosing cycles are performed.B. Combination Therapy

[0367] Also provided are methods of combination therapy that includes administration and uses, such as therapeutic and prophylactic uses, of the engineered cells expressing tire transmembrane proteins (and optionally, recombinant receptors), plurality of the engineered cells, and / or compositions comprising the same.

[0368] In some embodiments, the engineered cells described herein are administered as part of a combination treatment or combination therapy, such as simultaneously with, sequentially with or intermittently with, in any order, one or more additional therapeutic intervention. In some embodiments, the one or more additional therapeutic intervention includes, for example, an antibody, an engineered cell, a receptor and / or an agent, such as a cell expressing a recombinant receptor, and / or cytotoxic or therapeutic agent, e.g.. a chemotherapeutic agent. In some embodiments, the combinationdierapy includes administration of one or more additional agents, therapies and / or treatments, e.g., any of the additional agents, therapy and / or treatments described herein. In some embodiments, the combination treatment or combination therapy includes an additional treatment, such as a surgical treatment, transplant, and / or radiation therapy. Also provided are methods of combination treatment or combination therapy that include the engineered cells and / or compositions described herein and one or more additional therapeutic interventions.

[0369] In some embodiments, the additional therapy, treatment or agent includes chemotherapy, radiation therapy, surgery, transplantation, adoptive cell therapy, antibodies, cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitory agents, anti-hormonal agents, kinase inhibitors, anti-angiogenic agents, cardioprotectants, immunostimulatory agents, immunosuppressive agents, immune checkpoint inhibitors, antibiotics, angiogenesis inhibitors, metabolic modulators or other therapeutic agents or any combination thereof. In some embodiments, the additional agent is a protein, a peptide, a nucleic acid, a small molecule agent, a cell, a toxin, a lipid, a carbohydrate or combinations thereof, or any other type of therapeutic agent, e.g. radiation. In some embodiments, the additional therapy, agent or treatment includes surgery, chemotherapy, radiation therapy, transplantation, administration of cells expressing a recombinant receptor, e.g., CAR, kinase inhibitor, immune checkpoint inhibitor, mTOR pathway inhibitor, immunosuppressive agents, immunomodulators, antibodies, immunoablative agents, antibodies and / or antigen binding fragments thereof, antibody conjugates, other antibody therapies, cytotoxins. steroids, cytokines, peptide vaccines, hormone therapy, antimetabolites, metabolic modulators, alkylating agents, anthracyclines, vinca alkaloids, proteasome inhibitors, protein kinase inhibitors, and / or other types of immunotherapy. In some embodiments, the additional agent or treatment is bone marrow transplantation, T cell ablative therapy using chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, and / or antibody therapy .

[0370] In some embodiments, the additional agent is a chemotherapeutic agent. In several embodiments, the chemotherapeutic agent comprises an antimetabolite, an alkylating agent, a topoisomerase inhibitor, a mitotic inhibitor, an antibiotic, a protein kinase inhibitor, a proteasome inhibitor, an inhibitor of poly (ADP-ribose) polymerase (PARP), or a combination thereof.

[0371] In several embodiments, the additional agent is a hormone therapy or a therapeutic agent that disrupts or modifies a hormone pathway. In several embodiments, the additional agent comprises hyperthermia. In several embodiments, the additional agent is an immunotherapy. In several embodiments, the additional agent comprises photodynamic therapy (PDT). In several embodiments, the additional agent comprises radiation therapy. In several embodiments, the additional agent comprises transplanted stem cells.

[0372] In some embodiments, the engineered cells and / or compositions thereof are administered in combination with other engineered cells. In some embodiments, the additional therapy or treatment is cell therapy, e.g.. adoptive cell therapy. In some embodiments, the additionaldierapy includes administration of engineered cells, e.g., CAR-expressing cells. In some embodiments, where the engineered cells express a CAR, the additional engineered cell is a CAR- expressing cell that expresses the same or different recombinant receptor as the engineered cells provided herein. In some embodiments, the recombinant receptor, e.g., CAR, expressed on the additional engineered cell, recognizes a different antigen, or a different epitope of the same antigen. In such embodiments, the additional engineered cell is administered prior to, concurrently with, or after administration (e.g., infusion) of the engineered cells described herein. In some embodiments, the additional engineered cell is allogeneic to the subject being treated. In some embodiments, the additional engineered cell is autologous to the subject being treated. In some embodiments, the additional agent includes any of the cells or plurality of cells described herein.

[0373] In several embodiments, the therapeutic agent is an NK cell engager (e.g., a molecule that binds both an antigen expressed by cells of the cancer and an antigen expressed by NK cells). In several embodiments, the NK cell engager binds to an activating receptor on an NK cell and an antigen expressed by cells of the cancer. In some embodiments, the activating receptor on the NK cell is selected from the group consisting of CD 16. NKp30. NKp46, NKG2D, and any combination thereof. In some embodiments, the NK cell engager binds to CD 16. In some embodiments, the NK cell engager binds to NKp30. In some embodiments, the NK cell engager binds to NKp46. In some embodiments, the NK cell engager binds to NKG2D.

