Tethered interleukin 2 recombinant receptors and methods of use

Abstract

A recombinant cytokine receptor is provided, comprising an IL-2 receptor polypeptide tethered to an IL-2 cytokine. Treg cells comprising the recombinant cytokine receptor and methods of use are also provided herein. In one aspect, the present application relates to a recombinant cytokine receptor comprising an interleukin-2 (IL-2) cytokine molecule tethered to the extracellular domain of IL-2Rβ. Cells expressing the IL-2-tethered IL-2Rβ recombinant cytokine receptor and methods of use are also provided herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 373,591, filed August 26, 2022, U.S. Provisional Application No. 63 / 498,803, filed April 27, 2023, and U.S. Provisional Application No. 63 / 499,954, filed May 3, 2023, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Reference to the electronic sequence listing The contents of the electronic sequence listing (237752000541SEQLIST.xml; size: 23,881 bytes; and creation date: August 18, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] The immune system plays an important role in maintaining the homeostasis of organisms, balancing between the elimination of foreign antigens and the self-tolerance of self-antigens.In particular, overactivated immune dysregulation can lead to various autoimmune disorders (such as irritable bowel syndrome, systemic lupus erythematosus, alopecia areata, multiple sclerosis), which is often the result of overactivated effector T lymphocytes or underactivated regulatory T lymphocytes (i.e., Treg).Current treatments for autoimmune diseases include the administration of steroids, which cause severe side effects in patients and often provide little relief to patients. Regulatory T cells are important players in maintaining the homeostasis of the organism to prevent the destruction of otherwise healthy tissues. Tregs are a unique subset of T cells that inhibit the cytotoxic or pro-inflammatory activity of effector CD4+ T cells or effector CD8+ T cells. Tregs differentiate from the parental T lymphocyte lineage upon upregulation of key Treg genes, particularly IL-2Rα and FOXP3 (see, e.g., Chen, ML et al. (2005), Proc Natl Accad Sci USA 102(2):419-424; and Liu, VC et al. (2007), J Immunol 178(5):2883-2892, which are incorporated herein by reference in their entireties). Upon T cell receptor (TCR) activation, these Tregs are responsible for directly suppressing effector T cell activity through cytokine production (e.g., TGF-β and IL-10) (see, e.g., Chen, J et al. (2019), Trends Mol Med; 25(11):1010-1023, incorporated herein by reference in its entirety) and engagement of immune checkpoint receptors (e.g., engagement of TIGIT or CTLA-4) (see, e.g., Knochelmann, HM et al. (2018), Cell Mol Immunol; 15(5):458-469, incorporated herein by reference in its entirety). While effector T cells can produce the cytokine IL-2 upon TCR activation to support their own expansion, Tregs cannot produce their own IL-2, and therefore Treg cells rely on exogenous IL-2 to promote Treg survival and maintenance. However, Treg cells, like all lineages of T lymphocytes, require IL-2 signaling for survival and proliferation.This biological mechanism ensures the maintenance of Tregs in tissue niches enriched for activated effector T cells, thus generating a cellular negative feedback mechanism by which increased IL-2 production by target effector T cells promotes Treg expansion, which then acts to inhibit effector T cell activity and, therefore, their own expansion and survival downstream (see, e.g., Shevyrev, D & Tereshchenko, V (2020), Front Immunol; 10: 3100, 1-13). Thus, targeting the IL-2 receptor signaling pathway in Tregs is one area of ​​recent interest in efforts to identify novel therapies for autoimmune disorders. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Chen, ML et al. (2005), Proc Natl Accad Sci USA 102(2):419-424; and Liu, VC et al. (2007), J Immunol 178(5):2883-2892 [Non-patent document 2] Chen, J et al. (2019),Trends Mol Med;25(11):1010-1023 [Non-patent document 3] Knochelmann, HM et al. (2018), Cell Mol Immunol;15(5):458-469 [Non-patent document 4] Shevyrev,D & Tereshchenko,V(2020),Front Immunol;10:3100,1-13 Summary of the Invention

[0005] In one aspect, the present application relates to a recombinant cytokine receptor comprising an interleukin-2 (IL-2) cytokine molecule tethered to the extracellular domain of IL-2Rβ. Cells expressing the IL-2-tethered IL-2Rβ recombinant cytokine receptor and methods of use are also provided herein.

[0006] In one aspect of the present invention, there is provided a regulatory T cell (Treg) comprising a recombinant cytokine receptor, wherein the recombinant cytokine receptor comprises an IL-2 cytokine; an IL-2 receptor β extracellular domain; a transmembrane domain; and an IL-2 receptor β intracellular domain; and the IL-2 receptor β extracellular domain is tethered to the IL-2 cytokine by a polypeptide linker. In some embodiments, the recombinant cytokine receptor forms a protein complex with IL-2Rγ. In some embodiments, the recombinant cytokine receptor participates in IL-2 signaling in the absence of exogenous IL-2.

[0007] In some embodiments according to any of the Tregs described above, the IL-2 cytokine comprises at least one amino acid substitution that reduces its affinity for IL-2Rα and / or IL-2Rγ by at least about two-fold. In some embodiments, the IL-2 cytokine comprises one or more amino acid substitutions selected from amino acid positions 18, 22, 126, 38, 43, 61, 15, 16, 19, 20, 22, 23, and 81. In some embodiments, the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of L18R, Q22E, Q126H, R38D, K43E, E61R, E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D. In some embodiments, the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126H; and / or R38D, K43E, and E61R; and / or E15S, H16Q, L19V, D20L, M23Q, and R81D; and / or E15S, H16Q, L19V, D20L, Q22K, and M23A.In some embodiments, Tregs comprise a. WT IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain; b. 3x IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain; c. REH IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain; d. 3x REH e. an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain comprising substitutions at positions E15S, H16Q, L19V, D20L, M23Q, and R81D; or f. an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain comprising substitutions at positions E15S, H16Q, L19V, D20L, Q22K, and M23A; in some embodiments, the polypeptide linker comprises glycine and serine.

[0008] In some embodiments according to any of the Tregs described above, the Treg comprises a recombinant cytokine receptor comprising: a. an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 4; b. an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 5; c. an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 6; or d. an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 12.

[0009] In some embodiments according to any of the Tregs described above, the Tregs comprise: a. an IL-2 cytokine comprising an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12; b. a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21; c. an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. a recombinant cytokine receptor comprising: an IL-2 receptor beta extracellular domain having 9%, or 100% sequence identity; d. an IL-2 receptor beta transmembrane domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14; and / or e. an IL-2 receptor beta intramembrane domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the IL-2 cytokine comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the recombinant cytokine receptor does not comprise a T cell receptor activation domain or a T cell costimulatory domain, and optionally, the T cell receptor activation domain or the T cell costimulatory domain is a CD28 signaling domain.

[0010] In some embodiments according to any of the Tregs described above, the Tregs comprise: a. an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 4, a polypeptide linker comprising the amino acid sequence of SEQ ID NO: 9, an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15; b. an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 5, a polypeptide linker comprising the amino acid sequence of SEQ ID NO: 9, an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15. a recombinant cytokine receptor comprising: an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 6, a polypeptide linker comprising the amino acid sequence of SEQ ID NO: 9, an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15; or d. an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 12, a polypeptide linker comprising the amino acid sequence of SEQ ID NO: 9, an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 11.

[0011] In some embodiments according to any of the Tregs described above, the Tregs comprise a recombinant cytokine receptor consisting of an IL-2 cytokine; an IL-2 receptor β extracellular domain; a transmembrane domain; and an IL-2 receptor β intracellular domain; wherein the IL-2 receptor β extracellular domain is tethered to the IL-2 cytokine by a polypeptide linker.

[0012] In some embodiments according to any of the Tregs described above, the Tregs are CD25+ and express FOXP3 and / or HELIOS. In some embodiments, the Tregs further comprise a chimeric antigen receptor (CAR).

[0013] In another aspect of the present invention, there is provided a recombinant cytokine receptor comprising an IL-2 cytokine, an IL-2 receptor β extracellular domain, a transmembrane domain, and an IL-2 receptor β intracellular domain, wherein the IL-2 receptor β extracellular domain is tethered to the IL-2 cytokine by a polypeptide linker. In some embodiments, the recombinant cytokine receptor does not comprise a T cell receptor activation domain or a T cell costimulatory domain. In some embodiments, the IL-2 receptor extracellular domain comprises the extracellular domain of IL-2Rβ. In some embodiments, the polypeptide linker comprises glycine and serine. In some embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21. In some embodiments, the recombinant cytokine receptor can form a protein complex with IL-2Rγ.

[0014] In some embodiments, the cytokine receptor participates in IL-2 signaling in the absence of exogenous IL-2.

[0015] In some embodiments according to any of the recombinant cytokine receptors described above, the IL-2 cytokine comprises at least one amino acid substitution that reduces affinity for IL-2Rα and / or IL-2Rγ by at least about 2-fold. In some embodiments, the IL-2 cytokine comprises one or more amino acid substitutions selected from amino acid positions 18, 22, 126, 38, 43, 61, 15, 16, 19, 20, 22, 23, and 81. In some embodiments, the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of L18R, Q22E, Q126H, R38D, K43E, E61R, E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D. In some embodiments, the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126H; and / or R38D, K43E, and E61R. In some embodiments, the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126H; and / or R38D, K43E, and E61R; and / or E15S, H16Q, L19V, D20L, M23Q, and R81D; and / or E15S, H16Q, L19V, D20L, Q22K, and M23A.In some embodiments, the recombinant cytokine receptor comprises: a. WT IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain; b. 3x IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain; c. REH IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain; d. 3x REH an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain; e. an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain comprising substitutions at positions E15S, H16Q, L19V, D20L, M23Q, and R81D; or f. an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain comprising substitutions at positions E15S, H16Q, L19V, D20L, Q22K, and M23A,

[0016] In some embodiments according to any of the recombinant cytokine receptors described above, the IL-2 cytokine comprises an amino acid sequence comprising 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 about 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12. In some embodiments, the IL-2 cytokine comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the recombinant cytokine receptor does not comprise a T cell receptor activation domain or a T cell costimulatory domain, and optionally, the T cell receptor activation domain or the T cell costimulatory domain is a CD28 signaling domain. In some embodiments, the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of F42A, F42K, R38D, R38A, E61R, R38D and E61R, K35D, K43E, K43D, E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D. In some embodiments, the IL-2 cytokine comprises the amino acid substitutions R38D, E61R, and K43E. In some embodiments, the IL-2 cytokine comprises the amino acid substitutions R38A, E61R, and K43E. In some embodiments, the IL-2 cytokine comprises the amino acid substitutions R38D, E61R, and K43D. In some embodiments, the IL-2 cytokine comprises the amino acid substitutions R38A, E61R, and K43D. In some embodiments, the IL-2 receptor comprises an IL-2 cytokine comprising one or more amino acid substitutions selected from the group consisting of L18R, Q22E, Q126K, Q126H, Q126M, and Q126R, hi some embodiments, the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126K.In some embodiments, the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126M. In some embodiments, the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126R. In some embodiments, the recombinant cytokine receptor comprises: a. an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 4; b. an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 5; c. an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 6; or d. an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 12.

[0017] In some embodiments according to any of the recombinant cytokine receptors described above, the recombinant cytokine receptor comprises an IL-2 receptor β extracellular domain that comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the recombinant cytokine receptor comprises an IL-2 receptor β transmembrane domain that comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the recombinant cytokine receptor comprises an IL-2 receptor β intracellular domain that comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the recombinant cytokine receptor comprises an IL-2 receptor beta polypeptide that comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:7.

[0018] In some embodiments according to any of the recombinant cytokine receptors described above, the recombinant cytokine receptor comprises: a. an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 4, a polypeptide linker comprising the amino acid sequence of SEQ ID NO: 9, an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15; b. an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 5, a polypeptide linker comprising the amino acid sequence of SEQ ID NO: 9, an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15. an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15; c. an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 6, a polypeptide linker comprising the amino acid sequence of SEQ ID NO: 9, an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15; or d. an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 12, a polypeptide linker comprising the amino acid sequence of SEQ ID NO: 9, an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence that comprises 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 about 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 11.

[0019] In some embodiments according to any of the recombinant cytokine receptors described above, the recombinant cytokine receptor consists of an IL-2 cytokine, an IL-2 receptor β extracellular domain, a transmembrane domain, and an IL-2 receptor β intracellular domain, wherein the IL-2 receptor β extracellular domain is tethered to the IL-2 cytokine by a polypeptide linker.

[0020] Also provided herein is a nucleic acid encoding an IL-2-tethered IL-2Rβ recombinant cytokine receptor. Also provided herein is a nucleic acid encoding any of the recombinant cytokine receptors described herein. Also provided herein is a vector comprising the nucleic acid provided herein. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector further comprises a marker gene. In some embodiments, the marker gene is a transmembrane protein. In some embodiments, the transmembrane protein is EGFR.

[0021] Also provided herein are T cells comprising any of the recombinant cytokine receptors described herein, nucleic acids encoding the recombinant cytokine receptors, or vectors comprising the nucleic acids. In some embodiments, the T cells are regulatory T cells (Treg), and the Treg are CD25+ and express FOXP3 and / or HELIOS. In some embodiments, the T cells (such as Treg cells) further comprise a chimeric antigen receptor (CAR). Also provided herein are compositions comprising a nucleic acid encoding any of the recombinant cytokine receptors described herein, a vector comprising a nucleic acid encoding a recombinant cytokine receptor(s), or a T cell, such as a Treg cell, expressing a recombinant cytokine receptor(s).

[0022] In another aspect of the present invention, there is provided a method of treating an immune-related disorder, comprising administering T cells, such as Treg cells (e.g., any of the Treg cells described herein) that express any of the recombinant cytokine receptors described herein, or a composition comprising the T cells, to an individual in need of the T cells or composition. In some embodiments, the cells are autologous to the individual. In some embodiments, the individual is human. In some embodiments, the individual expresses IL-2. Also provided herein is the use of T cells, such as Tregs, comprising a recombinant cytokine receptor provided herein for treating an immune-related disorder in an individual. In some embodiments, the Tregs prevent, ameliorate, or cure the immune-related disorder.

[0023] In another aspect of the present invention, there is provided a method for expanding transduced Treg cells in the absence of exogenous IL-2, comprising introducing into Treg cells a nucleic acid encoding an IL-2-tethered IL-2Rβ recombinant cytokine receptor or a vector comprising said nucleic acid, and culturing the cells.

[0024] In some embodiments according to any of the recombinant cytokine receptors described above, the recombinant cytokine receptor does not activate IL-2 signaling in cells that do not contain the recombinant cytokine receptor. In some embodiments, the recombinant cytokine receptor does not activate IL-2 signaling in Tregs that do not contain the recombinant cytokine receptor. In some embodiments, the recombinant cytokine receptor does not activate signaling of an IL-2 receptor that comprises a different amino acid sequence.

[0025] In some embodiments according to any of the recombinant cytokine receptors described above, the recombinant cytokine receptor does not cause the transduced Treg cells to secrete one or more cytokines more than Treg cells cultured with exogenous IL-2 cytokine. In some embodiments, the recombinant cytokine receptor does not cause the transduced Treg cells to secrete one or more cytokines at a level(s) higher than the level(s) of cytokine secretion(s) from Treg cells cultured with exogenous IL-2 cytokine.

[0026] In some embodiments according to any of the Tregs described above, at least one Treg marker selected from the group consisting of CD4+, CD25+, and CD1271 is detected in Treg cells comprising a recombinant cytokine receptor. In some embodiments, the level of FOXP3 and / or HELIOS is detected in Treg cells comprising a recombinant cytokine receptor. In some embodiments, the methylation pattern of a Treg-specific differentially regulated gene is detected in Treg cells comprising a recombinant cytokine receptor. In some embodiments, the Treg-specific differentially regulated gene is FOXP3.

[0027] In some embodiments according to any of the Tregs described above, the Treg cells comprising the recombinant cytokine receptor are capable of dividing and proliferating in the absence of IL-2.

[0028] In some embodiments according to any of the Tregs described above, one or more markers of endogenous IL-2 signaling are detected in Treg cells comprising a recombinant cytokine receptor. In some embodiments, the one or more markers of endogenous IL-2 signaling comprise phosphorylated STAT5.

[0029] In some embodiments according to any of the Tregs described above, the suppressive activity of the Tregs against CD8+ T cells and / or CD4+ T cells is increased. In some embodiments, the suppressive activity of the Tregs against CD8+ T cells and / or CD4+ T cells is increased compared to Treg cells that do not comprise a recombinant cytokine receptor. In some embodiments, the suppressive activity comprises a decrease in the rate of division of CD4+ T cells and / or CD8+ T cells.

[0030] In some embodiments according to any of the Tregs described above, the relative number of Treg cells in a composition comprising a population of Treg cells transduced with a recombinant cytokine receptor increases over time.

[0031] In some embodiments according to any of the Tregs described above, a composition comprising a population of recombinant cytokine receptor-transduced Treg cells expanded without IL-2 contains a similar number of viable cells compared to a composition comprising the same population of Treg cells not transduced with recombinant cytokine receptors expanded with IL-2.

[0032] In some embodiments according to any of the Tregs described above, at least about 80% of the cells in the population of Treg cells transduced with the recombinant cytokine maintain expression of FOXP3 and / or HELIOS for about 14 days after transduction, hi some embodiments, at least about 80% of the cells in the population of Treg cells transduced with the recombinant cytokine maintain expression of FOXP3 and / or HELIOS for about 23 days after transduction.

[0033] In some embodiments according to any of the Tregs described above, the Treg cells comprise a recombinant cytokine receptor, and i. the in vitro and / or in vivo suppressive activity of the Tregs against CD8+ T cells and / or CD4+ T cells is increased compared to control Tregs that have not been transduced with the recombinant cytokine receptor; ii. the rate of division of CD4+ T cells and / or CD8+ T cells when cultured in the presence of Tregs is increased compared to control Tregs that have not been transduced with the recombinant cytokine receptor; the relative amount of Treg cells in a composition comprising a population of recombinant cytokine receptor-transduced Treg cells increases over time; iv. a composition comprising a population of recombinant cytokine receptor-transduced Treg cells expanded without IL-2 contains a similar number of viable cells compared to a composition comprising the same population of Treg cells not transduced with recombinant cytokine receptors expanded with IL-2; v. at least 80% of the cells in a population of recombinant cytokine receptor-transduced Treg cells maintain expression of FOXP3 and / or HELIOS for about 14 days after transduction; vi. the cells in a population of recombinant cytokine receptor-transduced Treg cells at least 80% of the Treg cells maintain expression of FOXP3 and / or HELIOS for about 23 days after transduction; vii. the population of Treg cells transduced with the recombinant cytokine receptor is expanded at least two-fold over the population of the same Treg cells not transduced with the recombinant cytokine receptor; viii. the IL-10 cytokine levels produced by Tregs in vitro and / or in vivo are equivalent to or increased compared to control Tregs not transduced with the recombinant cytokine receptor; ix. the IFN-γ cytokine levels produced by Tregs in vitro and / or in vivo are equivalent to or increased compared to control Tregs not transduced with the recombinant cytokine receptor; x.The levels of Gr-B cytokine produced by Tregs in vitro and / or in vivo are comparable to or increased compared to control Tregs not transduced with the recombinant cytokine receptor; xi. Tregs are functionally reactivated in vivo and / or in vitro to levels higher than control Tregs not transduced with the recombinant cytokine receptor; and / or xii. The level of Treg proliferation is increased one or more times in vitro and / or in vivo following restimulation.