[0374] In some embodiments, the additional agent includes an immune checkpoint inhibitor. Immune checkpoint inhibitors include any agent that blocks or inhibits in a statistically significant manner, the inhibitory’ pathways of the immune system. Such inhibitors may include small molecule inhibitors or may include antibodies, or antigen binding fragments thereof, that bind to and block or inhibit immune checkpoint receptors, ligands and / or receptor-ligand interaction. In some embodiments, modulation, enhancement and / or stimulation of particular receptors can overcome immune checkpoint pathway components. Illustrative immune checkpoint molecules that may be targeted for blocking, inhibition, modulation, enhancement and / or stimulation include, but are not limited to, PD-1 (CD279), PD-L1 (CD274, B7-H1), CTLA-4, LAG-3 (CD223), TIM-3, 4-1BB (CD137), 4-1BBL (CD137L), GITR (TNFRSF18. AITR), CD40, 0X40 (CD134, TNFRSF4), B7-H3, B7-H4, B7H3, B7H4, VISTA, KIR. 2B4. CEACAM (e.g.. CEACAM-1, CEACAM-3 and / or CEACAM-5), TIGIT, and LAIR1.

[0375] In some embodiments, the additional agent is a CD 19 inhibitor, e.g., an anti-CD19 antibody (e.g., an anti-CD19 mono- or bi-specific antibody) or a fragment thereof, antibody-drug conjugate (ADC), engineered toxin body (ETB), or a small molecule. In some embodiments, the additional agent is a CD20 inhibitor, e.g., an anti-CD20 antibody (e.g.. an anti-CD20 mono- or bi- specific antibody) or a fragment thereof, antibody-drug conjugate (ADC), or engineered toxin body (ETB), or a small molecule. In some embodiments, the additional agent is a CD22 inhibitor, e.g., an anti-CD22 antibody (e.g.. an anti-CD22 mono- or bi-specific antibody) or a fragment thereof.antibody-drug conjugate (ADC), engineered toxin body (ETB), or a small molecule. In some embodiments, the additional agent is an EGFR inhibitor, e.g., an anti-EGFR antibody (e.g., an anti- EGFR mono- or bi-specific antibody) or a fragment thereof, antibody-drug conjugate (ADC), or engineered toxin body (ETB), or a small molecule. In some embodiments, the additional agent a BCMA inhibitor, e.g., an anti-BCMA antibody (e.g., an anti-BCMA mono- or bi-specific antibody) or a fragment thereof, antibody-drug conjugate (ADC), or engineered toxin body (ETB). or a small molecule. In some embodiments, the additional agent is a SLAMF7 inhibitor, e.g., an anti-SLAMF7 antibody (e.g., an anti-SLAMF7 mono- or bi-specific antibody) or a fragment thereof, antibody-drug conjugate (ADC), engineered toxin body (ETB), or a small molecule. In some embodiments, the additional agent is a CD38 inhibitor, e.g., an anti-CD38 antibody (e.g.. an anti-CD38 mono- or bi- specific antibody) or a fragment thereof, antibody-drug conjugate (ADC), engineered toxin body (ETB), or a small molecule. In some embodiments, the additional agent is a C1D38 inhibitor, e.g., an anti-CD138 antibody (e.g., an anti-CD138 mono- or bi-specific antibody) or a fragment thereof, antibody -drug conjugate (ADC), engineered toxin body (ETB), or a small molecule.

[0376] In some embodiments, the additional agent is an immunomodulatory agent (IMiD). In some embodiments, the additional agent is or comprises a steroid. In some embodiments, the steroid is a corticosteroid. In some embodiments, the steroid is a glucocorticoid. In some embodiments, the steroid comprises dexamethasone or prednisone. In some embodiments, the steroid is or comprises dexamethasone. In some embodiments, the steroid is or comprises prednisone.

[0377] Any of the additional agents described herein can be prepared and administered as combination therapy with the engineered cells described herein, such as in pharmaceutical compositions comprising one or more agents of the combination therapy and a pharmaceutically acceptable carrier, such as any described herein. In some embodiments, the engineered cells described herein can be administered simultaneously, concurrently or sequentially, in any order with the additional agents, therapy or treatment, wherein such administration provides therapeutically effective levels each of die agents in the body of the subject. In some embodiments, the additional agent can be co-administered with the engineered cells and / or compositions described herein, for example, as part of the same pharmaceutical composition or using the same method of deliver}'. In some embodiments, the additional agent is administered simultaneously with the engineered cells and / or compositions described herein, but in separate compositions. In some embodiments, the additional agent is an additional engineered cell, e.g., cell engineered to express a different recombinant receptor, and is administered in the same composition or in a separate composition. In some embodiments, the additional agent is incubated with die engineered cells prior to administration of the cells.