[0034] In some embodiments according to any of the Tregs described above, the Treg cells are cultured in a composition comprising a cytokine other than IL-2.

[0035] In some embodiments according to any of the Tregs described above, the population of Treg cells transduced with the recombinant cytokine receptor is expanded at least about two-fold greater than the population of the same Treg cells not transduced with the recombinant cytokine receptor. In some embodiments, the population of Treg cells transduced with the recombinant cytokine receptor is expanded at least about two-fold, and the population of Treg cells transduced with the recombinant cytokine receptor maintains expression of at least one Treg marker selected from the group consisting of CD25, FOXP3, and HELIOS. [Brief explanation of the drawings]

[0036] [Figure 1]Fluorescence-activated cell sorting (FACS) plots of regulatory T (Treg) cells transduced with an empty vector control (MND control), wild-type IL-2 fused to IL-2Rβ, or mutant IL-2 fused to IL-2Rβ are shown. Cells were transduced, expanded for 14 days, and then stained for IL-2Rα and CD28. The REH IL-2 mutant is an IL-2 cytokine with amino acid substitutions L18R, Q22E, and Q126H. The 3x IL-2 mutant is an IL-2 cytokine with amino acid substitutions R38D, K43E, and E61R. The 3x REH IL-2 3x is an IL-2 cytokine with amino acid substitutions L18R, Q22E, Q127H, R38D, K43E, and E61R. [Figure 2A] Figure 1 shows a FACS plot of Treg cells transduced with an empty vector control. Cells were transduced and expanded in the presence of IL-2 for 14 days, then assayed for the presence of FOXP3, HELIOS, CD28, IL-2Rα, and EGFRt. EGFR+ is a marker for cells transduced with the construct. [Figure 2B] Figure 1 shows a FACS plot of Treg cells transduced with a construct expressing 3x-IL-2 / IL-2Rβ. Cells were transduced, expanded in the presence of IL-2 for 14 days, and then assayed for the presence of FOXP3, HELIOS, CD28, IL-2Rα, and EGFRt. EGFR+ is a marker for successful transduction. [Figure 2C] Figure 1 shows a FACS plot of Treg cells transduced with a construct expressing 3x IL-2 / IL-2Rβ. Cells were transduced, expanded for 14 days without IL-2, and then assayed for the presence of FOXP3, HELIOS, CD28, IL-2Rα, and EGFRt. EGFR+ is a marker for successful transduction. [Figure 3A]Figure 1 shows a FACS plot of Treg cells transduced with an empty vector control. Cells were transduced and expanded in the presence of IL-2 for 14 days, then assayed for the presence of FOXP3, HELIOS, CD28, IL-2Rα, and EGFRt. EGFR+ is a marker for successful transduction. [Figure 3B] Figure 1 shows a FACS plot of Treg cells transduced with a construct expressing a 3x REH-IL-2 / IL-2Rβ fusion construct. Cells were transduced and expanded in the presence of IL-2 for 14 days and then assayed for the presence of FOXP3, HELIOS, CD28, IL-2Rα, and EGFRt. REH 3x IL-2 is an IL-2 cytokine with the amino acid substitutions L18R, Q22E, Q127H, R38D, K43E, and E61R. [Figure 3C] Figure 1 shows a FACS plot of Treg cells transduced with a construct expressing a 3x REH-IL-2 / IL-2Rβ fusion construct. Cells were transduced, grown for 14 days without IL-2, and then assayed for the presence of FOXP3, HELIOS, CD28, IL-2Rα, and EGFRt. EGFR+ is a marker for successful transduction. REH 3x IL-2 is an IL-2 cytokine with the amino acid substitutions L18R, Q22E, Q127H, R38D, K43E, and E61R. [Figure 4] A chart showing the percentage of cells positive for EGFRt days post-transduction when grown with or without exogenous IL-2 is shown. The mean EGFRt percentage was calculated from five donors. The percentage was determined by FACS staining for EGFRt. EGFR+ is a marker for cells transduced with the construct. [Figure 5A]Shown is the fold change in Treg expansion over a period of days after transduction with control or 3x IL-2 / IL-2Rβ constructs when grown with or without exogenous IL-2. Fold expansion was normalized for EGFRt expression across the four donors. EGFR+ is a marker for successful transduction. [Figure 5B] Bar graphs depicting in vitro cell fold expansion for control Tregs cultured in the presence of IL-2 cytokine, 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor-expressing Tregs cultured in the presence of IL-2, and 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor-expressing Tregs cultured without IL-2 are shown. [Figure 6-1] Figure 1 shows FACS plots of Treg cells transduced with an empty vector control or a 3x IL-2 / IL-2Rβ recombinant cytokine receptor construct. Cells were transduced, expanded with IL-2 for 23 days, and then assayed for the presence of FOXP3, HELIOS, CD28, IL-2Rα, and EGFRt. EGFR+ is a marker for successful transduction. [Figure 6-2] Figure 1 shows a FACS plot of Treg cells transduced with a 3x IL-2 / IL-2Rβ recombinant cytokine receptor construct. Cells were transduced and grown with or without IL-2 for 23 days, then assayed for the presence of FOXP3, HELIOS, CD28, IL-2Rα, and EGFRt. EGFR+ is a marker for successful transduction. [Figure 7A] We show that Tregs expressing various mutant IL-2 fused to IL-2Rβ can suppress the expansion of CD4+ and CD8+ cells when co-cultured at various ratios. Peripheral blood mononuclear cells (PBMCs) are used as a negative control. [Figure 7B] Figure 1 shows the suppressive activity of Tregs expressing 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor on effector CD4+ T cells and effector CD8+ T cells. [Figure 8A-1]FACS plots sorting for STAT5 phosphorylation (pSTAT5) in Tregs expressing various 3x IL-2 / IL-2Rβ recombinant cytokine receptor constructs are shown. The concentration of exogenous IL-2 pulse is indicated next to the corresponding curve. EGFR+ is a marker for successful transduction. [Figure 8A-2] FACS plots sorting for STAT5 phosphorylation (pSTAT5) in Tregs expressing various 3x IL-2 / IL-2Rβ recombinant cytokine receptor constructs are shown. The concentration of exogenous IL-2 pulse is indicated next to the corresponding curve. EGFR+ is a marker for successful transduction. [Figure 8B] Shown is a histogram (B) and bar graph (C) representation of pSTAT5 levels in EGFR-positive control Tregs and 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor-expressing Tregs cultured in vitro with or without IL-2. [Figure 8C] Shown is a histogram (B) and bar graph (C) representation of pSTAT5 levels in EGFR-positive control Tregs and 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor-expressing Tregs cultured in vitro with or without IL-2. [Figure 9A-1] Figure 1 shows FACS plots sorting for pSTAT5 in expanded Treg EGFRt-negative cells expressing various 3x IL-2 / IL-2Rβ recombinant cytokine receptor constructs. Cells were pulsed with various concentrations of exogenous IL-2 prior to analysis. The concentration of the exogenous IL-2 pulse is indicated next to the corresponding curve. EGFR+ is a marker for successful transduction. [Figure 9A-2] Figure 1 shows FACS plots sorting for pSTAT5 in expanded Treg EGFRt-negative cells expressing various 3x IL-2 / IL-2Rβ recombinant cytokine receptor constructs. Cells were pulsed with various concentrations of exogenous IL-2 prior to analysis. The concentration of the exogenous IL-2 pulse is indicated next to the corresponding curve. EGFR+ is a marker for successful transduction. [Figure 9B] Shown is a histogram (B) and bar graph (C) representation of pSTAT5 levels in EGFR-negative control Tregs and 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor-expressing Tregs cultured in vitro with or without IL-2. [Figure 9C] Shown is a histogram (B) and bar graph (C) representation of pSTAT5 levels in EGFR-negative control Tregs and 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor-expressing Tregs cultured in vitro with or without IL-2. [Figure 10A] Shown is the percent methylation at the indicated CpG sites within the FOXP3 locus for various 3x IL-2 / IL-2Rβ recombinant cytokine receptor constructs cultured with or without exogenous IL-2 cytokine. 200,000 cells were harvested on day 14 and then sent for TSDR analysis. Tregs derived from two donors were analyzed. [Figure 10B] 1 provides a bar graph depicting quantification of percent methylation at the TSDR FOXP3 locus in conventional CD4 T cells, control Tregs, and Tregs expressing the 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor. [Figure 11] Figure 1 shows the concentrations of cytokines secreted into the culture medium by Tregs expressing various 3x IL-2 / IL-2Rβ constructs. Cells were restimulated on day 16 at a bead-to-cell ratio of 1:5. Supernatants were collected on day 19, and cytokine production was measured by ELISA. [Figure 12-1] Levels of Treg lineage and activation markers, including FOXP3 (A) and CD25 (B), are shown for control Tregs and 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor-expressing Tregs cultured with or without PBMCs at PBMC:Treg ratios of 1:1, 1:2, and 1:4. [Figure 12-2]Levels of markers of Treg lineage and activation, including CTLA4 (C) and GARP (D), are shown for control Tregs and 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor-expressing Tregs cultured with or without PBMCs at PBMC:Treg ratios of 1:1, 1:2, and 1:4. [Figure 13A] 1 provides a schematic overview of Treg (re)stimulation experiments testing Treg responses to anti-CD3 / anti-CD28 antibody stimulation versus CD19 antigen stimulation in vitro. [Figure 13B-1] Figure 1 shows the time course of proliferation of control Tregs and 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor-expressing Tregs cultured in vitro with or without IL-2. [Figure 13B-2] Figure 1 shows the time course of proliferation of control Tregs and 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor-expressing Tregs cultured in vitro with or without IL-2. [Figure 13C-1] Representative FACS plots comparing FOXP3+ and HELIOS+ levels in control Tregs vs. Tregs expressing 3x REH IL-2 / IL-2Rβ recombinant cytokine receptor after 1st vs. 3rd round of stimulation with anti-CD3 / anti-CD28 antibodies are shown. [Figure 13C-2] Representative FACS plots comparing FOXP3+ and HELIOS+ levels in control Tregs vs. Tregs expressing 3x REH IL-2 / IL-2Rβ recombinant cytokine receptor after 1st vs. 3rd round of stimulation with CD19 antigen-specific activation are shown. [Figure 13D-1]Representative FACS plots of FOXP3 and HELIOS levels in control Tregs and Tregs expressing the 3x REH IL-2 / IL-2Rβ recombinant cytokine receptor are shown for cells expanded in vitro under control conditions at day 3. Cells expanded in pro-inflammatory conditions were treated with 50 ng / mL IL-1β, 50 ng / mL IL-6, 50 ng / mL IL-23, and 300 IU IL-2. Cells were further activated by polyclonal anti-CD3 / anti-CD28 antibody stimulation or CD19 antigen stimulation. [Figure 13D-2] Representative FACS plots of FOXP3 and HELIOS levels in control Tregs and Tregs expressing the 3x REH IL-2 / IL-2Rβ recombinant cytokine receptor are shown for cells expanded in vitro under control conditions at day 17. Cells expanded in pro-inflammatory conditions were treated with 50 ng / mL IL-1β, 50 ng / mL IL-6, 50 ng / mL IL-23, and 300 IU IL-2. Cells were further activated by polyclonal anti-CD3 / anti-CD28 antibody stimulation or CD19 antigen stimulation. [Figure 13D-3] Representative FACS plots of FOXP3 and HELIOS levels in control Tregs and Tregs expressing the 3x REH IL-2 / IL-2Rβ recombinant cytokine receptor for cells expanded in vitro under pro-inflammatory conditions on day 3 are shown. Cells expanded under pro-inflammatory conditions were treated with 50 ng / mL IL-1β, 50 ng / mL IL-6, 50 ng / mL IL-23, and 300 IU IL-2. Cells were further activated by polyclonal anti-CD3 / anti-CD28 antibody stimulation or CD19 antigen stimulation. [Figure 13D-4]Representative FACS plots of FOXP3 and HELIOS levels in control Tregs and Tregs expressing the 3x REH IL-2 / IL-2Rβ recombinant cytokine receptor for day 17 cells expanded in vitro under pro-inflammatory conditions. Cells expanded in pro-inflammatory conditions were treated with 50 ng / mL IL-1β, 50 ng / mL IL-6, 50 ng / mL IL-23, and 300 IU IL-2. Cells were further activated by polyclonal anti-CD3 / anti-CD28 antibody stimulation or CD19 antigen stimulation. [Figure 13E] Graphical representation of FOXP3 mean fluorescence intensity (MFI) over time for control, IL-12, and pro-inflammatory conditions is shown upon either anti-CD3 / anti-CD28 antibody activation or CD19 antigen-specific activation. The IL-12 condition included cell incubation with 20 ng / mL IL-12 and 300 IU IL-2. [Figure 14A] Shown is the mean fluorescence intensity (MFI) over time of a marker of Treg proliferation, activation, or exhaustion (CD71) for control Tregs and 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor-expressing Tregs cultured in vitro with or without IL-2 using the stimulation conditions shown in Figure 13-1. [Figure 14B] Figure 13-1 shows the mean fluorescence intensity (MFI) over time of a marker of Treg proliferation, activation, or exhaustion (ICOS) for control Tregs and 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor-expressing Tregs cultured in vitro with or without IL-2 using the stimulation conditions shown in Figure 13-1. [Figure 14C] Figure 13-1 shows the mean fluorescence intensity (MFI) of a marker of Treg proliferation, activation, or exhaustion (PD-1) over time for control Tregs and 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor-expressing Tregs cultured in vitro with or without IL-2 using the stimulation conditions shown in Figure 13-1. [Figure 15A]The percentage of cytokine IL-10 producing Tregs at days 2 and 19 for control Tregs and 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor expressing Tregs cultured in vitro with IL-2 is shown. [Figure 15B] The percentage of cytokine IFN-γ producing Tregs at days 2 and 19 for control Tregs and 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor expressing Tregs cultured in vitro with IL-2 is shown. [Figure 15C] The percentage of cytokine GrB-producing Tregs at days 2 and 19 for control Tregs and 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor-expressing Tregs cultured in vitro with IL-2 is shown. [Figure 16A] Absolute numbers of Tregs engrafted and expanded in vivo in an HGD mouse model are shown. Mice were immunized with CD19 or vehicle and then intravenously injected with control Tregs or 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor-expressing Tregs with or without CD19-CAR expression, as indicated in the bottom panel schematic. The left panel shows the results of cell expansion in the liver at day 30 after Treg adoptive transfer, and the right panel shows the results of cell expansion in the liver at day 60. [Figure 16B-1] Treg phenotype according to FOXP3 and HELIOS levels in control and 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor-expressing Tregs found in the liver or lung at 30 days after Treg adoptive transfer or 60 days in vivo is shown. [Figure 16B-2] Treg phenotype according to FOXP3 and HELIOS levels in control and 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor-expressing Tregs found in bone marrow or spleen at day 30 after Treg adoptive transfer or day 60 in vivo is shown. [Figure 16C]Graphical representation shows the number of human immune cells circulating in the blood and the percentage of immune cells that are Treg cells 15 days after adoptive cell transfer with vs. without exogenous IL-2. [Figure 16D] FACS plots show the number of human immune cells circulating in the blood and the percentage of immune cells that are Treg cells 15 days after adoptive cell transfer with vs. without exogenous IL-2. [Figure 17] Figure 1 shows the in vivo suppressive activity of Tregs expressing the 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptor in a humanized mouse model of graft-versus-host disease (GVHD). PBMC, peripheral blood mononuclear cells; 5M, 5 million (5 × 10). [Figure 18] 1 shows the progression of EAE scores over time as an indicator of neurological autoimmune severity (i.e., multiple sclerosis) in control mice and mice with constitutively activated STAT5b (CA-STAT5b) in cells expressing FOXP3 (i.e., Tregs). [Figure 19A] Fold amplification is shown as a bar graph for wild-type (WT) Tregs with or without IL-2 and CA-STAT5b Tregs without IL-2. [Figure 19B] Fold amplification is shown as FACS plots for wild-type (WT) Tregs with or without IL-2 and CA-STAT5b Tregs without IL-2. [Figure 19C] Figure 1 shows the suppressive activity of WT Tregs and CA-STAT5b Tregs in vitro. [Figure 19D] Figure 1 shows the suppressive activity of WT and CA-STAT5b Tregs in an in vivo graft-versus-host disease (GVHD) mouse model. [Figure 20A] Figure 1 shows the persistence of IL-2-secreting Tregs. The percentage of IL-2-secreting Tregs cultured with or without IL-2 that persist over time is shown. [Figure 20B] Figure 1 shows the expansion of IL-2-secreting Tregs. The percentage of expanded IL-2-secreting Tregs is shown when cultured with (lower panel) or without (upper panel) IL-2. [Figure 21A] Cell counts over time are shown for control Tregs and Tregs expressing IL-2 tethered protein tags cultured with or without IL-2. [Figure 21B] Figure 1 shows FACS analysis of IL-2 and EGFR protein tag levels over time in IL-2-tethered EGFR-expressing Tregs expanded in vitro without IL-2. [Figure 21C] 1 provides a bar graph showing the amount of soluble IL-2 that accumulates over time in the culture medium of Tregs expressing IL-2-tethered protein tags grown in vitro without IL-2. [Figure 22] A heatmap of RNA sequencing analysis was performed on wild-type and CA-STAT5b Tregs. 278 differentially expressed genes (DEGs) were identified, of which 99 were down-regulated and 179 were up-regulated in CA-STAT5b Tregs compared to wild-type Tregs. [Figure 23] Heatmap of RNA-sequencing analysis performed on wild-type Tregs and Tregs expressing 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptors (both cultured with IL-2) shows that 34 differentially expressed genes (DEGs) were identified, 13 of which were down-regulated and 21 of which were up-regulated in Tregs expressing 3x / REH IL-2 / IL-2Rβ recombinant cytokine receptors compared to wild-type Tregs. DETAILED DESCRIPTION OF THE INVENTION

[0037] Successful adoptive cell therapy requires the steady expansion and persistence of administered cells, and environmental signals received by cells strongly contribute to these behaviors. The present invention relates to recombinant cytokine receptors that enhance IL-2 signaling in target cells (e.g., Tregs) in the absence of exogenous IL-2. This IL-2 signaling cascade enhances cell survival, proliferation, and function. In particular, the present invention relates to IL-2Rβ protein or polypeptide chains tethered to IL-2 at the N-terminus of the IL-2Rβ extracellular domain. In some instances, the tethered IL-2 molecule is mutated to reduce or abolish IL-2 cytokine binding to the IL-2Rα and / or IL-2Rγ chains, as described herein. In some embodiments, provided herein is a system in which the proliferation and proliferation of immune cells (e.g., T cells, such as Tregs), as well as their in vivo persistence, immune-stimulating cytokine production, and immune function, can be enhanced by the introduction of recombinant cytokine receptors. These recombinant cells can be used, for example, as a cell therapy for autoimmune disorders.