[0378] In some examples, the additional agent is administered subsequent to or prior to the administration of the engineered cells and / or compositions described herein, separated by a selected time period. In some examples, the time period is 1 day, 2 days. 3 days. 4 days. 5 days. 6 days. 1 week. 2 weeks, 3 weeks, 1 month. 2 months, or 3 months. In some examples, the additional agent isadministered multiple times and / or die engineered cells and / or compositions described herein, is administered multiple times. For example, in some embodiments, the additional agent is administered prior to die engineered cells and / or compositions described herein, e.g., two weeks. 12 days, 10 days, 8 days, one week, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day before tire administration. For example, in some embodiments, the additional agent is administered after the engineered cells and / or compositions described herein, e g., two weeks, 12 days, 10 days, 8 days, one week, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day after the administration.

[0379] The dose of the additional agent can be any therapeutically effective amount, e.g., any dose amount described herein, and the appropriate dosage of the additional agent may depend on the type of disease to be treated, the type, dose and / or frequency of the additional agent, the severity and course of the disease, whether the additional agent is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history, and the discretion of the attending physician. The engineered cells and / or compositions thereof and the additional agent can be administered to the patient at one time, repeated or administered over a series of treatments.VI. Articles of Manufacture and Kits

[0380] Also provided are articles of manufacture or kits containing the provided cells and / or compositions comprising the same. The articles of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, test tubes. IV solution bags. etc. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container has a sterile access port. Exemplary containers include intravenous solution bags and vials, including those with stoppers pierceable by a needle for injection. The article of manufacture or kit may further include a package insert indicating that the composition can be used to treat a particular condition such as a condition described herein (e.g., cancer, infectious disease, or autoimmune disease). Alternatively, or additionally, the article of manufacture or kit may further include another or the same container comprising a pharmaceutically -acceptable buffer. It may further include other materials such as other buffers, diluents, fdters, needles, and / or syringes.

[0381] The label or package insert may indicate that the composition is used for treating a disease, disorder, or condition in an individual. The label or a package insert, which is on or associated with the container, may indicate directions for reconstitution and / or use of the formulation. The label or package insert may further indicate that the formulation is useful or intended for subcutaneous, intravenous, or other modes of administration for treating or preventing a disease, disorder, or condition in an individual.

[0382] The container in some embodiments holds a composition which is by itself or combined with another composition effective for treating, preventing and / or diagnosing the condition. The article of manufacture or kit may include (a) a first container with a composition containeddierein (i.e., first medicament), wherein the composition includes the provided cells; and (b) a second container with a composition contained therein (i.e., second medicament), wherein the composition includes a further agent, such as a cytotoxic or otherwise therapeutic agent, and which article or kit further comprises instructions on the label or package insert for treating the subject with the second medicament, in an effective amount.DEFINITIONS

[0383] The terms described below, or elsewhere herein, shall be understood to have their ordinary' meaning and shall also be understood to have the meanings specifically described herein, unless otherwise specifically indicated.

[0384] The term “Fc region” herein is used to define a C -terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy' chain Fc region extends from Cys226. or from Pro230, to the carbo.xvl-tcrminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0385] The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0386] An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g. . SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g. . ion exchange or reverse phase HPLC).

[0387] An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0388] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including die progeny of such cells. Host cells include “transformants” and “transformed cells,” which include die primary transformed cell and progeny derived therefrom without regard to die number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but maycontain substitutions. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0389] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides, including the transmembrane proteins, antigen-targeting antibodies and antibody chains and oilier peptides, e.g., linkers, may include amino acid residues including natural and / or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through substitutions of hosts which produce the proteins or errors due to PCR amplification.

[0390] As used herein, “percent (%) amino acid sequence identity” and “percent identity” and “sequence identity” when used with respect to an amino acid sequence (reference polypeptide sequence) is defined as the percentage of amino acid residues in a candidate sequence (e.g. , the subject antibody or fragment) that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2. ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0391] An amino acid substitution may include replacement of one amino acid in a polypeptide with another amino acid. Amino acid substitutions may be introduced into a transmembrane protein or a component thereof of interest and the products screened for a desired activity, e g., improved binding and / or stability.

[0392] Amino acids generally can be grouped according to the following common sidechain properties:(1) hydrophobic: Norleucine, Met, Ala, Vai. Leu, He;(2) neutral hydrophilic: Cys, Ser, Thr. Asn, Gin;(3) acidic: Asp, Glu;(4) basic: His, Lys. Arg;(5) residues that influence chain orientation: Gly, Pro;(6) aromatic: Trp, Tyr. Phe.

[0393] Non-conservative amino acid substitutions will involve exchanging a member of one of these classes for another class.

[0394] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0395] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0396] As used herein, the singular forms “a.” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or "an” means “at least one” or "one or more.” It is understood that aspects, embodiments, and variations described herein include “comprising.” “consisting of.” and / or “consisting essentially of’ aspects, embodiments, and variations.

[0397] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and arc also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of tire breadth of the range.

[0398] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.

[0399] As used herein, a “composition” refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.

[0400] As used herein, a statement that a cell or population of cells is "positive” for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically asurface marker. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotypc- matched control under otherwise identical conditions and / or at a level substantially similar to that for cell known to be positive for the marker, and / or at a level substantially higher than that for a cell known to be negative for the marker.

[0401] As used herein, a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and / or at a level substantially lower than that for cell known to be positive for the marker, and / or at a level substantially similar as compared to that for a cell known to be negative for the marker.

[0402] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary' skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity7and / or for ready7reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0403] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety7for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary7to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0404] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.NON-LIMITING EMBODIMENTS

[0405] Among the embodiments provided herein are:1. A transmembrane protein comprising: (a) an extracellular domain comprising an interleukin; and (2) a transmembrane domain comprising a fragment of interleukin- 15 receptor alpha (IL15Ra).2. A transmembrane protein comprising: (a) an extracellular domain comprising an interleukin; and (b) a transmembrane domain comprising a fragment of CD25.3. The transmembrane protein of embodiment 1 or embodiment 2, wherein the interleukin is interleukin- 15 (IL15) or interleukin-2 (IL2).4. A transmembrane protein comprising: (a) an extracellular domain comprising interleukin- 15 (IL 15); and (b) a transmembrane domain comprising a fragment of interleukin- 15 receptor alpha (IL15Ra).5. A transmembrane protein comprising: (a) an extracellular domain comprising interleukin- 15 (IL15); and (b) a transmembrane domain comprising a fragment of CD25.6. The transmembrane protein of any one of embodiments 3-5, wherein IL15 is full- length IL 15.7. The transmembrane protein of any one of embodiments 3-6, wherein IL 15 is human ILI 5.8. The transmembrane protein of any one of embodiments 3-7, wherein IL 15 comprises the amino acid sequence of SEQ ID NO:25.9. The transmembrane protein of any one of embodiments 3-7. wherein IL 15 comprises a K86R substitution, a N112A substitution, or both.10. The transmembrane protein of embodiment 9, wherein IL15 comprises the amino acid sequence of SEQ ID NO:46.11. A transmembrane protein comprising: (a) an extracellular domain comprising interleukin-2 (IL2); and (b) a transmembrane domain comprising a fragment of interleukin- 15 receptor alpha (IL15Ra).12. A transmembrane protein comprising: (a) an extracellular domain comprising interleukin-2 (IL2); and (b) a transmembrane domain comprising a fragment of CD25.13. The transmembrane protein of any one of embodiments 3, 11, and 12, wherein IL2 is full-length IL2.14. The transmembrane protein of any one of embodiments 3 and 11-13, wherein IL2 is human IL2.15. The transmembrane protein of any one of embodiments 3 and 11-14, wherein IL2 comprises the amino acid sequence of SEQ ID NO:44.16. The transmembrane protein of any one of embodiments 3 and 11-14, wherein IL2 comprises a L80F substitution, a R81D substitution, a L85V substitution, a I86V substitution, a I92F substitution, or any combination thereof.17. The transmembrane protein of embodiment 16, wherein IL2 comprises the amino acid sequence of SEQ ID NO:45.18. The transmembrane protein of any one of embodiments 1, 3, 4. 6-11, and 13-17. wherein the extracellular domain comprises a IL15Ra stalk.19. The transmembrane protein of embodiments, wherein the IL15Ra stalk of the extracellular domain is C-terminal to IL15.20. The transmembrane protein of any one of embodiments 1-19, wherein the extracellular domain does not comprise the IL15Ra sushi domain or a functional fragment thereof.21. The transmembrane protein of any one of embodiments 1-20, wherein the extracellular domain does not comprise SEQ ID NO:61.22. The transmembrane protein of any one of embodiments 18-21, wherein the IL15Ra stalk comprises the amino acid sequence of SEQ ID NO:35.23. The transmembrane protein of any one of embodiments 1, 3, 4, 6-11, and 13-22. wherein the fragment of IL15Ra comprises a IL15Ra transmembrane region and a IL15Ra intracellular region.24. The transmembrane protein of embodiment 23, wherein the lL15Ra transmembrane region comprises the amino acid sequence of SEQ ID NO:36 and / or the IL15Ra intracellular region comprises the amino acid sequence of SEQ ID NO:37.25. The transmembrane protein of any one of embodiments 1. 3, 4, 6-11, and 13-24, wherein the fragment of IL15Ra comprises the amino acid sequence of SEQ ID NO:38.26. The transmembrane protein of any one of embodiments 18-25, wherein the extracellular domain comprises a linker between the interleukin and the IL15Ra stalk.27. The transmembrane protein of any one of embodiments 18-26, wherein the extracellular domain comprises, from N-tenninus to C-tenninus: (i) the interleukin; (ii) a linker, and (iii) the IL15Ra stalk.28. The transmembrane protein of any one of embodiments 18-27, wherein the extracellular domain comprises, from N-tenninus to C-tenninus: (i) IL15; (ii) a linker, and (iii) the IL15Ra stalk.29. The transmembrane protein of embodiment 24, wherein the extracellular domain consists of, from N-terminus to C-tenninus: (i) SEQ ID NO:25; (ii) a linker; and (iii) SEQ ID NO:35.30. The transmembrane protein of any one of embodiments 26-29, wherein the linker comprises the amino acid sequence of SEQ ID NO:21.31. The transmembrane protein of any one of embodiments 1, 3, 4, 6-11, and 13-30, wherein the fragment of IL15Ra comprises a IL15Ra transmembrane region and a IL15Ra intracellular region.32. The transmembrane protein of embodiment 31, wherein the IL15Ra transmembrane region comprises the amino acid sequence of SEQ ID NO:36 and / or wherein the IL15Ra intracellular region comprises the amino acid sequence of SEQ ID NO:37.33. The transmembrane protein of any one of embodiments 1, 3, 4. 