[0038] Furthermore, recombinant cytokine receptors exhibit different functional properties depending on the tethered IL-2 cytokine or its variant. For example, Treg cells expressing 3x IL-2 / IL-2Rβ or 3x REH IL-2 / IL-2Rβ recombinant cytokine receptors exhibit the highest degree of Treg activation. Tregs expressing 3x REH IL-2 / IL-2Rβ recombinant cytokine receptors exhibit the highest percentage of FOXP3+ and HELIOS+ co-expression (i.e., Treg stability) and suppressive activity against CD4+ and CD8+ conventional T cells. The level of STAT-5 activation, as measured by the level of phosphorylated STAT-5 species, is approximately similar among each of the recombinant cytokine receptors described herein. As described herein, each of the four IL-2 molecules (i.e., wild-type, 3x, REH, or 3x REH) tethered to the recombinant cytokine receptor possesses unique IL-2 receptor chain binding properties and results in unique biological responses upon activation of the IL-2 receptor and downstream signaling cascade. Thus, the in vitro and in vivo functional characteristics of the recombinant cytokine receptor are surprising, and these differences can be exploited to meet the biological constraints of different diseases where targeting Tregs may prove beneficial for therapeutic purposes.

[0039] In some embodiments, the recombinant cytokine receptor lacks a TCR activation or TCR coactivation domain (e.g., lacks the activation or coactivation domain of CD3 or CD28) to enhance cell function in the absence of T cell receptor or CAR stimulation. Cells (e.g., Tregs) containing these recombinant cytokine receptors are still activated, for example, by the canonical TCR activation pathway in the presence of a target antigen (including CAR activation), to induce (i.e., turn on or increase) their suppressive activity. Thus, Tregs are not capable of becoming activated in the absence of target activation (e.g., target antigen presentation by an antigen-presenting cell (APC) or the presence of one or more polypeptides containing the required activation domain). In some embodiments, the recombinant cytokine receptor lacking a coactivation or activation domain comprises a tethered wild-type IL-2 molecule.

[0040] Various proposed methods for activating the IL-2 signaling pathway in Tregs have not demonstrated functional success. See Example 9, where three separate approaches each failed to support Treg function. That is, the constitutively activated STAT5 Treg model, the IL-2-secreting Treg model, and the IL-2-tethered tagged protein Treg model each failed to enhance Treg activity relative to conventional T cells, thereby demonstrating that not all approaches are successful when transferred to Treg cells. Treg cells are known to be completely different from conventional T cells in terms of function, molecular profile, and genetic profile (see, e.g., Grinberg-Bleyer et al. Cell 170(6):1096-1108, 2017; Grinberg-Bleyer et al. J Immunol 200(7):2362-71, 2018, both of which are incorporated herein by reference in their entireties), thus necessitating a Treg-specific tailored approach, as described herein. Furthermore, when comparing T cells as a genus with NK cells as a genus, the biological processes of cell differentiation, survival, proliferation, and activation are more unique, which can be readily recognized by those skilled in the art, thereby confirming the importance of a Treg-specific tailored approach. Of particular note is the IL-2-tethered tagged protein Treg model, in which IL-2 was tethered to a membrane-bound tagged protein. In this model, the IL-2 cytokine would be in close proximity to the extracellular domain of the IL-2 receptor to initiate IL-2 binding and intracellular signaling. This model was unable to support long-term Treg survival, nor Treg function, demonstrating the surprising and beneficial technical features of the present invention.

[0041] The present invention relates generally to the field of immunology and in part to compositions and methods for growing, modifying, and amplifying cells, including recombinant cytokine receptors that enable immune cells, such as Treg cells, to proliferate in the absence of exogenous IL-2. Provided herein are recombinant cytokine receptors that activate IL-2Rβ signaling by tethering the IL-2 cytokine to the extracellular domain of the IL-2Rβ (also referred to as "IL-2RB") polypeptide chain. These recombinant IL-2 cytokine receptors are not dependent on exogenous cytokines for activation. Thus, compared to untransduced cells, cells transduced with the IL-2 recombinant cytokine receptors described herein have certain advantages, including independence from exogenous IL-2. In some embodiments, the IL-2 molecule tethered to the IL-2 recombinant cytokine receptor expressed by cells transduced with the IL-2 recombinant cytokine receptor does not activate the native IL-2 receptor of the untransduced cells. In some embodiments of the methods and compositions provided herein, cells are engineered to contain the IL-2 recombinant cytokine receptor described herein. The IL-2 recombinant cytokine receptor described herein provides a stimulatory cytokine signal to cells, improving the efficacy of cell therapy. In some embodiments, the recombinant cytokines of the present application contain amino acid substitutions in the cytokine domain. These recombinant cytokine receptors can form complexes with IL-2Rγ, allowing constitutive IL-2R signaling in cells. In some embodiments, the recombinant cytokine receptors do not activate signaling of a different IL-2 receptor and cause the cells to secrete more cytokines than cells cultured with exogenous IL-2 cytokine.

[0042] In some embodiments, the IL-2 recombinant cytokine receptor comprises an IL-2 cytokine. In some embodiments, the IL-2 recombinant cytokine receptor comprises an IL-2 cytokine with one or more amino acid substitutions that result in reduced or abolished binding affinity for IL-2RA (also "IL-2Rα") and / or IL-2Rγ. In some embodiments, the IL-2 cytokine comprises at least one or more amino acid substitutions at amino acid positions selected from 18, 22, 126, 38, 43, and 61. In some embodiments, the IL-2 cytokine comprises at least one amino acid substitution selected from the group consisting of L18R, Q22E, Q126H, R38D, K43E, and E61R. In some embodiments, the mutant IL-2 molecule comprises the amino acid substitutions L18R, Q22E, and Q126H; and / or R38D, K43E, and E61R. In some embodiments, the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of F42A, F42K, R38D, R38A, E61R, R38D and E61R, K35D, K43E, and K43D. In some embodiments, the IL-2 receptor comprises an IL-2 cytokine comprising one or more amino acid substitutions selected from the group consisting of L18R, Q22E, Q126K, Q126H, Q126M, and Q126R. In some embodiments, the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126K. In some embodiments, the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126M. In some embodiments, the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126R. In some embodiments, the IL-2 cytokine with one or more amino acid substitutions exhibits reduced binding to IL-2Rα and / or reduced binding to IL-2Rγ. In some embodiments, the recombinant cytokine receptor forms a complex with IL-2Rγ. In some embodiments, the cytokine receptor participates in IL-2 signaling in the absence of exogenous IL-2 cytokine.In some embodiments, the recombinant cytokine receptor does not activate IL-2 signaling in a cell that does not contain the recombinant cytokine receptor. In some embodiments, the recombinant cytokine receptor does not activate signaling of a different IL-2 receptor. In some embodiments, the cell is a T cell (e.g., a Treg).

[0043] All publications referenced in this application (including patent documents, scientific articles, and databases) are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. To the extent that a definition set forth herein contradicts or otherwise conflicts with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

[0044] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0045] I. Recombinant Cytokine Receptors The present invention provides a recombinant cytokine receptor that transduces intracellular interleukin-2 (IL-2) signaling in the absence of exogenous IL-2. In some embodiments, the recombinant cytokine receptor is an engineered molecule comprising (I) an IL-2 cytokine, (II) a polypeptide linker, (III) an extracellular domain, (IV) a transmembrane domain, and (V) an intracellular domain. In some embodiments, the recombinant cytokine receptor does not comprise a TCR activation domain or a costimulatory domain (e.g., an activation or costimulatory domain of CD3 or CD28 (such as the CD28 signaling domain)). In some embodiments, the IL-2 cytokine is a naturally occurring IL-2 cytokine and / or a naturally occurring IL-2 receptor polypeptide (e.g., naturally occurring IL-2Rβ). In some embodiments, the recombinant cytokine receptor comprises a naturally occurring IL-2 cytokine but does not comprise a CD28 signaling domain (e.g., the activation or costimulatory domain of CD28). In some embodiments, the IL-2 cytokine comprises one or more amino acid substitutions. In some embodiments, the IL-2 cytokine comprises at least one or more amino acid substitutions at a position selected from amino acid positions 18, 22, 126, 38, 43, 61, 29, 15, 16, 19, 20, 22, 23, and 81. In some embodiments, the IL-2 cytokine comprises one or more of the following substitutions: L18R, Q22E, Q126H. In some embodiments, the IL-2 cytokine comprises one or more of the following substitutions: R38D, K43E, and E61R. In some embodiments, the IL-2 cytokine comprises one or more substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, M23Q, and R81D. In some embodiments, the IL-2 cytokine comprises one or more substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, and M23A.In some embodiments, the IL-2 cytokine comprises L18R, Q22E, and Q126H; and / or R38D, K43E, and E61R; and / or E15S, H16Q, L19V, D20L, M23Q, and R81D; and / or E15S, H16Q, L19V, D20L, Q22K, and M23A. In some embodiments, the IL-2 cytokine comprises L18R, Q22E, Q126H, R38D, K43E, and E61R. In some embodiments, the IL-2 cytokine comprises L18R, Q22E, Q126H, R38D, K43E, E61R, E15S, H16Q, L19V, D20L, M23Q, and R81D. In some embodiments, the IL-2 cytokine comprises L18R, Q22E, Q126H, R38D, K43E, E61R, E15S, H16Q, L19V, D20L, Q22K, and M23A. In some embodiments, the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of F42A, F42K, R38D, R38A, E61R, R38D and E61R, K35D, K43E, K43D, E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D. In some embodiments, the IL-2 receptor comprises an IL-2 cytokine comprising one or more amino acid substitutions selected from the group consisting of L18R, Q22E, Q126K, Q126H, Q126M, and Q126R. In some embodiments, the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126K. In some embodiments, the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126M. In some embodiments, the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126R. In some embodiments, the IL-2 cytokine comprises an amino acid sequence comprising 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 about 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 or 12.In some embodiments, the IL-2 cytokine is tethered to an IL-2 receptor extracellular domain (e.g., an IL-2Rβ extracellular domain) by a polypeptide linker. In some embodiments, the polypeptide linker comprises glycine and serine. In some embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21. In some embodiments, the IL-2 receptor extracellular domain comprises an IL-2Rβ extracellular domain. In some embodiments, the IL-2 receptor extracellular domain is an IL-2Rβ extracellular domain. In some embodiments, the IL-2 receptor extracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, provided herein is a receptor comprising an IL-2Rβ polypeptide tethered at its N-terminus to an IL-2 cytokine, such that it can be activated in the absence of any exogenous IL-2 cytokine. The recombinant cytokine receptor described herein is derived from an interleukin-2 cytokine receptor (e.g., IL-2Rβ), wherein the IL-2-tethered receptor is the interleukin-2 receptor β (IL-2Rβ or IL-2RB) chain of the IL-2R complex or a derivative thereof (e.g., an IL-2Rβ polypeptide containing at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations, such as substitutions, deletions, or additions, compared to the naturally occurring IL-2Rβ polypeptide sequence). In some instances, amino acid variations can be introduced into the IL-2Rβ intracellular domain to, for example, optimize, promote, enhance, increase, etc., intracellular IL-2 signaling (e.g., STAT5 phosphorylation).In other instances, amino acid variations can be introduced into the IL-2Rβ extracellular domain to alter binding affinity to, for example, an IL-2 cytokine, e.g., to reduce binding to a naturally occurring IL-2 cytokine while simultaneously increasing binding to a non-naturally occurring IL-2 cytokine (such as any of the non-naturally occurring (e.g., "mutant" or "mutated") IL-2 cytokines described herein). In some instances, amino acid variations can be introduced into both the IL-2Rβ intracellular domain and the IL-2Rβ extracellular domain. Canonical IL-2 signaling leads to STAT5 phosphorylation, thereby activating STAT5 nuclear translocation and initiating target gene transcription. In some cells (e.g., T cells, including Treg cells), IL-2 signaling is required for cell survival and proliferation. Thus, a recombinant cytokine receptor comprising an IL-2Rβ polypeptide tethered to an IL-2 cytokine can activate cell survival and proliferation in the absence of exogenous IL-2 through preferential binding to the tethered IL-2 cytokine.

[0046] In some embodiments, the IL-2-tethered recombinant cytokine receptors described herein are described with reference to the tethered IL-2 cytokine. Thus, for example, a "wild-type IL-2-tethered" receptor comprises a wild-type IL-2 molecule linked to an IL-2Rβ polypeptide. In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine with one or more amino acid substitutions that result in reduced or eliminated binding affinity for the IL-2Rα (alternatively, "IL-2RA") polypeptide chain, but minimal or no disruption of binding to the IL-2Rβ polypeptide chain, e.g., a reduction in binding affinity for IL-2Rα of at least about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less than the binding affinity of WT IL-2 for IL-2Rα. In some embodiments, the IL-2 cytokine contains at least one amino acid substitution that reduces its affinity for IL-2Rα by at least about any of the following: 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or more, compared to the binding affinity of WT IL-2 for IL-2Rα. In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine with one or more amino acid substitutions that result in reduced or eliminated binding affinity for the IL-2Rγ polypeptide chain, but minimal or no disruption of binding to the IL-2Rβ polypeptide chain, e.g., a reduction in binding affinity for IL-2Rγ of at least about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less than the binding affinity of WT IL-2 for IL-2Rγ.In some embodiments, the IL-2 cytokine comprises at least one amino acid substitution that reduces affinity for IL-2Rγ by at least about any of the following: 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or more, compared to the binding affinity for IL-2Rγ of WT IL-2. In some embodiments, the recombinant cytokine receptor exhibits reduced or abolished binding affinity for either or both of the IL-2Rα and / or IL-2Rγ polypeptide chains, with minimal or no disruption of binding to the IL-2Rβ polypeptide chain, e.g., a binding affinity for IL-2Rα that is reduced to at least about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less than the binding affinity of WT IL-2 for IL-2Rα, and / or a binding affinity for IL-2Rγ that is reduced to at least about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less than the binding affinity of WT IL-2 for IL-2Rα. The present invention also includes IL-2 cytokines with one or more amino acid substitutions that exhibit a reduction in the binding affinity of IL-2 for IL-2Rγ of at least about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less.In some embodiments, the IL-2 cytokine has an affinity for IL-2Rα that is at least about any of the following: 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or more, compared to the binding affinity of WT IL-2 for IL-2Rα and / or IL-2Rγ. and / or comprises at least one amino acid substitution that reduces affinity for IL-2Rγ by at least about any of the following: 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or more.

[0047] In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine with one or more amino acid substitutions that result in reduced or abolished binding affinity for naturally occurring IL-2Rβ but enhanced binding affinity for non-naturally occurring IL-2Rβ. Thus, in some embodiments, the recombinant cytokine receptor comprises both an IL-2 cytokine with one or more amino acid substitutions and an IL-2Rβ polypeptide with one or more amino acid substitutions. For example, in some embodiments, the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at positions 133 and / or 134. See, e.g., Zhang et al., Sci Transl Med 13, eabg6986 (2021); and Sockolosky et al., Science 359, 1037-1042 (2018), both of which are incorporated herein by reference in their entireties. Human IL-2 can be mutated at positions i. E15S, H16Q, L19V, D20L, M23Q, and R81D, or ii. E15S, H16Q, L19V, D20L, Q22K, and M23A, and binds to human IL-2Rβ containing mutations H133D and Y134F. In some embodiments, the IL-2 cytokine contains at least one amino acid substitution that reduces the affinity for naturally occurring IL-2Rβ to at least about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less than the binding affinity of WT IL-2 for naturally occurring IL-2Rβ.In some embodiments, the IL-2 cytokine comprises at least one amino acid substitution that reduces the affinity for naturally occurring IL-2Rβ by at least about any of the following: 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or more, compared to the binding affinity of WT IL-2 for naturally occurring IL-2Rβ. In some embodiments, the IL-2Rβ comprises at least one amino acid substitution that reduces the affinity for a naturally occurring IL-2 cytokine by at least about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less than the binding affinity of WT IL-2Rβ for a naturally occurring IL-2 cytokine. In some embodiments, the IL-2Rβ comprises at least one amino acid substitution that reduces the affinity for a naturally occurring IL-2 cytokine by at least about any of the following: 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or more, compared to the binding affinity of WT IL-2Rβ for a naturally occurring IL-2 cytokine.

[0048] In some embodiments, the recombinant cytokine receptor is more effective in maintaining immune cell survival. In some embodiments, the recombinant cytokine receptor allows the transduced Treg cells to proliferate without exogenous IL-2. In some embodiments, the recombinant cytokine receptor increases the relative amount of Treg cells in a composition comprising a population of Treg cells transduced with the recombinant cytokine receptor over time. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are Tregs.

[0049] In some embodiments, recombinant cytokine receptors are more effective at maintaining Treg persistence compared to other strategies for generating exogenous IL-2-independent Tregs (e.g., IL-2-secreting Tregs or Tregs comprising a membrane-bound IL-2-tethered tag protein). In some embodiments, cells transduced with the recombinant cytokine receptors provided herein can maintain the Treg phenotype and / or have increased persistence compared to IL-2-secreting Treg cells or compared to Tregs comprising a membrane-bound IL-2-tethered tag protein.

[0050] In some embodiments, the recombinant cytokine receptors provided herein have significant advantages, including, but not limited to, (1) expression in regulatory T cells (Tregs); (2) increased survival and proliferation of Tregs in the absence of IL-2; (3) increased IL-2 receptor signaling via STAT5 phosphorylation; (4) the ability to suppress effector T cells in the absence of IL-2 to about or nearly the same extent as wild-type Tregs expanded in the presence of IL-2; (5) support of Treg expansion and survival for at least about 14 days and / or at least about 23 days without exogenous IL-2; and (6) support of Treg identity for at least about 14 days and / or at least about 23 days without exogenous IL-2. [Table 1]

[0051] Provided herein is a recombinant cytokine receptor (e.g., a recombinant IL-2 receptor) that comprises an IL-2Rβ polypeptide tethered at its N-terminus to an IL-2 cytokine, and thus can be activated in the absence of any exogenous cytokine (e.g., exogenous IL-2). In some embodiments, the recombinant cytokine receptor is capable of signaling in the absence of exogenous IL-2. In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine, a linker polypeptide, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain. In some embodiments, the recombinant cytokine receptor does not comprise a TCR activation domain or a costimulatory domain (e.g., an activation domain or costimulatory domain of CD3 or CD28 (such as the CD28 signaling domain)). In some embodiments, the IL-2 receptor binds to an IL-2 cytokine linked to the IL-2Rβ polypeptide by a polypeptide linker sequence. Thus, in some embodiments, a recombinant cytokine receptor is provided, comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine, (II) a polypeptide linker, (III) an IL-2 receptor extracellular domain, (IV) an IL-2 receptor transmembrane domain, and (V) an IL-2 receptor intracellular domain. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134.For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F. In some embodiments, a recombinant cytokine receptor is provided, comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain. In some embodiments, a recombinant cytokine receptor is provided, comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine, (II) a polypeptide linker, and (III) an IL-2Rβ polypeptide. In some embodiments, a recombinant cytokine receptor is provided, comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain. In some embodiments, a recombinant cytokine receptor is provided, comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12, (II) a polypeptide linker, and (III) an IL-2Rβ polypeptide. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 11. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence that comprises 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 about 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 11.In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine having an amino acid sequence comprising 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 about 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12. In some embodiments, the IL-2 cytokine is tethered to the IL-2 cytokine receptor extracellular domain by a polypeptide linker. In some embodiments, the polypeptide linker comprises glycine and serine. In some embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21. In some embodiments, the extracellular domain comprises an IL-2Rβ extracellular domain. In some embodiments, the extracellular domain is an IL-2Rβ extracellular domain. In some embodiments, the extracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the transmembrane domain comprises an IL-2Rβ transmembrane domain. In some embodiments, the transmembrane domain is an IL-2Rβ extracellular domain. In some embodiments, the transmembrane domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the intracellular domain comprises an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain is an IL-2Rβ intracellular domain.In some embodiments, the intracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, a recombinant cytokine receptor is provided comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, (III) an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, (IV) an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and (V) an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, a recombinant cytokine receptor is provided that comprises, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, and (III) an IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO: 7, and (III) an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 7.