6-11, and 13-32. wherein the transmembrane domain comprising the fragment of IL15Ra comprises the amino acid sequence of SEQ ID NO:38.34. A transmembrane protein comprising:(a) an extracellular domain comprising, from N-terminus to C-terminus: IL15; a linker; and an interleukin- 15 receptor alpha (IL15Ra) stalk comprising the amino acid sequence of SEQ ID NO:35; and(b) a transmembrane domain comprising a fragment of IL15Ra, the fragment of IL15Ra comprising, from N-terminus to C-terminus, a IL15Ra transmembrane region comprising the amino acid sequence of SEQ ID NO:36 and a IL15Ra intracellular region comprising the amino acid sequence of SEQ ID NO:37.35. The transmembrane protein of embodiment 34, wherein IL15 comprises the amino acid sequence of SEQ ID NO:25.36. The transmembrane protein of embodiment 34 or embodiment 35, wherein the linker comprises the amino acid sequence of SEQ ID NO:21.37. The transmembrane protein of any one of embodiments 1, 3, 4. 6-11, and 13-36. wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO:38.38. The transmembrane protein of any one of embodiments 1-37, wherein the extracellular domain does not comprise the IL15Ra sushi domain or a functional fragment thereof.39. The transmembrane protein of any one of embodiments 1-38, wherein the extracellular domain does not comprise SEQ ID NO:61.40. A transmembrane protein comprising interleukin- 15 (IL15) and a fragment of interleukin- 15 receptor alpha (IL15Ra), wherein the transmembrane protein does not comprise the IL15Ra sushi domain or a functional fragment thereof.41. The transmembrane protein of embodiment 40, wherein the transmembrane protein does not comprise SEQ ID NO:61.42. The transmembrane protein of any one of embodiments 1, 3-10 and 18-41 comprising die amino acid sequence of SEQ ID NO:47 or SEQ ID NO:51.43. The transmembrane protein of any one of embodiments 1, 3, 11-25, 27, and 30 comprising the amino acid sequence of SEQ ID NO:54 or SEQ ID NO:57.44. The transmembrane protein of any one of embodiments 2, 3, 5-10, and 12-17, wherein the extracellular domain comprises a CD25 stalk.45. The transmembrane protein of embodiment 44, wherein the CD25 stalk comprises the amino acid sequence of SEQ ID NO:29.46. The transmembrane protein of any one of embodiments 2, 3, 5-10, 12-17, 44, and 45, wherein the fragment of CD25 comprises a CD25 transmembrane region and a CD25 intracellular region.47. The transmembrane protein of embodiment 46, wherein the CD25 transmembrane region comprises the amino acid sequence of SEQ ID NO:31 and / or the CD25 intracellular region comprises the amino acid sequence of SEQ ID NO:32.48. The transmembrane protein of any one of embodiments 2, 3, 5-10, 12-17, and 44-47, wherein the fragment of CD25 comprises the amino acid sequence of SEQ ID NO:33.49. The transmembrane protein of any one of embodiments 44-48, wherein the extracellular domain comprises a linker betw een the interleukin and the CD25 stalk.50. The transmembrane protein of any one of embodiments 44-49, wherein the extracellular domain comprises, from N-tenninus to C-terminus: (i) the interleukin; (ii) a linker; and (iii) the CD25 stalk.51. The transmembrane protein of embodiment 49 or embodiment 50, wherein the linker comprises the amino acid sequence of SEQ ID NO:21.52. The transmembrane protein of any one of embodiments 2, 3, 5-10, 44-49, and 51 comprising the amino acid sequence of SEQ ID NO:34 or SEQ ID NO:50.53. The transmembrane protein of any one of embodiments 2, 3, 12-17. 44-48, 50, and 51 comprising the amino acid sequence of SEQ ID NO:53 or SEQ ID NO:56.54. A nucleic acid sequence encoding the transmembrane protein of any one of embodiments 1-53.55. A polynucleotide comprising:(a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising:(i) an extracellular antigen-binding domain;(ii) a transmembrane region; and(iii) an intracellular signaling region; and(b) a second nucleic acid sequence encoding the transmembrane protein of any one of embodiments 1-53.56. The polynucleotide of embodiment 55, wherein the nucleic acid sequences encoding die CAR and the transmembrane protein arc separated by a nucleic acid sequence encoding a ribosomal skip element.57. The polynucleotide of embodiment 56, wherein the ribosomal skip element is a 2A peptide, optionally a T2A peptide.58. The polynucleotide of embodiment 56 or embodiment 57, wherein the ribosomal skip element comprises the amino acid sequence of SEQ ID NO:23.59. The polynucleotide of any one of embodiments 55-58, wherein the extracellular antigen-binding domain binds to an antigen expressed by cells of a disease or condition.60. The polynucleotide of any one of embodiments 55-59, wherein the disease or condition comprises a cancer, an infectious disease, or an autoimmune disease.61. The polynucleotide of embodiment 60, wherein the CAR does not comprise interleukin-15 receptor alpha (IL15Ra) or a fragment thereof.62. The polynucleotide of embodiment 60 or embodiment 61, wherein the transmembrane region of the CAR does not comprise IL15Ra or a fragment thereof.63. The polynucleotide of any one of embodiments 55-62, wherein the transmembrane region of the CAR comprises a CD8 alpha (CD8a) transmembrane protein or a CD28 transmembrane protein.64. The polynucleotide of any one of embodiments 55-63, wherein the intracellular signaling region of the CAR comprises a co-stimulatory signaling domain and a CD3zeta domain.65. The polynucleotide of embodiment 64, wherein the co-stimulatory signaling domain of the CAR comprises a CD28 protein, an ICOS protein, a CD27 protein, a 4-1BB protein, an 0X40 protein, or a CD40L protein.66. A polypeptide encoded by the polynucleotide of any one of embodiments 55-65.67. A vector comprising the nucleic acid sequence of embodiment 54 or the polynucleotide of any one of embodiments 55-65.68. The vector of embodiment 67, wherein the vector is a retroviral vector.69. A cell expressing tire transmembrane protein of any one of embodiments 1-53, the nucleic acid sequence of embodiment 54, the polynucleotide of any one of embodiments 55-65. or the vector of embodiment 67 or embodiment 68.70. A cell expressing die transmembrane protein of any one of embodiments 1-53 and a recombinant receptor.71. The cell of embodiment 70, wherein the recombinant receptor comprises a T cell receptor (TCR) or a chimeric antigen receptor (CAR).72. The cell of embodiment 70 or embodiment 71, wherein the recombinant receptor comprises a chimeric antigen receptor (CAR) comprising: (i) an extracellular...