[0052] In some embodiments, a recombinant IL-2 receptor (i.e., "recombinant cytokine receptor") binds to a wild-type IL-2 cytokine linked to an IL-2Rβ polypeptide by a polypeptide linker sequence. Thus, in some embodiments, a recombinant cytokine receptor (i.e., "recombinant IL-2 receptor") is provided that comprises, from N-terminus to C-terminus, (I) a wild-type IL-2 cytokine; (II) a polypeptide linker; (III) an IL-2Rβ extracellular domain; (IV) a transmembrane domain; and (V) an IL-2Rβ intracellular domain. In some embodiments, the recombinant cytokine receptor does not comprise a T cell receptor (TCR) activation domain or a costimulatory domain (e.g., an activation or costimulatory domain of CD3 or CD28 (such as the CD28 signaling domain)). In some embodiments, a recombinant cytokine receptor is provided that comprises, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 4; (II) a polypeptide linker; (III) an IL-2Rβ extracellular domain; (IV) a transmembrane domain; and (V) an IL-2Rβ intracellular domain. In some embodiments, a recombinant cytokine receptor is provided that comprises, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 4, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain, wherein the recombinant cytokine receptor does not comprise a T cell receptor activation domain or a T cell costimulatory domain, and optionally, the T cell receptor activation domain or the T cell costimulatory domain is a CD28 signaling domain. In some embodiments, a recombinant cytokine receptor is provided that comprises, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 4, (II) a polypeptide linker, and (III) an IL-2Rβ polypeptide.In some embodiments, a recombinant cytokine receptor is provided comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 4, (II) a polypeptide linker, and (III) an IL-2Rβ polypeptide, wherein the recombinant cytokine receptor does not comprise a T cell receptor activation domain or a T cell costimulatory domain, and optionally, the T cell receptor activation domain or the T cell costimulatory domain is a CD28 signaling domain. In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the recombinant cytokine receptor comprises a wild-type IL-2 cytokine having an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the IL-2 cytokine is tethered to the IL-2 cytokine receptor extracellular domain by a polypeptide linker. In some embodiments, the polypeptide linker comprises glycine and serine. In some embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:9 and 16-21. In some embodiments, the extracellular domain comprises an IL-2Rβ extracellular domain. In some embodiments, the extracellular domain is an IL-2Rβ extracellular domain.In some embodiments, the extracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the transmembrane domain comprises an IL-2Rβ transmembrane domain. In some embodiments, the transmembrane domain is an IL-2Rβ extracellular domain. In some embodiments, the transmembrane domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the intracellular domain comprises an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain is an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, a recombinant cytokine receptor is provided comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 4, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, (III) an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, (IV) an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and (V) an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15.In some embodiments, a recombinant cytokine receptor is provided comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO:4, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:9 and 16-21, (III) an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO:13, (IV) an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO:14, and (V) an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO:15, wherein the recombinant cytokine receptor does not comprise a T cell receptor activation domain or a T cell costimulatory domain, and optionally, the T cell receptor activation domain or the T cell costimulatory domain is a CD28 signaling domain. In some embodiments, a recombinant cytokine receptor is provided comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO:4, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:9 and 16-21, and (III) an IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO:7. In some embodiments, a recombinant cytokine receptor is provided comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 4, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, and (III) an IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO: 7, wherein the recombinant cytokine receptor does not comprise a T cell receptor activation domain or a T cell costimulatory domain, and optionally, the T cell receptor activation domain or the T cell costimulatory domain is a CD28 signaling domain.

[0053] In some embodiments, the recombinant IL-2 receptor (i.e., "recombinant cytokine receptor") binds to the IL-2 cytokine and comprises one or more amino acid substitutions. In some embodiments, the one or more substitutions reduce binding or affinity between i) the IL-2Rα cytokine receptor extracellular domain and the IL-2 cytokine, and / or ii) the IL-2Rγ cytokine receptor extracellular domain and the IL-2 cytokine. In some embodiments, the one or more substitutions do not reduce or only minimally reduce binding or affinity between the IL-2Rβ extracellular domain and the IL-2 cytokine. In some embodiments, the IL-2 cytokine comprises at least one or more amino acid substitutions at a position selected from amino acid positions 42, 38, 61, 35, 18, 22, 126, and 43. In some embodiments, the IL-2 cytokine comprises at least one or more amino acid substitutions at a position selected from amino acid positions 18, 22, 126, 38, 43, and 61. In some embodiments, the IL-2 cytokine is linked to the IL-2Rβ polypeptide by a polypeptide linker sequence, and the amino acid substituted IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of R38D, K43E, and E61R. In some embodiments, the amino acid substituted IL-2 cytokine may comprise additional amino acid substitutions. In some embodiments, a recombinant IL-2 receptor binds to an amino acid substituted IL-2 cytokine linked to the IL-2Rβ polypeptide by a polypeptide linker sequence, and the mutant IL-2 cytokine comprises the amino acid substitutions R38D, K43E, and E61R. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134.For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F. Thus, in some embodiments, a recombinant cytokine receptor (i.e., "recombinant IL-2 receptor") is provided that comprises, from N-terminus to C-terminus, (I) a mutant IL-2 cytokine ("3x IL-2") comprising amino acid substitutions R38D, K43E, and E61R, (II) a polypeptide linker, (III) an IL-2 receptor extracellular domain, (IV) a transmembrane domain, and (V) an IL-2 receptor intracellular domain. In some embodiments, a recombinant cytokine receptor is provided, comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 5, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain. In some embodiments, a recombinant cytokine receptor is provided, comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 5, (II) a polypeptide linker, and (III) an IL-2Rβ polypeptide. In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2.In some embodiments, the recombinant cytokine receptor comprises a 3x-IL-2 cytokine having an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the IL-2 cytokine is tethered to the IL-2 cytokine extracellular domain by a polypeptide linker. In some embodiments, the polypeptide linker comprises glycine and serine. In some embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:9 and 16-21. In some embodiments, the extracellular domain comprises an IL-2Rβ extracellular domain. In some embodiments, the extracellular domain is an IL-2Rβ extracellular domain. In some embodiments, the extracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the transmembrane domain comprises an IL-2Rβ transmembrane domain. In some embodiments, the transmembrane domain comprises an IL-2Rβ transmembrane domain. In some embodiments, the transmembrane domain is an IL-2Rβ extracellular domain. In some embodiments, the transmembrane domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the intracellular domain comprises an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain comprises an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain is an IL-2Rβ intracellular domain.In some embodiments, the intracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the 3x IL-2 cytokine exhibits reduced or no binding to IL-2Rα but does not exhibit reduced or abolished binding to IL-2Rβ. In some embodiments, a recombinant cytokine receptor is provided comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 5, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, (III) an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, (IV) an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and (V) an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, a recombinant cytokine receptor is provided, comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 5, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, and (III) an IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO: 7.

[0054] In some embodiments, the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of F42A, F42K, R38D, R38A, E61R, R38D and E61R, K35D, K43E, and K43D. In some embodiments, the IL-2 cytokine is tethered to the IL-2 cytokine receptor extracellular domain by a polypeptide linker. In some embodiments, the polypeptide linker comprises glycine and serine. In some embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21. In some embodiments, the extracellular domain comprises an IL-2Rβ extracellular domain. In some embodiments, the transmembrane domain comprises an IL-2Rβ transmembrane domain. In some embodiments, the intracellular domain comprises an IL-2Rβ intracellular domain. In some embodiments, the cytokine exhibits reduced or no binding to IL-2Rα but does not exhibit reduced or abolished binding to IL-2Rβ. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F.

[0055] In some embodiments, the recombinant IL-2 receptor (i.e., "recombinant cytokine receptor") comprises an IL-2 cytokine comprising one or more amino acid substitutions selected from the group consisting of L18R, Q22E, and Q126H. In some embodiments, the one or more substitutions reduce binding or affinity between i) the IL-2Rα cytokine receptor extracellular domain and the IL-2 cytokine, and / or ii) the IL-2Rγ cytokine receptor extracellular domain and the IL-2 cytokine. In some embodiments, the one or more substitutions do not reduce or only minimally reduce binding or affinity between the IL-2Rβ extracellular domain and the IL-2 cytokine. In some embodiments, the mutant IL-2 cytokine may comprise additional amino acid substitutions. In some embodiments, the recombinant IL-2 receptor binds to a mutant IL-2 cytokine linked to an IL-2Rβ polypeptide by a polypeptide linker sequence, and the mutant IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126H. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F.Thus, in some embodiments, a recombinant cytokine receptor (i.e., a "recombinant IL-2 receptor") is provided that comprises, from N-terminus to C-terminus, (I) a mutant IL-2 cytokine ("REH IL-2") comprising amino acid substitutions L18R, Q22E, and Q126H, (II) a polypeptide linker, (III) an IL-2 receptor extracellular domain, (IV) a transmembrane domain, and (V) an IL-2 receptor intracellular domain. In some embodiments, a recombinant cytokine receptor is provided that comprises, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO:6, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain. In some embodiments, a recombinant cytokine receptor is provided that comprises, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO:6, (II) a polypeptide linker, and (III) an IL-2Rβ polypeptide. In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the recombinant cytokine receptor comprises an REH IL-2 molecule having an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the IL-2 cytokine is tethered to the IL-2 cytokine extracellular domain by a polypeptide linker. In some embodiments, the polypeptide linker comprises glycine and serine. In some embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:9 and 16-21.In some embodiments, the extracellular domain comprises an IL-2Rβ extracellular domain. In some embodiments, the extracellular domain is an IL-2Rβ extracellular domain. In some embodiments, the extracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the transmembrane domain comprises an IL-2Rβ transmembrane domain. In some embodiments, the transmembrane domain comprises an IL-2Rβ transmembrane domain. In some embodiments, the transmembrane domain is an IL-2Rβ extracellular domain. In some embodiments, the transmembrane domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the intracellular domain comprises an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain comprises an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain is an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the REH IL-2 cytokine exhibits reduced or no binding to IL-2Rα and / or IL-2Rγ, but not reduced or abolished binding to IL-2Rβ.In some embodiments, a recombinant cytokine receptor is provided that comprises, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 6, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, (III) an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, (IV) an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and (V) an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, a recombinant cytokine receptor is provided that comprises, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 6, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, and (III) an IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO: 7.

[0056] In some embodiments, the recombinant IL-2 receptor comprises an IL-2 cytokine comprising one or more amino acid substitutions selected from the group consisting of L18R, Q22E, Q126K, Q126H, Q126M, and Q126R. In some embodiments, the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126K. In some embodiments, the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126M. In some embodiments, the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126R. In some embodiments, the IL-2 cytokine is tethered to the IL-2 cytokine receptor extracellular domain by a polypeptide linker. In some embodiments, the polypeptide linker comprises glycine and serine. In some embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21. In some embodiments, the extracellular domain comprises an IL-2Rβ extracellular domain. In some embodiments, the transmembrane domain comprises an IL-2Rβ transmembrane domain. In some embodiments, the intracellular domain comprises an IL-2Rβ intracellular domain. In some embodiments, the IL-2 cytokine exhibits reduced or no binding to IL-2Rα and / or IL-2Rγ, but does not exhibit reduced or abolished binding to IL-2Rβ. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134.For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F.

[0057] In some embodiments, the recombinant IL-2 receptor (i.e., "recombinant cytokine receptor") comprises an IL-2 cytokine comprising one or more amino acid substitutions selected from the group consisting of L18R, Q22E, Q126H, R38D, K43E, and E61R. In some embodiments, the IL-2 cytokine may comprise additional amino acid substitutions. In some embodiments, the recombinant IL-2 receptor binds to a mutant IL-2 cytokine linked to an IL-2Rβ polypeptide by a polypeptide linker sequence, wherein the mutant IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, Q126H, R38D, K43E, and E61R. In some embodiments, the one or more substitutions decrease binding or affinity between i) the IL-2Rα cytokine receptor extracellular domain and the IL-2 cytokine, and / or ii) the IL-2Rγ cytokine receptor extracellular domain and the IL-2 cytokine. In some embodiments, the one or more substitutions do not reduce or only minimally reduce binding or affinity between the IL-2Rβ extracellular domain and the IL-2 cytokine. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F.Thus, in some embodiments, a recombinant cytokine receptor (i.e., a "recombinant IL-2 receptor") is provided that comprises, from N- to C-terminus, (I) a mutant IL-2 cytokine ("3x REH IL-2") comprising amino acid substitutions L18R, Q22E, Q126H, R38D, K43E, and E61R, (II) a polypeptide linker, (III) an IL-2 receptor extracellular domain, (IV) a transmembrane domain, and (V) an IL-2 receptor intracellular domain. In some embodiments, a recombinant cytokine receptor is provided that comprises, from N- to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 12, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain. In some embodiments, a recombinant cytokine receptor is provided that comprises, from N- to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 12, (II) a polypeptide linker, and (III) an IL-2Rβ polypeptide. In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the recombinant cytokine receptor comprises a 3x REH IL-2 cytokine having an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the IL-2 cytokine is tethered to the IL-2 cytokine receptor extracellular domain by a polypeptide linker. In some embodiments, the polypeptide linker comprises glycine and serine.In some embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21. In some embodiments, the extracellular domain comprises an IL-2Rβ extracellular domain. In some embodiments, the extracellular domain is an IL-2Rβ extracellular domain. In some embodiments, the extracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the transmembrane domain comprises an IL-2Rβ transmembrane domain. In some embodiments, the transmembrane domain comprises an IL-2Rβ transmembrane domain. In some embodiments, the transmembrane domain is an IL-2Rβ extracellular domain. In some embodiments, the transmembrane domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the intracellular domain comprises an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain comprises an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain is an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the 3x REH IL-2 cytokine exhibits reduced or no binding to IL-2Rα and / or IL-2Rγ, but not reduced or abolished binding to IL-2Rβ.In some embodiments, a recombinant cytokine receptor is provided that comprises, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 12, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, (III) an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, (IV) an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and (V) an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, a recombinant cytokine receptor is provided that comprises, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 12, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, and (III) an IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO: 7.

[0058] In some embodiments, cells such as T cells (e.g., Treg cells) comprise a recombinant cytokine receptor described herein. Thus, in some embodiments, T cells (e.g., Treg cells) are provided that comprise a recombinant cytokine receptor comprising, from N-terminus to C-terminus, (I) a wild-type IL-2 cytokine, a 3x IL-2 cytokine, an REH IL-2 cytokine, or a 3x REH IL-2 cytokine; (II) a polypeptide linker; (III) an IL-2 receptor extracellular domain; (IV) a transmembrane domain; and (V) an IL-2 receptor intracellular domain. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions at positions 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at positions 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F. In some embodiments, T cells (e.g., Treg cells) are provided that comprise a recombinant cytokine receptor comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain.In some embodiments, T cells (e.g., Treg cells) are provided that comprise a recombinant cytokine receptor comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, (III) an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, (IV) an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and (V) an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, T cells (e.g., Treg cells) are provided that comprise a recombinant cytokine receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 11. In some embodiments, the recombinant cytokine receptor does not comprise a CD28 signaling domain.

[0059] In some embodiments, cells expressing a recombinant cytokine receptor survive and proliferate in the absence of exogenous IL-2. In some embodiments, at least about 60% (e.g., at least about any of 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of cells transduced with the recombinant cytokine receptor remain viable in the absence of exogenous IL-2. In some embodiments, the cells are T cells. In some embodiments, the cells are Treg cells.

[0060] In some embodiments, Tregs expressing recombinant cytokine receptors survive and proliferate in the absence of exogenous IL-2. In some embodiments, at least about 60% (e.g., at least about any of 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of Tregs transduced with recombinant cytokine receptors remain viable in vitro for at least about 2 to about 23 days after transduction. In some embodiments, at least about 60% to 99% of Tregs transduced with recombinant cytokine receptors remain viable in vitro for at least about 14 to about 23 days after transduction. In some embodiments, at least about 60% to 90% of Tregs transduced with recombinant cytokine receptors remain viable in vitro for at least about 14 to about 23 days after transduction. In some embodiments, at least about 60% to 80% of Tregs transduced with recombinant cytokine receptors remain viable in vitro for at least about 14 to about 23 days after transduction. In some embodiments, at least about 60% to 99% of Tregs transduced with recombinant cytokine receptors remain viable in vitro for at least about 14 days after transduction. In some embodiments, at least about 60% to 90% of Tregs transduced with recombinant cytokine receptors remain viable in vitro for at least about 14 days after transduction. In some embodiments, at least about 60% to 80% of Tregs transduced with recombinant cytokine receptors remain viable in vitro for at least about 14 days after transduction. In some embodiments, the survival rate of Tregs transduced with the recombinant cytokine receptor is increased compared to Treg cells that are not transduced with the recombinant cytokine receptor.In some embodiments, the viability of Tregs transduced with a recombinant cytokine receptor is increased compared to Treg cells not transduced with a recombinant cytokine receptor when cultured without the cognate receptor cytokine (i.e., IL-2 cytokine). In some embodiments, the viability of Tregs is increased by at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 3.2-fold, at least about 4-fold, at least about 10-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 45-fold, at least about 50-fold, or more, compared to non-transduced T cells (e.g., Tregs).

[0061] In some embodiments, Tregs expressing recombinant cytokine receptors survive and proliferate in the absence of exogenous IL-2. In some embodiments, at least about 60% (e.g., at least about any of 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of Tregs transduced with recombinant cytokine receptors persist in vivo for at least 3 days or longer. In some embodiments, at least about 60%-99% of Tregs transduced with recombinant cytokine receptors persist in vivo for at least 3 days or longer. In some embodiments, at least about 60%-90% of Tregs transduced with recombinant cytokine receptors persist in vivo for at least about 3 days or more, hi some embodiments, at least about 60%-80% of Tregs transduced with recombinant cytokine receptors persist in vivo for at least about 3 days or more.

[0062] In some embodiments, cells transduced with a recombinant cytokine receptor have increased or sustained proliferation after transduction and in vivo. In some embodiments, cells transduced with a recombinant cytokine receptor have a higher number of cells in in vitro culture compared to the number of cells before transduction. In some embodiments, the cells are T cells. In some embodiments, the cells are Treg cells.

[0063] In some embodiments, Tregs transduced with recombinant cytokine receptors have increased or sustained proliferation after transduction. In some embodiments, Tregs transduced with recombinant cytokine receptors have a higher number of cells compared to the number of cells before transduction. In some embodiments, Tregs transduced with recombinant cytokine receptors have approximately the same number of cells compared to Tregs not expressing the recombinant cytokine receptors expanded long-term with IL-2. In some embodiments, the period is about 5-14 days. In some embodiments, the period is about 9-14 days. In some embodiments, Tregs transduced with recombinant cytokine receptors have a higher rate of proliferation compared to untransduced Treg cells. In some embodiments, the number of cells after transduction is increased by at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 75-fold, or more compared to untransduced cells. In some embodiments, the cells are cultured without cytokines after transduction.

[0064] In some embodiments, cell viability, transduction of intracellular signaling, activation of intracellular signaling, or ability to proliferate is increased upon transduction with a recombinant cytokine receptor.

[0065] In some embodiments, cell viability, transduction of intracellular signaling, activation of intracellular signaling, or ability to proliferate is increased upon transduction with a recombinant cytokine receptor. In some embodiments, the recombinant cytokine receptor expressed in the cell transduces intracellular IL-2 signaling within the cell. In some embodiments, the recombinant cytokine receptor activates intracellular IL-2 signaling in the cell. In some embodiments, the recombinant cytokine receptor enhances intracellular IL-2 signaling in the cell. In some embodiments, the recombinant cytokine receptor increases intracellular IL-2 signaling in the cell compared to a cell without the recombinant cytokine receptor. In some embodiments, the recombinant cytokine receptor provided herein comprises an activating IL-2Rβ polypeptide. In some embodiments, the recombinant cytokine receptor comprises an activating IL-2Rβ polypeptide tethered to a wild-type IL-2 molecule (hereinafter "WT IL-2"). In some embodiments, the recombinant cytokine receptor comprises an activated IL-2Rβ polypeptide tethered to a mutant IL-2 cytokine comprising one or more amino acid substitutions selected from the group consisting of L18R, Q22E, Q126H, R38D, K43E, and E61R. In some embodiments, the recombinant cytokine receptor comprises an activated IL-2Rβ polypeptide tethered to a mutant IL-2 cytokine comprising amino acid substitutions R38D, K43E, and E61R (hereinafter "3x IL-2"). In some embodiments, the recombinant cytokine receptor comprises an activated IL-2Rβ polypeptide tethered to a mutant IL-2 cytokine comprising amino acid substitutions L18R, Q22E, and Q126H (hereinafter "REH IL-2"). In some embodiments, the recombinant cytokine receptor comprises an activated IL-2Rβ polypeptide tethered to a mutant IL-2 cytokine comprising the amino acid substitutions L18R, Q22E, Q126H, R38D, K43E, and E61R (hereinafter "3x REH IL-2").In some embodiments, the mutant IL-2 cytokine may comprise additional amino acid substitutions. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F. In some embodiments, the recombinant cytokine receptor does not include a T cell receptor activation domain or a T cell costimulatory domain, and optionally, the T cell receptor activation domain or the T cell costimulatory domain is a CD28 signaling domain. In some embodiments, the tethered recombinant cytokine receptor provided herein is capable of participating in downstream signal transduction. In some embodiments, the recombinant cytokine receptor includes an IL-2Rβ polypeptide that phosphorylates STAT5 via activation of JAK1 kinase upon recombinant cytokine receptor activation. In some embodiments, the IL-2Rβ polypeptide phosphorylates STAT5 via activation of JAK1 kinase upon recombinant cytokine receptor activation. In some embodiments, the recombinant cytokine receptor phosphorylates Shc upon recombinant cytokine receptor activation, thereby activating the downstream PI3K-AKT pathway. In some embodiments, the recombinant cytokine receptor phosphorylates Shc upon recombinant cytokine receptor activation, thereby activating the downstream Ras / MAPK pathway.In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine. In some embodiments, the IL-2 cytokine is a mutant IL-2 molecule. In some embodiments, mutant IL-2 molecules with one or more amino acid substitutions exhibit reduced or no binding to IL-2Rα and / or polypeptides. In some embodiments, mutant IL-2 cytokines known as "3x IL-2" exhibit reduced or no binding to IL-2Rα. In some embodiments, mutant IL-2 cytokines known as "REH IL-2" and "3x REH IL-2" exhibit reduced or no binding to IL-2Rα and / or IL-2Rγ polypeptides. In some embodiments, the cell is a T cell. In some embodiments, the cell is a Treg cell.

[0066] In some embodiments, the recombinant cytokine receptor phosphorylates STAT5 via activation of JAK1 kinase upon recombinant cytokine receptor activation. In some embodiments, the recombinant cytokine receptor phosphorylates Shc upon recombinant cytokine receptor activation, thereby activating the downstream PI3K-AKT pathway. In some embodiments, the recombinant cytokine receptor phosphorylates Shc upon recombinant cytokine receptor activation, thereby activating the downstream Ras / MAPK pathway. In some embodiments, the recombinant cytokine receptor comprises IL-2Rβ tethered to the IL-2 cytokine. In some embodiments, the recombinant cytokine receptor is IL-2Rβ tethered to the IL-2 cytokine.

[0067] In some embodiments, recombinant cytokine receptor has the ability to stimulate STAT5 phosphorylation in cells (for example, Treg).STAT5 signaling can be measured by any suitable method known in the art, for example, by the phosphorylation of STAT5.For example, STAT5 phosphorylation can be measured by using antibodies specific for the phosphorylated versions of these molecules, in combination with flow cytometry analysis as described herein.

[0068] receptor polypeptides IL-2 is a class I cytokine. Class I cytokine receptors generally have a large extracellular domain (ED) containing multiple all-β Ig-like domains and an Fn3 domain, a single-pass modular transmembrane domain (TD), and a highly dynamic intracellular domain that transduces signaling events (see, e.g., Metcalfe, RD et al. (2020), Front Immunol;11:1424). These domains maintain a β-sandwich structure with two antiparallel β-sheets. The two Fn3 domains form a cytokine-binding homology region at the domain junction. The class I cytokine receptor ED contains a conserved WSXWS (Trp-Ser-X-Trp-Ser, where X is any amino acid) motif that acts to stabilize the receptor and undergoes conformational changes upon cytokine binding and can be extensively glycosylated. Class I cytokine receptor chains, including IL-2Rβ (also called IL-2Rβ), are most often found in heterodimers or heterotrimers. For example, IL-2Rβ is involved in the regulation of IL-2Rγ. c Heterodimers of IL-2Rα and IL-2Rγ c (also called IL-2RA and CD132, respectively), where the IL-2 binding affinity to IL-2Rβ / IL-2RB alone is K d Approximately 100 nm, and in heterodimeric form K d Approximately 1 nM, and K in the heterotrimeric form dApproximately 10 pM (see, e.g., Wang, X et al. (2009), Annu Rev Immunol; 27:29-60, incorporated herein by reference in its entirety).

[0069] The IL-2Rβ polypeptide is primarily expressed in hematopoietic cells, and its involvement in immune cell-mediated immune responses has been well documented. IL-2Rβ interacts with JAK-1 through its C-terminal cytoplasmic domain, encompassing amino acids 240-525. The IL-2Rβ intracellular domain (ID) lacks intrinsic catalytic activity and therefore depends on constitutive interaction with JAK1 at the box 1 and box 3 motifs in the juxtamembrane region to enhance IL-2 signaling (see, e.g., Wang, X et al. (2009), Annu Rev Immunol;27:29-60). In activated T cells, IL-2 signaling through IL-2Rβ leads to the activation of three pathways: (1) STAT5 phosphorylation by JAK1 kinase, (2) PI3K-AKT pathway activation by Shc phosphorylation, and (3) Ras / MAPK pathway activation by Shc phosphorylation (see, e.g., Ye, C et al. (2018), Signal Transduct Target Ther; 3:2, incorporated herein by reference in its entirety). Of these three pathways, JAK1 / STAT5 signaling is predominant. Activation of the IL-2 pathway across all T cell lineages has been shown to induce T cell proliferation and survival. However, IL-2Rβ signaling only participates in the AK1 / STAT5 pathway in CD4+IL-2Rα+ Treg cells (see, e.g., Bensinger, SJ et al. (2010), J Immunol; 172(9):5287-5296, incorporated herein by reference in its entirety), which is required for Treg suppressive activity (see, e.g., Ye, C et al. (2018), Signal Transduct Target Ther; 3:2). While CD4+ effector T cells, and to a lesser extent CD8+ effector T cells, are capable of producing IL-2 to signal their own proliferation and survival after activation, Tregs are not. Thus, Treg survival depends on IL-2 produced by effector T cells.

[0070] The polypeptide chains IL-2Rα (i.e., IL-2RA) and IL-2Rγ (i.e., CD132) engage with the IL-2Rβ chain (i.e., IL-2RB) to generate receptors with varying degrees of affinity for the IL-2 molecule. The IL-2Rα chain (i.e., IL-2RA) is upregulated on activated T cells and constitutively expressed on Tregs. This chain contributes by increasing the binding affinity of IL-2 to the IL-2 receptor and is therefore involved in tolerance regulation and T cell expansion (see, e.g., Goudy, K et al. (2013), Clin Immun; 146:248-261, incorporated herein by reference in its entirety), but is not involved in intracellular signaling following the IL-2 binding event (see, e.g., Bezrodnik, L et al. (2014), Clin Exp. Immun.; 175:227-234, incorporated herein by reference in its entirety). On the other hand, the IL-2Rγ chain (i.e., CD132) can be found in heterodimeric complexes with multiple cytokine receptor-α chains (e.g., IL-4Rα, IL-7Rα, IL-15Rα, and IL-21Rα, and IL-2Rα and / or IL-2Rβ; see, e.g., Brandt, K et al. (2007), Cytokine Growth Factor Rev;18:223-232, incorporated herein by reference in its entirety). The IL-2Rγ chain is important for the formation of high-affinity and intermediate-affinity IL-2 receptors: the high-affinity heterotrimer (i.e., a complex of IL-2Rα, IL-2Rβ, and IL-2Rγ) and the intermediate-affinity heterodimer (i.e., a complex of IL-2Rβ and IL-2Rγ). Therefore, this subunit is important for a functional and sensitive IL-2 receptor (see, e.g., Takeshita, T et al. (1992), Science;257:379-382), which in turn is important for T-cell biological activity (such as T-cell proliferation).

[0071] In some embodiments, the recombinant cytokine receptor is capable of signaling in the absence of exogenous IL-2. In some embodiments, the recombinant cytokine receptor provided herein comprises an activated IL-2Rβ polypeptide with a wild-type extracellular domain, transmembrane domain, and intracellular domain. In some embodiments, the intracellular domain of the recombinant cytokine receptor provided herein is capable of participating in downstream signal transduction. In some embodiments, the IL-2Rβ intracellular domain phosphorylates STAT5 via activation of JAK1 kinase upon IL-2Rβ activation. In some embodiments, the IL-2β intracellular domain phosphorylates Shc upon IL-2Rβ receptor activation, thereby activating the downstream PI3K-AKT pathway. In some embodiments, the IL-2Rβ intracellular domain phosphorylates Shc upon IL-2Rβ activation, thereby activating the downstream Ras / MAPK pathway. In some embodiments, the IL-2Rβ intracellular domain phosphorylates STAT5 via activation of JAK1 kinase upon recombinant cytokine receptor activation. In some embodiments, the IL-2Rβ intracellular domain phosphorylates Shc upon recombinant cytokine receptor activation, thereby activating the downstream PI3K-AKT pathway. In some embodiments, the IL-2Rβ intracellular domain phosphorylates Shc upon recombinant cytokine receptor activation, thereby activating the downstream Ras / MAPK pathway. In some embodiments, the recombinant cytokine receptor comprises IL-2Rβ tethered to wild-type IL-2, mutant 3x IL-2, mutant REH IL-2, or mutant 3x+REH IL-2 by a polypeptide linker. In some embodiments, the recombinant cytokine receptor is wild-type IL-2 / IL-2Rβ (“IL-2”), mutant 3x IL-2 / IL-2Rβ (“3x”), mutant REH IL-2 / IL-2Rβ (“REH”), or mutant 3x+REH IL-2 / IL-2Rβ (“3x REH”).In some embodiments, the recombinant cytokine receptor comprises an IL-2Rβ polypeptide, a polypeptide linker, and an IL-2 cytokine. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F. Thus, in some embodiments, a recombinant cytokine receptor is provided, comprising: (I) an IL-2 cytokine; (II) a polypeptide linker; and (III) IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 11. In some embodiments, the IL-2 cytokine comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12. In some embodiments, a recombinant cytokine receptor is provided, comprising: (I) a wild-type IL-2 cytokine, (II) a polypeptide linker, (III) an extracellular domain, (IV) a transmembrane domain, and (V) an intracellular domain. In some embodiments, the recombinant cytokine receptor does not comprise a TCR activation domain or a costimulatory domain (e.g., an activation domain or costimulatory domain of CD3 or CD28 (such as a CD28 signaling domain)). In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the IL-2 cytokine comprises the amino acid sequence set forth in SEQ ID NO: 4.In some embodiments, a recombinant cytokine receptor is provided, comprising: (I) a 3x IL-2 cytokine, (II) a polypeptide linker, (III) an IL-2 receptor extracellular domain, (IV) a transmembrane domain, and (V) an IL-2 receptor intracellular domain. In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IL-2 cytokine comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, a recombinant cytokine receptor is provided, comprising: (I) an REH IL-2 cytokine, (II) a polypeptide linker, (III) an IL-2 receptor extracellular domain, (IV) a transmembrane domain, and (V) an IL-2 receptor intracellular domain. In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-2 cytokine comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, a recombinant cytokine receptor is provided, comprising: (I) a 3x REH IL-2 cytokine, (II) a polypeptide linker, (III) an IL-2 receptor extracellular domain, (IV) a transmembrane domain, and (V) an IL-2 receptor intracellular domain. In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the IL-2 cytokine comprises the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the recombinant cytokine receptor comprises an activated IL-2Rβ polypeptide tethered to a mutant IL-2 cytokine comprising one or more amino acid substitutions selected from the group consisting of L18R, Q22E, Q126H, R38D, K43E, and E61R. In some embodiments, the mutant IL-2 cytokine may comprise additional amino acid substitutions. In some embodiments, the IL-2Rβ polypeptide further comprises (a) an IL-2 receptor extracellular domain (ED), (b) a transmembrane domain (TD), and (c) an IL-2 receptor intracellular domain (ID). In some embodiments, the ED of the IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 13.In some embodiments, the full-length IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the full-length IL-2β polypeptide comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, an IL-2 cytokine comprising one or more amino acid substitutions selected from the group consisting of L18R, Q22E, Q126H, R38D, K43E, and E61R exhibits reduced or no binding affinity for IL-2Rα and / or IL-2Rγ, but does not exhibit reduced or abolished binding to IL-2Rβ.

[0072] In some embodiments, the recombinant cytokine receptor comprises a wild-type IL-2 cytokine. In some embodiments, the wild-type IL-2 cytokine comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the wild-type IL-2 cytokine comprises an amino acid sequence that is 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 about 100% identical to SEQ ID NO: 4. In some embodiments, the recombinant cytokine receptor comprises, from N-terminus to C-terminus, (I) a wild-type IL-2 cytokine, (II) a polypeptide linker, (III) an extracellular domain, (IV) a transmembrane domain, and (V) an intracellular domain. In some embodiments, the recombinant cytokine receptor comprises, from N-terminus to C-terminus, (I) a wild-type IL-2 cytokine, (II) a polypeptide linker, (III) an IL-2 receptor extracellular domain, (IV) a transmembrane domain, and (V) an IL-2 receptor intracellular domain. In some embodiments, the recombinant cytokine receptor does not comprise a TCR activation domain or costimulatory domain (e.g., an activation domain or costimulatory domain of CD3 or CD28 (such as a CD28 signaling domain)). In some embodiments, the extracellular region comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the extracellular region comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the transmembrane of the IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the TD comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14.In some embodiments, the ID comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the ID comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F.

[0073] In some embodiments, the recombinant cytokine receptor comprises a mutant 3x IL-2 cytokine. In some embodiments, the mutant 3x IL-2 cytokine comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the mutant 3x IL-2 cytokine comprises an amino acid sequence that is 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 about 100% identical to SEQ ID NO: 5. In some embodiments, the recombinant cytokine receptor comprises, from N-terminus to C-terminus, (I) a mutant 3x IL-2 cytokine, (II) a polypeptide linker, (III) an IL-2 receptor extracellular domain, (IV) a transmembrane domain, and (V) an IL-2 receptor intracellular domain. In some embodiments, the extracellular domain comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the ED comprises an amino acid sequence that has 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the TD comprises an amino acid sequence that has 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the intracellular domain comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the ID comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the full-length IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO:7.In some embodiments, the full-length IL-2β polypeptide comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine comprising one or more amino acid substitutions selected from the group consisting of R38D, K43E, and E61R. In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine comprising the amino acid substitutions R38D, K43E, and E61R and one or more additional amino acid substitutions. In some embodiments, an IL-2 cytokine comprising one or more amino acid substitutions selected from the group consisting of R38D, K43E, and E61R exhibits reduced or no binding affinity for IL-2Rα and / or IL-2Rγ, but does not exhibit reduced or abolished binding to IL-2Rβ. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F.

[0074] In some embodiments, the recombinant cytokine receptor comprises a mutant REH IL-2 molecule. In some embodiments, the mutant REH IL-2 molecule comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the mutant REH IL-2 molecule comprises an amino acid sequence that is 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 about 100% identical to SEQ ID NO: 6. In some embodiments, the recombinant cytokine receptor comprises, from N-terminus to C-terminus, (I) a mutant REH IL-2 molecule, (II) a polypeptide linker, (III) an IL-2 receptor extracellular domain, (IV) a transmembrane domain, and (V) an IL-2 receptor intracellular domain. In some embodiments, the full-length IL-2β polypeptide comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine comprising one or more amino acid substitutions selected from the group consisting of L18R, Q22E, and Q126H. In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine comprising the amino acid substitutions L18R, Q22E, and Q126H. In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine comprising the amino acid substitutions L18R, Q22E, and Q126H and one or more additional amino acid substitutions. In some embodiments, an IL-2 cytokine comprising one or more amino acid substitutions selected from the group consisting of L18R, Q22E, and Q126H exhibits reduced or no binding affinity for IL-2Rα and / or IL-2Rγ, but does not exhibit reduced or abolished binding to IL-2Rβ.In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F.

[0075] In some embodiments, the recombinant cytokine receptor comprises a mutant 3x REH IL-2 cytokine. In some embodiments, the mutant 3x REH IL-2 cytokine comprises the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the mutant 3x REH IL-2 cytokine comprises an amino acid sequence that is 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 about 100% identical to SEQ ID NO: 12. In some embodiments, the recombinant cytokine receptor comprises, from N- to C-terminus: (I) a 3x REH IL-2 cytokine, (II) a polypeptide linker, (III) an IL-2 receptor extracellular domain, (IV) a transmembrane domain, and (V) an IL-2 receptor intracellular domain. In some embodiments, the full-length IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the full-length IL-2β polypeptide comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine comprising one or more amino acid substitutions selected from the group consisting of L18R, Q22E, Q126H, R38D, K43E, and E61R. In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine comprising amino acid substitutions L18R, Q22E, Q126H, R38D, K43E, and E61R, and one or more additional amino acid substitutions.In some embodiments, an IL-2 cytokine comprising one or more amino acid substitutions selected from the group consisting of L18R, Q22E, Q126H, R38D, K43E, and E61R exhibits reduced or no binding affinity for IL-2Rα and / or IL-2Rγ, but does not exhibit reduced or abolished binding to IL-2Rβ. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F.

[0076] In some embodiments, the recombinant cytokine receptor comprising the IL-2 cytokine-tethered IL-2Rβ polypeptide is expressed in a cell. In some embodiments, the recombinant cytokine receptor comprising the IL-2 cytokine-tethered IL-2Rβ polypeptide is expressed in an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a Treg cell.