Claims

WHAT IS CLAIMED:

1. A transmembrane protein comprising: (a) an extracellular domain comprising interleukin- 15 (IL 15); and (b) a transmembrane domain comprising a fragment of interleukin- 15 receptor alpha (IL15Ra).

2. The transmembrane protein of claim 1, wherein IL 15 is full-length IL15.

3. The transmembrane protein of claim 1, wherein IL15 is human IL15.

4. The transmembrane protein of claim 1, wherein IL15 comprises the amino acid sequence of SEQ ID NO:25.

5. The transmembrane protein of claim 1, wherein IL 15 comprises a K86R substitution, a N112A substitution, or both.

6. The transmembrane protein of claim 5, wherein IL15 comprises the amino acid sequence of SEQ ID NO:46.

7. The transmembrane protein of claim 1, wherein the extracellular domain comprises a IL15Ra stalk.

8. The transmembrane protein of claim 7, wherein the IL15Ra stalk of the extracellular domain is C-terminal to IL15.

9. The transmembrane protein of claim 1, wherein the extracellular domain does not comprise the IL15Ra sushi domain or a functional fragment thereof.

10. The transmembrane protein of claim 1, wherein the extracellular domain does not comprise SEQ ID NO:61.

11. The transmembrane protein of claim 7, wherein the IL15Ra stalk comprises the amino acid sequence of SEQ ID NO:35.

12. The transmembrane protein of claim 7, wherein the extracellular domain comprises a linker between IL 15 and the IL15Ra stalk.

13. The transmembrane protein of claim 7. wherein the extracellular domain comprises, from N-terminus to C-terminus: (i) IL 15 ; (ii) a linker; and (iii) the IL15Ra stalk.

14. The transmembrane protein of claim 7, wherein the extracellular domain consists of, from N-terminus to C-terminus: (i) SEQ ID NO:25; (ii) a linker; and (iii) SEQ ID NO:35.

15. The transmembrane protein of claim 12, wherein the linker comprises the amino acid sequence of SEQ ID NO:21.

16. The transmembrane protein of claim 1, wherein the fragment of IL15Ra comprises a IL15Ra transmembrane region and a IL15Ra intracellular region.

17. The transmembrane protein of claim 16, wherein the IL15Ra transmembrane region comprises the amino acid sequence of SEQ ID NO:36 and / or wherein the IL15Ra intracellular region comprises the amino acid sequence of SEQ ID NO:37.

18. The transmembrane protein of claim 1, wherein the transmembrane domain comprising the fragment of IL15Ra comprises the amino acid sequence of SEQ ID NO:38.