[0077] In some embodiments, the recombinant cytokine receptor comprising the IL-2 cytokine-tethered IL-2Rβ polypeptide is expressed in Tregs. In some embodiments, Treg cells are CD4+, CD25+, and CD127lo. In some embodiments, Tregs expressing the recombinant cytokine receptor comprising the IL-2 cytokine-tethered IL-2Rβ polypeptide also express FOXP3 and HELIOS. In some embodiments, Tregs expressing the recombinant cytokine receptor comprising the IL-2 cytokine-tethered IL-2Rβ polypeptide also express high levels of FOXP3 and HELIOS. In some embodiments, Tregs expressing the recombinant cytokine receptor comprising the IL-2 cytokine-tethered IL-2Rβ polypeptide survive and proliferate in the absence of exogenous IL-2. In some embodiments, at least about 60% (e.g., at least about any of 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of Tregs transduced with a recombinant cytokine receptor comprising an IL-2 cytokine-tethered IL-2Rβ polypeptide as described above remain viable in vitro for 2-23 days after transduction. In some embodiments, at least about 60% (e.g., at least about any of at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the Tregs transduced with a recombinant cytokine receptor comprising an IL-2 cytokine-tethered IL-2Rβ polypeptide as described above persist in vivo for at least 3 days or longer.In some embodiments, at least about 60% (e.g., at least about any of at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the Tregs transduced with a recombinant cytokine receptor comprising an IL-2 cytokine-tethered IL-2Rβ polypeptide as described above persist in vivo for at least 14 days or longer. In some embodiments, at least about 60% (e.g., at least about any of 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of Tregs transduced with a recombinant cytokine receptor comprising an IL-2 cytokine-tethered IL-2Rβ polypeptide persist in vivo for at least 23 days or longer. In some embodiments, the recombinant cytokine receptor comprising an IL-2 cytokine-tethered IL-2Rβ polypeptide expressed in Tregs transduces intracellular IL-2 signaling within the Tregs. In some embodiments, the recombinant cytokine receptor comprising an IL-2 cytokine-tethered IL-2Rβ polypeptide is capable of signaling in the absence of exogenous IL-2. In some embodiments, the recombinant cytokine receptor comprising the IL-2 cytokine-tethered IL-2Rβ polypeptide is capable of signaling in the absence of exogenous interleukin cytokine. In some embodiments, signaling through the recombinant cytokine receptor comprising the IL-2 cytokine-tethered IL-2Rβ polypeptide expressed in Tregs induces phosphorylation of STAT5. In some embodiments, the recombinant cytokine receptor does not activate IL-2 signaling in cells that do not contain the recombinant cytokine receptor.In some embodiments, the recombinant cytokine receptor does not activate IL-2 signaling in Tregs that do not contain the recombinant cytokine receptor.

[0078] In some embodiments, the recombinant cytokine receptor does not activate signaling of a different IL-2 receptor (such as IL-2Rα and / or IL-2Rγ).

[0079] Stable imprinting of the suppressive phenotype has been suggested to be mediated by the Treg-specific demethylated region (TSDR, also known as CNS2) in the FOXP3 gene. This epigenetic switch region is selectively activated by DNA demethylation in Tregs, sustaining FOXP3 protein expression through epigenetic regulation. In some embodiments, the recombinant cytokine receptor allows transduced Treg cells to proliferate without exogenous IL-2. In some embodiments, the relative amount of Treg cells in a composition comprising a population of Treg cells transduced with the recombinant cytokine receptor increases over time. In some embodiments, a population of Treg cells transduced with the recombinant cytokine receptor expanded without IL-2 contains a similar number of viable cells compared to a composition comprising the same population of Treg cells not transduced with the recombinant cytokine receptor expanded with IL-2. In some embodiments, at least 80% of the population of Treg cells transduced with the recombinant cytokine receptor maintains expression of FOXP3 and / or HELIOS for 14 days after transduction. In some embodiments, at least 80% of the cells in a population of recombinant cytokine-transduced Treg cells maintain expression of FOXP3 and / or HELIOS for 23 days post-transduction.

[0080] In some embodiments, the population of Treg cells transduced with the recombinant cytokine receptor expands at least two-fold over the population of the same Treg cells not transduced with the recombinant cytokine receptor. In some embodiments, the population of Treg cells transduced with the recombinant cytokine receptor expands at least two-fold, and the population of Treg cells transduced with the recombinant cytokine receptor maintains expression of at least one Treg marker selected from the group consisting of CD25, FOXP3, and HELIOS.

[0081] In some embodiments, the relative amount of Treg cells in a composition comprising a population of Treg cells transduced with a recombinant cytokine receptor increases over time when cultured without IL-2.

[0082] In some embodiments, Tregs expressing a recombinant cytokine receptor comprising an IL-2 cytokine-tethered IL-2Rβ polypeptide as described above are capable of suppressing effector T cell activity in the absence of exogenous IL-2 to at least the same extent as, or better than, wild-type Tregs cultured with exogenous IL-2. In some embodiments, the suppressed effector T cells are CD4+ effector T cells. In some embodiments, the suppressed effector T cells are CD8+ effector T cells. In some embodiments, the Tregs expressing a recombinant cytokine receptor comprising an IL-2 cytokine-tethered IL-2Rβ polypeptide as described above also express a chimeric antigen receptor (CAR).

[0083] Tethered interleukin-2 cytokine and polypeptide linker As described above, IL-2 is a Class I cytokine and can bind to IL-2Rα, IL-2Rβ, and IL-2Rγ with different binding affinities, generating IL-2 receptor complexes that exhibit varying sensitivity to the presence of IL-2. Thus, in some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine, wherein the IL-2 cytokine is a wild-type IL-2 molecule. In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine that includes at least one or more amino acid substitutions at a position selected from amino acid positions 18, 22, 126, 38, 43, 61, 15, 16, 19, 20, 22, 23, and 81. In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of L18R, Q22E, Q126H, R38D, K43E, E61R, E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in an IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F. In some embodiments, the IL-2 cytokine is mammalian. In some embodiments, the IL-2 cytokine is human.In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine that does not activate IL-2 signaling in cells that do not contain the recombinant cytokine receptor. In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine that does not activate IL-2 signaling of a different IL-2 receptor. In some embodiments, the recombinant cytokine receptor does not comprise a T cell receptor activation domain or a T cell costimulatory domain, and optionally, the T cell receptor activation domain or the T cell costimulatory domain is a CD28 signaling domain. In some embodiments, the recombinant cytokine receptor comprises, from N-terminus to C-terminus, (I) an IL-2 cytokine, (II) a polypeptide linker, (III) an extracellular domain, (IV) a transmembrane domain, and (V) an intracellular domain. In some embodiments, the IL-2 cytokine tethered to the IL-2Rβ polypeptide of the recombinant cytokine receptor by a polypeptide linker comprises any of SEQ ID NOs: 4-6 and 12. In some embodiments, the IL-2 cytokine comprises an amino acid sequence comprising 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 about 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12. In some embodiments, a recombinant cytokine receptor is provided comprising, from N-terminus to C-terminus: (I) an IL-2 cytokine comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain.In some embodiments, the IL-2 tethered IL-2Rβ recombinant cytokine receptor comprises an amino acid sequence that has 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 about 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 11, and the cytokine receptor has 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 about 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12. and a polypeptide linker having an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the ED of the IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the ED of the IL-2β polypeptide comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the TD of the IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the TD of the IL-2β polypeptide comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the ID of the IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 15.In some embodiments, the ID of the IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the full-length IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the full-length IL-2β polypeptide comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:7.

[0084] In some embodiments, the recombinant cytokine receptor comprises a wild-type IL-2 cytokine tethered to an IL-2Rβ polypeptide. In some embodiments, the recombinant cytokine receptor comprises a wild-type IL-2 cytokine tethered to the N-terminus of the extracellular domain of a full-length IL-2Rβ polypeptide. In some embodiments, the recombinant cytokine receptor comprises, from N-terminus to C-terminus, (I) a wild-type IL-2 cytokine, (II) a polypeptide linker, (III) an extracellular domain, (IV) a transmembrane domain, and (V) an intracellular domain. In some embodiments, the recombinant cytokine receptor does not comprise a TCR activation domain or a costimulatory domain (e.g., an activation domain or costimulatory domain of CD3 or CD28 (such as the CD28 signaling domain)). In some embodiments, the wild-type IL-2 comprises the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, wild-type IL-2 comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, a recombinant cytokine receptor is provided comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 4, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain. In some embodiments, a recombinant cytokine receptor is provided comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 4, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain, wherein the recombinant cytokine receptor does not comprise a T cell receptor activation domain or a T cell costimulatory domain, and optionally, the T cell receptor activation domain or the T cell costimulatory domain is a CD28 signaling domain.In some embodiments, the wild-type IL-2 tethered IL-2Rβ recombinant cytokine receptor comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:1, and the cytokine receptor has at least about 80%, at least about 85%, at least about 98%, at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4. and a polypeptide linker having an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the extracellular domain of the IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the ED of the IL-2β polypeptide comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the transmembrane domain of the IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the transmembrane domain of the IL-2β polypeptide comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the intracellular domain of the IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 15.In some embodiments, the ID of the IL-2β polypeptide comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the full-length IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the full-length IL-2β polypeptide comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, a wild-type IL-2 cytokine tethered to an IL-2Rβ polypeptide also binds to an IL-2Rα polypeptide and / or an IL-2Rγ polypeptide to which IL-2 is complexed with the IL-2-tethered IL-2Rβ polypeptide. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F.

[0085] In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine comprising at least one or more amino acid substitutions at a position selected from amino acid positions 18, 22, 126, 38, 43, and 61. In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of R38D, K43E, and E61R. In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine wherein the IL-2 cytokine comprises amino acid substitutions R38D, K43E, and E61R ("3x IL-2"). In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine wherein the IL-2 cytokine comprises amino acid substitutions R38D, K43E, and E61R, as well as one or more additional amino acid substitutions. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F. In some embodiments, the recombinant cytokine receptor comprises a mutant 3x IL-2 cytokine tethered to an IL-2Rβ polypeptide. In some embodiments, the recombinant cytokine receptor comprises a mutant 3x IL-2 cytokine tethered to the N-terminus of the extracellular domain of a full-length IL-2Rβ polypeptide.In some embodiments, the recombinant cytokine receptor comprises, from N-terminal to C-terminal, (I) 3x IL-2 cytokine; (II) a polypeptide linker; (III) an extracellular domain; (IV) a transmembrane domain; and (V) an intracellular domain. In some embodiments, the recombinant cytokine receptor comprises, from N-terminal to C-terminal, (I) 3x IL-2 cytokine; (II) a polypeptide linker; and (III) a full-length IL-2Rβ polypeptide. In some embodiments, the mutant 3x IL-2 comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the mutant 3x IL-2 comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, a recombinant cytokine receptor is provided that comprises, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 5, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain.In some embodiments, the mutant 3x IL-2 tethered IL-2Rβ recombinant cytokine receptor comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:2, and the cytokine receptor is a mutant 3x IL-2 tethered IL-2Rβ recombinant cytokine receptor comprising an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:5. The IL-2Rβ polypeptide comprises an IL-2 cytokine and a polypeptide linker having an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the ED of the IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the ED of the IL-2β polypeptide comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the TD of the IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the TD of the IL-2β polypeptide comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the ID of the IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 15.In some embodiments, the ID of the IL-2β polypeptide comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the full-length IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the full-length IL-2β polypeptide comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the mutant 3x IL-2 cytokine exhibits reduced or no binding to IL-2Rα and / or IL-2Rγ, but does not reduce or abolish binding to IL-2Rβ.

[0086] In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of L18R, Q22E, and Q126H. In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine, wherein the IL-2 cytokine comprises amino acid substitutions L18R, Q22E, and Q126H (“REH IL-2”). In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine, wherein the IL-2 cytokine comprises amino acid substitutions L18R, Q22E, and Q126H and one or more additional amino acid substitutions. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F. In some embodiments, the recombinant cytokine receptor comprises a mutant REH IL-2 cytokine tethered to an IL-2Rβ polypeptide. In some embodiments, the recombinant cytokine receptor comprises a mutant REH IL-2 cytokine tethered to the N-terminus of the extracellular domain of a full-length IL-2Rβ polypeptide chain. In some embodiments, the recombinant cytokine receptor comprises, from N-terminus to C-terminus, (I) the REH IL-2 cytokine, (II) a polypeptide linker, (III) an extracellular domain, (IV) a transmembrane domain, and (V) an intracellular domain.In some embodiments, the recombinant cytokine receptor comprises, from N-terminus to C-terminus, (I) an REH IL-2 cytokine; (II) a polypeptide linker; and (III) a full-length IL-2Rβ polypeptide. In some embodiments, the mutant REH IL-2 comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the mutant REH IL-2 comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, a recombinant cytokine receptor is provided comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 6, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain. In some embodiments, the mutant REH IL-2 tethered IL-2Rβ recombinant cytokine receptor comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, and the cytokine receptor comprises a mutant REH IL-2 tethered IL-2Rβ recombinant cytokine receptor comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:6. The ED comprises an IL-2 cytokine and a polypeptide linker having an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the ED of the IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 13.In some embodiments, the ED of an IL-2β polypeptide comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the TD of an IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the TD of an IL-2β polypeptide comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the ID of an IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the ID of the IL-2β polypeptide comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the full-length IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the full-length IL-2β polypeptide comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the mutant REH IL-2 cytokine exhibits reduced or no binding to IL-2Rα and / or IL-2Rγ, but does not reduce or abolish binding to IL-2Rβ.

[0087] In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of L18R, Q22E, Q126H, R38D, K43E, and E61R. In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine, wherein the IL-2 cytokine comprises amino acid substitutions L18R, Q22E, Q126H, R38D, K43E, and E61R ("3x REH IL-2"). In some embodiments, the recombinant cytokine receptor comprises an IL-2 cytokine, wherein the IL-2 cytokine comprises amino acid substitutions L18R, Q22E, Q126H, R38D, K43E, and E61R, and one or more additional amino acid substitutions. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F. In some embodiments, the recombinant cytokine receptor comprises a mutant 3x REH IL-2 cytokine tethered to an IL-2Rβ polypeptide. In some embodiments, the recombinant cytokine receptor comprises a mutant 3x REH IL-2 cytokine tethered to the N-terminus of the extracellular domain of a full-length IL-2Rβ polypeptide chain.In some embodiments, the recombinant cytokine receptor comprises, from N-terminus to C-terminus, (I) a 3x REH IL-2 cytokine, (II) a polypeptide linker, (III) an extracellular domain, (IV) a transmembrane domain, and (V) an intracellular domain. In some embodiments, the recombinant cytokine receptor comprises, from N-terminus to C-terminus, (I) a 3x REH IL-2 cytokine; (II) a polypeptide linker; and (III) a full-length IL-2Rβ polypeptide. In some embodiments, the mutant 3x REH IL-2 comprises the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the mutant 3x REH IL-2 comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, a recombinant cytokine receptor is provided, comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 12, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain.In some embodiments, the mutant 3x REH IL-2 tethered IL-2Rβ recombinant cytokine receptor comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:11, wherein the cytokine receptor is a mutant 3x REH IL-2 tethered IL-2Rβ recombinant cytokine receptor comprising an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:11. The IL-2Rβ polypeptide comprises an IL-2 cytokine and a polypeptide linker having an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the ED of the IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the ED of the IL-2β polypeptide comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the TD of the IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the TD of the IL-2β polypeptide comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the ID of the IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 15.In some embodiments, the ID of the IL-2β polypeptide comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the full-length IL-2Rβ polypeptide comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the full-length IL-2β polypeptide comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the mutant 3x REH IL-2 cytokine exhibits reduced binding affinity to IL-2Rα and / or IL-2Rγ. In some embodiments, the mutant 3x REH IL-2 cytokine exhibits no binding to IL-2Rα and / or IL-2Rγ, but does not reduce or abolish binding to IL-2Rβ.

[0088] The polypeptide linker can be any length. In some embodiments, the polypeptide linker can be about 1 to about 10 amino acids in length, about 2 to about 15 amino acids in length, about 3 to about 12 amino acids in length, about 4 to about 10 amino acids in length, about 5 to about 9 amino acids in length, about 6 to about 8 amino acids in length, about 1 to about 20 amino acids in length, about 21 to about 30 amino acids in length, about 1 to about 30 amino acids in length, about 2 to about 20 amino acids in length, about 10 to about 30 amino acids in length, about 2 to about 19 amino acids in length, or about 2 to about 18 amino acids in length. The peptide linker may be about 2 to about 17 amino acids in length, about 2 to about 16 amino acids in length, about 2 to about 10 amino acids in length, about 2 to about 14 amino acids in length, about 2 to about 13 amino acids in length, about 2 to about 12 amino acids in length, about 2 to about 11 amino acids in length, about 2 to about 9 amino acids in length, about 2 to about 8 amino acids in length, about 2 to about 7 amino acids in length, about 2 to about 6 amino acids in length, about 2 to about 5 amino acids in length, about 2 to about 4 amino acids in length, or about 2 to about 3 amino acids in length. In some embodiments, the peptide linker is any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the polypeptide linker is any of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. For example, in some embodiments, the polypeptide linker is about 5 amino acids in length. In some embodiments, the N-terminus of the polypeptide linker is covalently linked to the C-terminus of the IL-2 cytokine and the C-terminus of the polypeptide linker is covalently linked to the N-terminus of the IL-2Rβ polypeptide.

[0089] In some embodiments, the IL-2 receptor extracellular domain is tethered to the IL-2 cytokine by a polypeptide linker. The polypeptide linker may have a naturally occurring or non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody may be used as a linker. See, e.g., WO 1996 / 34103. In some embodiments, the linker is a flexible linker. In some embodiments, an exemplary flexible linker is (GGGGS) n (SEQ ID NO: 20), wherein n is at least one integer, for example, (GGGGS) when n is 1 to 5. n (SEQ ID NO: 20). Glycine and glycine-serine polymers are relatively unstructured and may therefore be able to act as neutral tethers between components. Glycine has access to significantly more φψ space than alanine and is much less restrictive than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Thus, exemplary flexible linkers include, but are not limited to, Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 19). In some embodiments, the linker comprises the amino acid sequence GGGGS (SEQ ID NO: 16). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGGSGGGGGS (SEQ ID NO: 17). In some embodiments, the linker comprises the amino acid sequence SGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 18). Those skilled in the art will recognize that the design of a recombinant cytokine receptor tethered to human IL-2 cytokine may include a fully or partially flexible linker, such that the linker includes a flexible linker portion and one or more portions that impart less flexible structure to provide the desired recombinant cytokine receptor structure.

[0090] In some embodiments, the linker between the IL-2 cytokine and the IL-2Rβ polypeptide is a stable, non-cleavable linker. In some embodiments, the linker between the IL-2 cytokine and the IL-2Rβ polypeptide is not cleavable by proteases. In some embodiments, the linker between the IL-2 cytokine and the IL-2Rβ polypeptide is a flexible linker.

[0091] II.Cells The present disclosure provides adoptive cellular immunotherapy compositions comprising the genetically modified cell preparations described herein (e.g., genetically modified immune cells, e.g., lymphocytes). These cells can be, for example, multipotent cells (e.g., hematopoietic stem cells), various progenitor or progenitor cells of the hematopoietic lineage, and various immune cells (e.g., human autologous cells, allogeneic T cells, natural killer (NK) cells, dendritic cells, or B cells). These cells can also be pluripotent stem cells (PSCs) (e.g., human embryonic stem cells and induced PSCs) and can be used to generate therapeutic cell populations. In some embodiments, pluripotent and multipotent cells are differentiated in vitro into the desired cell type before being implanted into a patient.

[0092] In some alternatives, the cell preparation is a T lymphocyte cell preparation. In some embodiments, the T lymphocyte cell preparation comprises CD4+ T cells bearing a chimeric receptor comprising an extracellular antibody variable domain specific for a ligand associated with a disease or disorder, a spacer region, a transmembrane domain, and an intracellular signaling domain of a T cell receptor, and a recombinant cytokine receptor described herein. In another alternative, the adoptive cellular immunotherapy composition further comprises a chimeric receptor-modified CD8+ cytotoxic T lymphocyte cell preparation that provides a cellular immune response, the cytotoxic T lymphocyte cell preparation comprising CD8+ T cells bearing a chimeric receptor comprising an extracellular single-chain antibody specific for a ligand associated with a disease or disorder, a spacer region, a transmembrane domain, and an intracellular signaling domain of a T cell receptor, and a recombinant cytokine receptor described herein. In some alternatives, the chimeric receptor-modified T cell population of the present disclosure persists in vivo for at least about 3 days or longer. In some alternatives, each of these populations may be combined with each other or other cell types to provide the composition, hi some alternatives, the host cells are Treg cells.

[0093] Regulatory T (Treg) cells are involved in maintaining immunological self-tolerance and attenuating harmful immune responses to both self and non-self (alio) antigens. Tregs include naturally occurring and inducible subtypes. Naturally occurring Tregs (nTregs) are cells that arise as a separate cell lineage during development. Peripheral or inducible Tregs (iTregs) differentiate from conventional T cells. In some embodiments, CD4+ T cells that are not nTregs and are not iTregs are engineered into Tregs using the methods and compositions of the present disclosure. In some embodiments, CD4+ T cells are used to generate Treg cells that express a recombinant cytokine receptor using the methods and compositions of the present disclosure.

[0094] In some embodiments, the cells expressing the recombinant cytokine receptors of the present disclosure are non-naturally occurring. In some embodiments, the cells are immune cells. In some embodiments, the cells are lymphocytes. In some embodiments, the cells are T cells. In some embodiments, the cells are regulatory T cells (Tregs). In some embodiments, the Tregs are compared to control Tregs that are not transduced with the recombinant cytokine receptor. In other embodiments, the Tregs are compared to control Tregs that are transduced with an empty vector lacking the recombinant cytokine receptor.

[0095] Thus, provided herein are regulatory T cells (Tregs) comprising a recombinant cytokine receptor comprising an IL-2Rβ polypeptide tethered at its N-terminus to an IL-2 cytokine, the recombinant cytokine receptor being activated in the absence of exogenous IL-2. In some embodiments, the recombinant cytokine receptor is capable of signaling within the Treg in the absence of exogenous IL-2. In some embodiments, the Tregs comprise a recombinant cytokine receptor comprising an IL-2 cytokine, a linker polypeptide, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain. In some embodiments, the IL-2 receptor binds to the IL-2 cytokine linked to the IL-2Rβ polypeptide by a polypeptide linker sequence. In some embodiments, the recombinant cytokine receptor does not comprise a T cell receptor activation domain or a T cell costimulatory domain, and optionally, the T cell receptor activation domain or the T cell costimulatory domain is a CD28 signaling domain. Thus, in some embodiments, Tregs are provided that comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) an IL-2 cytokine, (II) a polypeptide linker, (III) an IL-2 receptor extracellular domain, (IV) an IL-2 receptor transmembrane domain, and (V) an IL-2 receptor intracellular domain. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) an IL-2 cytokine, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) an IL-2 cytokine, (II) a polypeptide linker, and (III) an IL-2Rβ polypeptide.In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) an IL-2 cytokine comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) an IL-2 cytokine comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12, (II) a polypeptide linker, and (III) an IL-2Rβ polypeptide. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 11. In some embodiments, the Treg comprises a recombinant cytokine receptor comprising an amino acid sequence that comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 11.In some embodiments, Tregs comprise a recombinant cytokine receptor comprising an IL-2 cytokine having an amino acid sequence comprising 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 about 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12. In some embodiments, the IL-2 cytokine is tethered to the IL-2 cytokine receptor extracellular domain by a polypeptide linker. In some embodiments, the polypeptide linker comprises glycine and serine. In some embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21. In some embodiments, the extracellular domain comprises an IL-2Rβ extracellular domain. In some embodiments, the extracellular domain is an IL-2Rβ extracellular domain. In some embodiments, the extracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the transmembrane domain comprises an IL-2Rβ transmembrane domain. In some embodiments, the transmembrane domain is an IL-2Rβ extracellular domain. In some embodiments, the transmembrane domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the intracellular domain comprises an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain is an IL-2Rβ intracellular domain.In some embodiments, the intracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the Treg comprises a recombinant cytokine receptor comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, (III) an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, (IV) an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and (V) an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the Treg comprises, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, and (III) an IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO: 7.

[0096] In some embodiments, Tregs are provided that comprise a recombinant cytokine receptor comprising a wild-type IL-2 cytokine linked to an IL-2Rβ polypeptide by a polypeptide linker sequence. Thus, in some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N-terminus to C-terminus, (I) a wild-type IL-2 cytokine; (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N-terminus to C-terminus, (I) a wild-type IL-2 cytokine; (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain, wherein the recombinant cytokine receptor does not comprise a T cell receptor activation domain or a T cell costimulatory domain, and optionally, the T cell receptor activation domain or the T cell costimulatory domain is a CD28 signaling domain.

[0097] In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 4, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 4, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain, wherein the recombinant cytokine receptor does not comprise a T cell receptor activation domain or a T cell costimulatory domain, and optionally, the T cell receptor activation domain or the T cell costimulatory domain is a CD28 signaling domain. In some embodiments, the Treg comprises, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 4, (II) a polypeptide linker, and (III) a recombinant cytokine receptor comprising an IL-2Rβ polypeptide.In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 4, (II) a polypeptide linker, and (III) an IL-2Rβ polypeptide, wherein the recombinant cytokine receptor does not comprise a T cell receptor activation domain or a T cell costimulatory domain, and optionally, the T cell receptor activation domain or the T cell costimulatory domain is a CD28 signaling domain. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the Treg comprises a recombinant cytokine receptor comprising a wild-type IL-2 cytokine having an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL-2 cytokine is tethered to the IL-2 cytokine receptor extracellular domain by a polypeptide linker. In some embodiments, the polypeptide linker comprises glycine and serine. In some embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21. In some embodiments, the extracellular domain comprises an IL-2Rβ extracellular domain. In some embodiments, the extracellular domain is an IL-2Rβ extracellular domain.In some embodiments, the extracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the transmembrane domain comprises an IL-2Rβ transmembrane domain. In some embodiments, the transmembrane domain is an IL-2Rβ extracellular domain. In some embodiments, the transmembrane domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the intracellular domain comprises an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain is an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 4, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, (III) an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, (IV) an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and (V) an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15.In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO:4, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:9 and 16-21, (III) an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO:13, (IV) an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO:14, and (V) an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO:15, wherein the recombinant cytokine receptor does not comprise a T cell receptor activation domain or a T cell costimulatory domain, and optionally, the T cell receptor activation domain or the T cell costimulatory domain is a CD28 signaling domain. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO:4, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:9 and 16-21, and (III) an IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO:7. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N-terminus to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 4, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, and (III) an IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO: 7, wherein the recombinant cytokine receptor does not comprise a T cell receptor activation domain or a T cell costimulatory domain, and optionally, the T cell receptor activation domain or the T cell costimulatory domain is a CD28 signaling domain. In some embodiments, Tregs are provided that comprise a recombinant cytokine receptor comprising an IL-2 cytokine comprising one or more amino acid substitutions. In some embodiments, the one or more substitutions decrease binding or affinity between i) the IL-2Rα cytokine receptor extracellular domain and the IL-2 cytokine and / or ii) the IL-2Rγ cytokine receptor extracellular domain and the IL-2 cytokine.In some embodiments, the one or more substitutions do not reduce or only minimally reduce binding or affinity between the IL-2Rβ extracellular domain and the IL-2 cytokine. In some embodiments, the IL-2 cytokine comprises at least one or more amino acid substitutions at a position selected from amino acid positions 42, 38, 61, 35, 18, 22, 126, and 43. In some embodiments, the IL-2 cytokine comprises at least one or more amino acid substitutions at a position selected from amino acid positions 18, 22, 126, 38, 43, and 61. In some embodiments, the IL-2 cytokine is linked to the IL-2Rβ polypeptide by a polypeptide linker, and the amino acid-substituted IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of R38D, K43E, and E61R. In some embodiments, the amino acid-substituted IL-2 cytokine may comprise additional amino acid substitutions. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F. In some embodiments, the Treg comprises a recombinant cytokine receptor comprising an amino acid substituted IL-2 cytokine linked to an IL-2Rβ polypeptide by a polypeptide linker sequence, wherein the mutant IL-2 cytokine comprises amino acid substitutions R38D, K43E, and E61R.Thus, in some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) a mutant IL-2 cytokine ("3x IL-2") comprising amino acid substitutions R38D, K43E, and E61R, (II) a polypeptide linker, (III) an IL-2 receptor extracellular domain, (IV) a transmembrane domain, and (V) an IL-2 receptor intracellular domain. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 5, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 5, (II) a polypeptide linker, and (III) an IL-2Rβ polypeptide. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the Treg is shown in SEQ ID NO:2. The Treg comprises a recombinant cytokine receptor comprising an amino acid sequence that has 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the Treg comprises a recombinant cytokine receptor comprising 3x-IL-2 cytokine having an amino acid sequence that has 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL-2 cytokine is tethered to the IL-2 cytokine extracellular domain by a polypeptide linker. In some embodiments, the polypeptide linker comprises glycine and serine. In some embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21. In some embodiments, the extracellular domain comprises an IL-2Rβ extracellular domain. In some embodiments, the extracellular domain is an IL-2Rβ extracellular domain. In some embodiments, the extracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the transmembrane domain comprises an IL-2Rβ transmembrane domain. In some embodiments, the transmembrane domain comprises an IL-2Rβ transmembrane domain. In some embodiments, the transmembrane domain is an IL-2Rβ extracellular domain. In some embodiments, the transmembrane domain comprises an amino acid sequence that comprises 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:14.In some embodiments, the intracellular domain comprises an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain comprises an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain is an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the 3x IL-2 cytokine exhibits reduced or no binding to IL-2Rα and / or IL-2Rγ, but does not exhibit reduced or abolished binding to IL-2Rβ. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 5, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, (III) an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, (IV) an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and (V) an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 5, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, and (III) an IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO: 7.

[0098] In some embodiments, Tregs are provided that comprise a recombinant cytokine receptor comprising an IL-2 cytokine comprising one or more amino acid substitutions selected from the group consisting of L18R, Q22E, and Q126H. In some embodiments, the one or more substitutions reduce binding or affinity between i) the IL-2Rα cytokine receptor extracellular domain and the IL-2 cytokine, and / or ii) the IL-2Rγ cytokine receptor extracellular domain and the IL-2 cytokine. In some embodiments, the one or more substitutions do not reduce or only minimally reduce binding or affinity between the IL-2Rβ extracellular domain and the IL-2 cytokine. In some embodiments, the mutant IL-2 cytokine may comprise additional amino acid substitutions. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F. In some embodiments, the Treg comprises a recombinant cytokine receptor comprising a mutant IL-2 cytokine linked to an IL-2Rβ polypeptide by a polypeptide linker sequence, wherein the mutant IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126H.Thus, in some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) a mutant IL-2 cytokine ("REH IL-2") comprising amino acid substitutions L18R, Q22E, and Q126H, (II) a polypeptide linker, (III) an IL-2 receptor extracellular domain, (IV) a transmembrane domain, and (V) an IL-2 receptor intracellular domain. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 6, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 6, (II) a polypeptide linker, and (III) an IL-2Rβ polypeptide. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising an amino acid sequence that has 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising an REH IL-2 molecule having an amino acid sequence that has 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the IL-2 cytokine is tethered to the IL-2 cytokine extracellular domain by a polypeptide linker. In some embodiments, the polypeptide linker comprises glycine and serine.In some embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21. In some embodiments, the extracellular domain comprises an IL-2Rβ extracellular domain. In some embodiments, the extracellular domain is an IL-2Rβ extracellular domain. In some embodiments, the extracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the transmembrane domain comprises an IL-2Rβ transmembrane domain. In some embodiments, the transmembrane domain comprises an IL-2Rβ transmembrane domain. In some embodiments, the transmembrane domain is an IL-2Rβ extracellular domain. In some embodiments, the transmembrane domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the intracellular domain comprises an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain comprises an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain is an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the REH IL-2 cytokine exhibits reduced or no binding to IL-2Rα and / or IL-2Rγ, but not reduced or abolished binding to IL-2Rβ.In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 6, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, (III) an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, (IV) an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and (V) an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 6, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, and (III) an IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO: 7.

[0099] In some embodiments, Tregs are provided that comprise a recombinant cytokine receptor comprising an IL-2 cytokine that comprises one or more amino acid substitutions selected from the group consisting of L18R, Q22E, Q126H, R38D, K43E, and E61R. In some embodiments, the IL-2 cytokine may comprise additional amino acid substitutions. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions at positions 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at positions 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in an IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F. In some embodiments, the Treg comprises a recombinant cytokine receptor comprising a mutant IL-2 cytokine linked to the IL-2Rβ polypeptide by a polypeptide linker sequence, where the mutant IL-2 cytokine comprises amino acid substitutions L18R, Q22E, Q126H, R38D, K43E, and E61R. In some embodiments, the one or more substitutions decrease binding or affinity between i) the IL-2Rα cytokine receptor extracellular domain and the IL-2 cytokine, and / or ii) the IL-2Rγ cytokine receptor extracellular domain and the IL-2 cytokine. In some embodiments, the one or more substitutions do not reduce or only minimally reduce the binding or affinity between the IL-2Rβ extracellular domain and the IL-2 cytokine.Thus, in some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) a mutant IL-2 cytokine ("3x REH IL-2") comprising amino acid substitutions L18R, Q22E, Q126H, R38D, K43E, and E61R, (II) a polypeptide linker, (III) an IL-2 receptor extracellular domain, (IV) a transmembrane domain, and (V) an IL-2 receptor intracellular domain. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 12, (II) a polypeptide linker, (III) an IL-2Rβ extracellular domain, (IV) a transmembrane domain, and (V) an IL-2Rβ intracellular domain. In some embodiments, a recombinant cytokine receptor is provided comprising, from N- to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 12, (II) a polypeptide linker, and (III) an IL-2Rβ polypeptide. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising 3x REH IL-2 cytokine having an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the IL-2 cytokine is tethered to the IL-2 cytokine receptor extracellular domain by a polypeptide linker. In some embodiments, the polypeptide linker comprises glycine and serine.In some embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21. In some embodiments, the extracellular domain comprises an IL-2Rβ extracellular domain. In some embodiments, the extracellular domain is an IL-2Rβ extracellular domain. In some embodiments, the extracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the transmembrane domain comprises an IL-2Rβ transmembrane domain. In some embodiments, the transmembrane domain comprises an IL-2Rβ transmembrane domain. In some embodiments, the transmembrane domain is an IL-2Rβ extracellular domain. In some embodiments, the transmembrane domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the intracellular domain comprises an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain comprises an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain is an IL-2Rβ intracellular domain. In some embodiments, the intracellular domain comprises an amino acid sequence comprising 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 about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the 3x REH IL-2 cytokine exhibits reduced or no binding to IL-2Rα and / or IL-2Rγ, but not reduced or abolished binding to IL-2Rβ.In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 12, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, (III) an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, (IV) an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and (V) an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, Tregs comprise a recombinant cytokine receptor comprising, from N- to C-terminus, (I) an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 12, (II) a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21, and (III) an IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO: 7.

[0100] In some embodiments, Tregs expressing a recombinant cytokine receptor of the present disclosure are not naturally occurring (i.e., are not nTregs and / or are not iTregs). In some embodiments, the cells express one or more markers characteristic of Tregs. Treg markers include high levels of IL-2Rα (IL-2Rα+), low levels of CD127 (CD127lo), or both high IL-2Rα and low CD127. The levels of IL-2Rα and CD127 are compared, for example, to CD4+ T cells that are not Tregs. In some embodiments, Treg cells expressing a recombinant cytokine receptor have a high IL-2Rα, high CD4, and low CD127 phenotype.

[0101] Also provided herein are cells expressing a recombinant cytokine receptor. In some embodiments, the recombinant cytokine receptor comprises an IL-2 receptor beta chain polypeptide, a linker polypeptide, and a human IL-2 cytokine. In some embodiments, the recombinant cytokine receptor comprises a human IL-2 cytokine selected from the group consisting of WT IL-2, 3x IL-2, REH IL-2, and 3x REH IL-2. In some embodiments, the recombinant cytokine receptor does not comprise a T cell receptor activation domain or a T cell costimulatory domain, and optionally, the T cell receptor activation domain or the T cell costimulatory domain is a CD28 signaling domain.

[0102] In some embodiments, the cells express one or more proteins associated with the Treg phenotype. In some embodiments, the cells express FOXP3. FOXP3 plays an important role in the development and function of Treg cells (Yagi et al., "Crucial role of FOXP3 in the development and function of human IL-2Rα+CD4+ regulatory T cells," Int Immunol. 2004 Nov;l6(l1):1643-56. Epub 2004 Oct 4; Sadlon et al., "Unravelling the molecular basis for regulatory T-cell plasticity and loss of function in disease," Clinical & Translational Immunology, 2018). FOXP3 is initially expressed during T cell expansion, followed by a loss of FOXP3 expression after polyclonal stimulation. This is in contrast to Tregs, in which FOXP3 expression is elevated and maintained over time. The expression level of FOXP3 can be assessed by conventional methods (such as Western blot, flow cytometry, or ELISA). Expression levels can also be assessed by analysis of mRNA-based techniques (such as RT-qPCR). In some embodiments, FOXP3 expression is increased compared to untransduced cells. In some embodiments, FOXP3 expression is increased compared to cells without the recombinant cytokine receptor.

[0103] In some embodiments, FOXP3 expression is increased by at least 2-fold, at least 3-fold, at least about 4-fold, or at least 5-fold compared to cells without the recombinant cytokine receptor. In some embodiments, FOXP3 expression is increased by at least 1.1-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold compared to cells not transduced with the recombinant cytokine receptor. In some embodiments, FOXP3 expression is increased in Tregs transduced with the recombinant cytokine receptor compared to untransduced T cells cultured without one or more cytokines. In some embodiments, FOXP3 expression is maintained at approximately the same level as the day of highest FOXP3 expression after transduction with the recombinant cytokine receptor.

[0104] In some embodiments, the Treg cells are CD4 positive (CD4+). In some embodiments, the cells are CD4+ / IL-2Rα+. In some embodiments, the cells are CD4+ / CD127low. In some embodiments, the cells are CD4+ / IL-2Rα+ / CD127low.

[0105] Tregs can be characterized by expression of IL-2Rα+. In some embodiments, the cells are IL-2Rα+. In some embodiments, the cells are CD4+ / IL-2Rα+. In some embodiments, the cells are IL-2Rα+ / CD127low. In some embodiments, the cells are CD4+ / IL-2Rα+ / CD127low.

[0106] In some embodiments, the cells express low levels of CD127 (CD127lo). In some embodiments, the cells are CD4+ / CD127low. In some embodiments, the cells are IL-2Rα+ / CD127low. In some embodiments, the cells are CD4+ / IL-2Rα+ / CD127low. In some embodiments, cells transduced with a recombinant cytokine receptor provided herein remain CD127low upon transduction.