19. A transmembrane protein comprising:(a) an extracellular domain comprising, from N-terminus to C-terminus: IL15; a linker; and an interleukin- 15 receptor alpha (IL15Ra) stalk comprising the amino acid sequence of SEQ ID NO:35; and(b) a transmembrane domain comprising a fragment of IL15Ra, the fragment of IL15Ra comprising, from N-terminus to C-terminus, a IL15Ra transmembrane region comprising the amino acid sequence of SEQ ID NO:36 and a IL15Ra intracellular region comprising the amino acid sequence of SEQ ID NO:37.

20. The transmembrane protein of claim 19. wherein IL15 comprises the amino acid sequence of SEQ ID NO:25.

21. The transmembrane protein of claim 19. wherein the linker comprises the ammo acid sequence of SEQ ID NO:21.

22. The transmembrane protein of claim 19, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO:38.

23. The transmembrane protein of claim 19, wherein the extracellular domain does not comprise the IL15Ra sushi domain or a functional fragment thereof.

24. The transmembrane protein of claim 19, wherein the extracellular domain does not comprise SEQ ID NO:61.

25. A transmembrane protein comprising interleukin- 15 (IL 15) and a fragment of interleukin- 15 receptor alpha (IL15Ra), wherein the transmembrane protein does not comprise the IL15Ra sushi domain or a functional fragment thereof.

26. The transmembrane protein of claim 25, wherein tire transmembrane protein does not comprise SEQ ID NO:61.

27. The transmembrane protein of any one of claims 1-4 and 7-26 comprising the amino acid sequence of SEQ ID NO:47.

28. The transmembrane protein of any one of claims 1-3 and 5-26 comprising the amino acid sequence of SEQ ID NO:51.

29. A nucleic acid sequence encoding the transmembrane protein of any one of claims 1- 28.

30. A polynucleotide comprising:(a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising:(i) an extracellular antigen-binding domain;(ii) a transmembrane region; and(iii) an intracellular signaling region; and(b) a second nucleic acid sequence encoding the transmembrane protein of any one of claims1-28.

31. The polynucleotide of claim 30, wherein the nucleic acid sequences encoding the CAR and the transmembrane protein are separated by a nucleic acid sequence encoding a ribosomal skip element.

32. The polynucleotide of claim 31, wherein the ribosomal skip element is a 2A peptide, optionally a T2A peptide.

33. The polynucleotide of claim 31. wherein the ribosomal skip element comprises the amino acid sequence of SEQ ID NO:23.

34. The polynucleotide of claim 30. wherein the extracellular antigen-binding domain binds to an antigen expressed by cells of a disease or condition.

35. The polynucleotide of claim 34. wherein the disease or condition comprises a cancer, an infectious disease, or an autoimmune disease.

36. The polynucleotide of claim 30, wherein the CAR does not comprise interleukin- 15 receptor alpha (IL15Ra) or a fragment thereof.

37. The polynucleotide of claim 30, wherein the transmembrane region of the CAR does not comprise IL15Ra or a fragment thereof.

38. The polynucleotide of any one of claims 30-37, wherein the transmembrane region of the CAR comprises a CD8 alpha (CD8a) transmembrane protein or a CD28 transmembrane protein.

39. The polynucleotide of any one of claims 30-38, wherein the intracellular signaling region of the CAR comprises a co-stimulatory signaling domain and a CD3zeta domain.

40. The polynucleotide of claim 39, wherein the co-stimulatory signaling domain of the CAR comprises a CD28 protein, an ICOS protein, a CD27 protein, a 4-1BB protein, an 0X40 protein, or a CD40L protein.

41. A polypeptide encoded by the polynucleotide of any one of claims 30-40.

42. A vector comprising the nucleic acid sequence of claim 29 or the poly nucleotide of any one of claims 30-40.

43. The vector of claim 42, wherein the vector is a retroviral vector, optionally a gamma retroviral vector or a lentiviral vector.

44. A cell expressing the transmembrane protein of any one of claims 1-28, the nucleic acid sequence of claim 29, the polynucleotide of any one of claims 30-40, the polypeptide of claim 41, or the vector of claim 42 or claim 43.

45. A cell expressing the transmembrane protein of any one of claims 1-28 and a recombinant receptor.

46. The cell of claim 45, wherein the recombinant receptor comprises a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

47. The cell of claim 45 or claim 46, wherein the recombinant receptor comprises a chimeric antigen receptor (CAR) comprising: (i) an extracellular antigen-binding domain; (ii) a transmembrane region; and (iii) an intracellular signaling region.

48. A cell expressing:(a) a chimeric antigen receptor (CAR) comprising:(i) an extracellular antigen binding domain;(ii) a transmembrane region; and(iii) an intracellular signaling region; and(b) the transmembrane protein of any one of claims 1-31.

49. The cell of any one of claims 44-48, wherein the CAR does not comprise interleukin- 15 receptor alpha (IL15Ra) or a fragment thereof, optionally wherein the transmembrane region of the CAR does not comprise IL15Ra or a fragment thereof.

50. The cell of any one of claims 44-49, wherein the cell is an immune cell.

51. The cell of claim 50, wherein the cell is a natural killer (NK) cell or a T cell.

52. The cell of claim 50 or claim 51. wherein the cell is a natural killer (NK) cell, optionally a primary NK cell.

53. The cell of any one of claims 50-52, wherein the cell is a primary NK cell obtained from a donor.

54. The cell of any one of claims 44-49, wherein the cell is a K562 cell or K562-derived cell.

55. A composition comprising a plurality of the cells of claim 54.

56. Use of the composition of claim 55 for expanding a population of immune cells, optionally wherein the immune cells are T cells or natural killer (NK) cells.

57. A composition comprising a plurality of the cells of any one of claims 44-53, wherein die plurality of cells comprises immune cells.

58. The composition of claim 57, wherein the plurality of cells comprises natural killer (NK) cells and / or T cells.

59. The composition of claim 57 or claim 58, wherein the plurality of cells comprises natural killer (NK) cells.

60. The composition of any one of claims 57-59, comprising a pharmaceutically acceptable excipient.

61. Use of the transmembrane protein of any one of claims 1-28, the nucleic acid sequence of claim 29, the polynucleotide of any one of claims 30-40. the polypeptide of claim 41, the vector of claim 42 or claim 43, the cell of any one of claims 44-53. or the composition of any one of claims 57-60 in the manufacture of a medicament for treatment of a subject having or suspected of having a disease or condition.

62. Use of the composition of any one of claims 57-60 for the treatment of a subject having or suspected of having a disease or condition.

63. A method of treating a subject having or suspected of having a disease or condition, the method comprising administering to the subject having or suspected of having the disease or condition the composition of any one of claims 57-60.

64. The use of claim 61 or claim 62 or the method of claim 63, wherein the disease or condition comprises a cancer, an infectious disease, or an autoimmune disease.

65. The use of any one of claims 61, 62, and 64 or the method of claim 63 or claim 64, wherein the cells are allogeneic to the subject.

66. A method of producing genetically engineered cells, the method comprising introducing into a plurality of cells a polynucleotide comprising a nucleic acid sequence encoding the transmembrane protein of any one of claims 1-28. the nucleic acid sequence of claim 29, the polynucleotide of any one of claims 30-40, or the vector of claim 42 or claim 43.

67. The method of claim 66, wherein the polynucleotide comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising: (i) an extracellular antigen-binding domain; (ii) a transmembrane region; and (iii) an intracellular signaling region.

68. The method of claim 67, wherein the nucleic acid sequences encoding the transmembrane protein and the CAR are separated by a nucleic acid sequence encoding a ribosomal skip element.

69. The method of any one of claims 66-68, wherein the plurality of cells comprises immune cells.

70. The method of any one of claims 66-69, wherein the plurality of cells comprises natural killer (NK) cells and / or T cells.

71. A composition comprising the genetically engineered cells produced by die method of any one of claims 66-70.

72. A method of increasing the persistence of a cell comprising introducing into the cell a polynucleotide encoding die transmembrane protein of any one of claims 1-28, the nucleic acid sequence of claim 29, the polynucleotide of any one of claims 30-40, or the vector of claim 42 or claim 43.

73. The method of claim 72, wherein the method increases the persistence of the cell, as compared to a cell in which the polynucleotide encoding the transmembrane protein has not been introduced.

74. The method of claim 72 or claim 73, wherein the method increases the persistence of the cell in the absence of interleukin-2 (IL2), as compared to a cell in the presence of IL2.

75. The method of any one of claims 72-74. wherein the persistence is in vitro or ex vivo persistence.

76. A method of increasing the proliferation of a cell comprising introducing into the cell a polynucleotide encoding the transmembrane protein of any one of claims 1-28, the nucleic acid sequence of claim 29, the polynucleotide of any one of claims 30-40, or the vector of claim 42 or claim 43.

77. The method of claim 76, wherein the method increases the proliferation of the cell, as compared to a cell in which the polynucleotide encoding the transmembrane protein has not been introduced.

78. The method of claim 76 or claim 77, wherein the method increases the proliferation of the cell in the absence of interleukin-2 (IL2), as compared to a cell in the presence of IL2.

79. The method of any one of claims 76-78. wherein the proliferation is in vitro or ex vivo proliferation.

80. A method of increasing the IL15 signaling of a cell, the method comprising introducing into the cell a polynucleotide encoding the transmembrane protein of any one of claims 1- 28, the nucleic acid sequence of claim 29, the polynucleotide of any one of claims 30-40, or the vector of claim 42 or claim 43.

81. The method of claim 80, wherein the method increases the IL 15 signaling of the cell in the absence of interleukin-2 (IL2), as compared to a cell in the presence of IL2.

82. A method of increasing the cytotoxicity of a cell, the method comprising introducing into the cell a polynucleotide encoding the transmembrane protein of any one of claims 1-28. the nucleic acid sequence of claim 29, the polynucleotide of any one of claims 30-40, or the vector of claim 42 or claim 43.

83. The method of any one of claims 72-82, wherein the cell is an immune cell.

84. The method of claim 83, wherein the immune cell is a natural killer (NK) cell or a T cell.

85. The method of claim 83 or claim 84, wherein the immune cell is a natural killer (NK) cell.

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