[0107] HELIOS is a transcription factor expressed in Tregs. In some embodiments, Treg cells of the present disclosure express HELIOS. In some embodiments, HELIOS expression is increased compared to non-Treg T cells. In some embodiments, HELIOS expression is detectable compared to non-Treg T cells. HELIOS expression levels can be assessed by conventional methods (such as Western blot, flow cytometry, or ELISA). Expression levels can also be assessed by analysis of mRNA-based techniques (such as RT-PCR). In some embodiments, HELIOS expression is increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 20-fold compared to non-Treg T cells. In some embodiments, HELIOS expression is increased in Tregs transduced with a recombinant cytokine receptor compared to cells cultured without cytokine. In some embodiments, HELIOS expression is increased in Tregs transduced with a recombinant cytokine receptor compared to non-transduced Tregs, hi some embodiments, Tregs transduced with a recombinant cytokine receptor maintain HELIOS expression after transduction.

[0108] In some embodiments, the relative amount of Treg cells in a composition comprising a population of recombinant cytokine receptor-transduced Treg cells increases over time. In some embodiments, a population of recombinant cytokine receptor-transduced Treg cells expanded without IL-2 contains a similar number of viable cells compared to a composition comprising the same population of Treg cells not transduced with recombinant cytokine receptor expanded with IL-2. In some embodiments, at least 80% of the population of recombinant cytokine-transduced Treg cells maintain expression of FOXP3 and / or HELIOS for 14 days post-transduction. In some embodiments, at least 80% of the cells in the population of recombinant cytokine-transduced Treg cells maintain expression of FOXP3 and / or HELIOS for 23 days post-transduction.

[0109] In some embodiments, the population of Treg cells transduced with the recombinant cytokine receptor expands at least two-fold over the population of the same Treg cells not transduced with the recombinant cytokine receptor. In some embodiments, the population of Treg cells transduced with the recombinant cytokine receptor expands at least two-fold, and the population of Treg cells transduced with the recombinant cytokine receptor maintains expression of at least one Treg marker selected from the group consisting of CD25, FOXP3, and HELIOS.

[0110] In some embodiments, the relative amount of Treg cells in a composition comprising a population of Treg cells transduced with a recombinant cytokine receptor increases over time when cultured without IL-2.

[0111] In some embodiments, the cells have an immunosuppressive phenotype. In one embodiment, the cells produce an immunosuppressive effect in an individual with an immune-related disorder. In some embodiments, the individual is human. In some embodiments, the cells are autologous to the individual.

[0112] In some embodiments, the cells suppress, block, or inhibit graft-versus-host disease (GvHD) in the individual. In one embodiment, the cells suppress, block, or inhibit an immune-related disorder in the individual. In some embodiments, the cells are administered prior to the onset of the immune-related disorder. In some embodiments, the individual is human. In some embodiments, the cells are autologous to the individual. In some embodiments, the cells are allogeneic to the individual.

[0113] Methods for measuring markers used to characterize Treg cells will be readily apparent to those skilled in the art. For example, Tregs, or a population of T cells containing Tregs, can be cultured using the methods described herein. Following culture, Tregs can be collected and stained using antibodies against Treg markers such as FOXP3 (PE), IL-2Rα (APC), and CD127 (BV421), and expression can be analyzed using fluorescence-activated flow cytometry (FACS) or fluorescence microscopy. Gene expression can be measured by methods such as RT-qPCR.

[0114] III. Method of Preparation The recombinant cytokine receptors described herein can be prepared and transduced into host cells (e.g., Treg cells). A sequence encoding the recombinant cytokine receptor can be synthesized. After obtaining such a sequence, it is cloned into a suitable expression vector and then transduced into a suitable host cell. The transduced host cell is recovered and cultured to obtain a viable host cell that stably expresses the recombinant cytokine receptor of the invention.

[0115] In some embodiments, the present application provides an isolated nucleic acid encoding one or more polypeptides of any one of the recombinant cytokine receptors. The isolated nucleic acid may be DNA. In some embodiments, the isolated nucleic acid is inserted into a vector (such as an expression vector, a viral vector, or a cloning vector). For nucleic acid expression, the vector may be introduced into a host cell to allow expression of the nucleic acid in the host cell. Expression vectors contain various elements for expression control, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection markers, and signal sequences. These elements may be selected appropriately by those skilled in the art. For example, a promoter sequence may be selected to enhance transcription of a polynucleotide in the vector. Suitable promoter sequences include, but are not limited to, MND, T7 promoter, T3 promoter, SP6 promoter, β-actin promoter, EF1a promoter, CMV promoter, and SV40 promoter. An enhancer sequence may be selected to promote transcription of the nucleic acid. The selectable marker is chosen to allow for the selection of host cells into which the vector has been inserted from those that have not; for example, the selectable marker can be a transmembrane gene such as EGFR, which can be identified by flow cytometry and FACS analysis. In some embodiments, the signal sequence comprises the amino acid sequence of SEQ ID NO:8.

[0116] In some embodiments, the nucleic acid (e.g., a vector such as an expression vector, a viral vector, or a cloning vector) expresses an antigen receptor and / or another additional polypeptide. The antigen receptor can be, for example, an antibody, an engineered antibody (such as an scFv), a CAR, an engineered TCR, a TCR mimetic or chimeric antibody T cell receptor, or a chimeric signaling receptor. The antigen receptor can target an antigen of interest (e.g., a tumor antigen or an antigen of a pathogen, or a target at a site of inflammation). Antigens include AFP (alpha-fetoprotein), αvβ6 or another integrin, BCMA, B7-H3, B7-H6, CA9 (carbonic anhydrase 9), CCL-1 (CC motif chemokine ligand 1), CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD45, CD47, CD56, CD66e, CD70, C D74, CD79a, CD79b, CD98, CD123, CD138, CD171, CD352, CEA (carcinoembryonic antigen), claudins, c-MET, DLL3 (delta-like protein 3), DLL4, ENPP3 (ectonucleotide pyrophosphatase / phosphodiesterase family member 3), EpCAM, EPG-2 (epithelial glycoprotein 2), EPG-40, ephrin B2, EPHa2 ephrine receptor A2), ERBB dimer, estrogen receptor, ETBR (endothelin B receptor), FAP-α (fibroblast activation protein α), fetal AchR (fetal acetylcholine receptor), FBP (folate binding protein), FCRL5, FR-α (folate receptor α), GCC (guanyl cyclase C), GD2, GD3, GPC2 (glypican-2), GPC3, gp100 (glycoprotein 100), GP NMB (glycoprotein NMB), GPRC5D (G protein-coupled receptor 5D), HER2, HER3, HER4, hepatitis B surface antigen, HLA-A1 (human leukocyte antigen A1), HLA-A2 (human leukocyte antigen A2), HMW-MAA (human high molecular weight melanoma-associated antigen), IGF1R (insulin-like growth factor 1 receptor), Igκ, Igλ, IL-22Ra (IL-22 receptor α), IL-13Ra2 (IL-13 receptor α2),KDR (kinase insert domain receptor), LI cell adhesion molecule (LI-CAM), Liv-1, LRRC8A (leucine-rich repeat-containing 8 family member A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MART-1 (melan-A), murine cytomegalovirus (MCMV), MCSP (melanoma-associated chondroitin sulfate proteoglycan), mesothelin, mucin 1 (MUC1), MUC16, MHC / peptide complexes (e.g., HLA-A complexed with peptides derived from AFP, KRAS, NY-ESO, MAGE-A, and WT1), NCAM (neural cell adhesion molecule), nectin-4, NKG2D (natural killer group 2 member D) ligand, NY-ESO, oncofetal antigen, PD-1, PD-L1, PRAME (preferentially expressed antigen of melanoma), progesterone receptor, PSA (prostate-specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), ROR1, ROR2, SIRPα (signal regulatory protein α), SLIT, SLITRK6 (NTRK-like protein 6), STEAP1 (prostate six-transmembrane epithelial antigen 1), survivin, TAG72 (tumor-associated glycoprotein 72), TPBG (trophoblast glycoprotein), Trop-2, VEGFR1 (vascular endothelial growth factor receptor 1), VEGFR2, and antigens from HIV, HBV, HCV, HPV and other pathogens, may be mentioned, but are not limited to.

[0117] In some embodiments, the constructs comprising the recombinant cytokine receptor described herein comprise a cleavable linker. In some embodiments, the cleavable linker is a 2A polypeptide. In some embodiments, the constructs comprising the recombinant cytokine receptor comprise a nucleotide sequence encoding a P2A linker between the nucleotide sequence encoding the recombinant cytokine receptor and a marker (e.g., EGFR). In some embodiments, the 2A-like sequence or "peptide bond skipping" 2A sequence is derived, for example, from a different virus (e.g., Thosea asigna). These sequences are sometimes also known as "peptide skipping sequences." When this type of sequence is placed within a cistron between two polypeptides intended to be separated, the ribosome appears to skip the peptide bond; in the case of Thosea asigna sequences, the bond between the Gly and Pro amino acids at the carboxy-terminal "PGP" is omitted. This can leave two to three polypeptides (e.g., an inducible chimeric pro-apoptotic polypeptide and a chimeric antigen receptor, or, for example, a marker polypeptide and an inducible chimeric pro-apoptotic polypeptide). When this sequence is used, the polypeptide encoded 5' of the 2A sequence can be terminated by additional amino acids at the carboxy terminus (including a Gly residue and any upstream residues in the 2A sequence). The peptide encoded 3' of the 2A sequence can be terminated by additional amino acids at the amino terminus (including a Pro residue and any downstream residues following the 2A sequence). In some embodiments, the cleavable linker is a 2A polypeptide derived from porcine teschovirus-1 (P2A). In some embodiments, the 2A cotranslational sequence is a 2A-like sequence. In some embodiments, the 2A cotranslational sequence is T2A (Thosea asigna virus 2A), F2A (foot-and-mouth disease virus 2A), P2A (porcine teschovirus-1 2A), BmCPV 2A (cytoplasmic polyhedrosis virus 2A), BmIFV 2A (flacheria virus of B. mori 2A), or E2A (equine rhinitis A virus 2A).In some embodiments, the 2A cotranslational sequence is T2A-GSG, F2A-GSG, P2A-GSG, or E2A-GSG. In some embodiments, the cotranslational sequence is selected from the group consisting of T2A, P2A, and F2A. By "cleavable linker" is meant that the linker is cleaved by any means, including, for example, non-enzymatic means (such as peptide skipping) or enzymatic methods (see, e.g., Donnelly, ML (2001), J. Gen. Virol. 82:1013-25, incorporated herein by reference in its entirety). In certain embodiments, P2A comprises (or consists of) a sequence disclosed herein. In certain embodiments, P2A comprises (or consists of) a sequence disclosed herein (e.g., a sequence disclosed in the Examples below).

[0118] In certain embodiments, the 2A linker comprises the amino acid sequence of SEQ ID NO: 10 (SGATNFSLLKQAGDVEENPGP). In certain embodiments, the 2A linker further comprises a GSG amino acid sequence at the amino terminus of the polypeptide, and in other embodiments, the 2A linker comprises a GSGPR amino acid sequence (SEQ ID NO: 21) at the amino terminus of the polypeptide. Thus, by "2A" sequence, the term can refer to the 2A sequences in the examples described herein, or can also refer to the 2A sequences listed herein that further comprise a GSG or GSGPR sequence (SEQ ID NO: 21) at the amino terminus of the linker.

[0119] In some embodiments, host cells (e.g., Treg cells) contain the vector described above. The vector can be introduced into the cell using any suitable method known in the art, including, but not limited to, DEAE-dextran-mediated delivery, calcium phosphate precipitation, cationic lipid-mediated delivery, liposome-mediated transfection, electroporation, microprojectile bombardment, receptor-mediated gene delivery, polylysine, histone, chitosan, and peptide-mediated delivery. Standard methods for transducing cells for expression of a vector of interest are well known in the art. In some embodiments, the host cell contains a vector comprising an isolated nucleic acid encoding a recombinant cytokine receptor.

[0120] In some embodiments, the present application provides a method for expressing any of the recombinant cytokine receptors described herein, comprising culturing isolated host cells containing a vector and recovering the recombinant cytokine receptor from the cell culture. The isolated host cells are cultured under conditions that allow expression of the isolated nucleic acid inserted into the vector. Suitable conditions for expression of the polynucleotide may include, but are not limited to, a suitable medium, a suitable density of host cells in the culture medium, the presence of necessary nutrients, the presence of supplementary factors, suitable temperature and humidity, and the absence of microbial contaminants. Those skilled in the art can select suitable conditions as appropriate for the purpose of expression.

[0121] vector In certain embodiments, the present disclosure provides nucleic acid molecules encoding any one or more of the recombinant cytokine receptors described herein. Such nucleic acid molecules can be inserted into a vector (e.g., a viral vector or a non-viral plasmid vector) suitable for introduction into host regulatory T cells (Tregs) of interest.

[0122] As used herein, the terms "recombinant" or "non-naturally occurring" refer to an organism, microorganism, cell, nucleic acid molecule, or vector that contains at least one genetic alteration or has been modified by the introduction of an exogenous nucleic acid molecule, where such alteration or modification is introduced by genetic engineering. Genetic alterations include, for example, modifications that introduce expressible nucleic acid molecules encoding proteins, fusion proteins, or enzymes, or the addition, deletion, substitution, or other functional disruption of other nucleic acid molecules in a cell's genetic material. Additional modifications include, for example, non-coding regulatory regions, where the modifications alter the expression of a gene or operon. In some embodiments, cells obtained from a subject (such as regulatory T cells (Tregs)) are converted into non-naturally occurring or recombinant regulatory T cells (Tregs) (e.g., non-naturally occurring or recombinant regulatory T cells (Tregs)) by the introduction of a nucleic acid encoding a recombinant cytokine receptor described herein, whereby the cells can express the recombinant cytokine receptor located on the cell surface.

[0123] Vectors encoding core viruses are referred to herein as "viral vectors." There are numerous available viral vectors suitable for use with the compositions of the present disclosure, including those identified for human gene therapy applications (see Pfeifer and Verma (2001), Ann. Rev. Genomics Hum. Genet. 2:177, incorporated herein by reference in its entirety). Suitable viral vectors include vectors based on RNA viruses (such as retrovirus-derived vectors, e.g., Maloney murine leukemia virus (MLV)-derived vectors), including more complex retrovirus-derived vectors (e.g., lentivirus-derived vectors). HIV-1-derived vectors fall into this category. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus). Methods for using retroviral and lentiviral viral vectors and packaging cells for transduction of mammalian host regulatory T cells (Tregs) with viral particles containing chimeric antigen receptor transgenes are known in the art and have been previously described, for example, in U.S. Pat. No. 8,119,772; Walchli et al. (2011), PLoS One 6:327930; Zhao et al. (2005), J. Immunol. 174:4415; Engels et al. (2003), Hum. Gene Ther. 14:1155; Frecha et al. (2010), Mol. Ther. 18:1748; and Verhoeyen et al. (2009), Methods Mol. Biol. 506:97. Retroviral and lentiviral vector constructs and expression systems are also commercially available.

[0124] In some embodiments, a viral vector is used to introduce a non-endogenous nucleic acid sequence encoding a recombinant cytokine receptor. The viral vector may be a retroviral or lentiviral vector. The viral vector may also contain a nucleic acid sequence encoding a marker for transduction. Transduction markers for viral vectors are known in the art and include selectable markers (which may confer drug resistance) or detectable markers (such as fluorescent markers or cell surface proteins that can be detected by methods such as flow cytometry). In certain embodiments, the viral vector further contains a genetic marker for transduction, including green fluorescent protein, the extracellular domain of human CD2, or a truncated human EGFR (huEGFRt; see Wang et al. (2011), Blood 118:1255). When the viral vector genome contains multiple nucleic acid sequences expressed in host cells (e.g., T cells such as Tregs) as separate transcripts, the viral vector may also contain additional sequences between the two (or more) transcripts to enable bicistronic or multicistronic expression. Examples of such sequences used in viral vectors include an internal ribosome entry site (IRES), a furin cleavage site, a viral 2A peptide, or any combination thereof.

[0125] Other vectors can also be used for polynucleotide delivery, including DNA viral vectors, such as adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; and vectors derived from herpes simplex virus (HSV), including amplicon vectors, replication-deficient HSV, and attenuated HSV (Krisky et al. (1998), Gene Ther. 5:1517).

[0126] Other vectors recently developed for gene therapy use can also be used with the compositions and methods of the present disclosure, including those derived from baculoviruses and alpha-viruses (Jolly, DJ (1999), Emerging Viral Vectors, pp. 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as Sleeping Beauty or other transposon vectors).

[0127] In certain embodiments, hematopoietic progenitor cells or embryonic stem cells are modified to contain a non-endogenous nucleic acid molecule encoding a recombinant cytokine receptor of the present disclosure. Hematopoietic progenitor cells may include induced pluripotent stem cells that may be derived from or originate from fetal liver tissue, bone marrow, umbilical cord blood, or peripheral blood. Hematopoietic progenitor cells may be from humans, mice, rats, or other mammals.

[0128] In certain embodiments, the host cells transfected to express the recombinant cytokine receptor of the present disclosure are functional regulatory T cells (Treg). One or more growth factor cytokines that promote the proliferation of regulatory T cells (Treg) expressing the recombinant cytokine receptor of the present disclosure can be added to the culture. The cytokines can be human or non-human. Exemplary growth factor cytokines that can be used to promote the proliferation of regulatory T cells (Treg) include IL-2, TGFβ, or similar.

[0129] IV. Methods for Expanding and Cultivating Cells In some embodiments, the cells transfected to express the recombinant cytokine receptor of the present disclosure are eukaryotic cells. In some embodiments, the cells transfected to express the recombinant cytokine receptor of the present disclosure are human cells. In certain embodiments, the cells transfected to express the recombinant cytokine receptor of the present disclosure are immune cells. In certain embodiments, the cells transfected to express the recombinant cytokine receptor of the present disclosure are T cells. In certain embodiments, the cells transfected to express the recombinant cytokine receptor of the present disclosure are functional regulatory T cells (Tregs).

[0130] In some embodiments, Treg cells are isolated from peripheral blood mononuclear (PBMC) cells. In some embodiments, Treg cells are isolated from PBMCs using density gradient centrifugation. In some embodiments, Treg cells are enriched by positive selection. In some embodiments, Treg cells are enriched by positive selection for IL-2Rα+. In some embodiments, Treg cells are enriched by positive selection for CD4+IL-2Rα+CD127lo cells. In some embodiments, enrichment occurs by FACS. In some embodiments, Tregs are stimulated with anti-CD3 and / or anti-CD28 on day 0 after positive selection. In some embodiments, Tregs are restimulated with anti-CD3 and / or anti-CD28 on day 9 of culture after positive selection.

[0131] One or more growth factor cytokines can be added to the culture to promote proliferation of T cells, such as Tregs, including Tregs expressing a recombinant cytokine receptor of the present disclosure. The cytokines can be human or non-human. Exemplary growth factor cytokines that can be used to promote proliferation of regulatory T cells (Tregs) include IL-4, IL-7, IL-9, IL-21, or the like. In some embodiments, cytokines are added to the medium prior to selection. In some embodiments, cytokines are added to the medium after cell isolation. In some embodiments, cytokines are added to the cell culture approximately every 12-60 (e.g., 24-48) hours. In some embodiments, cytokines are added to the medium for the duration of the culture.

[0132] In some embodiments, cells expressing a recombinant cytokine receptor of the present disclosure are cultured for a time sufficient to induce proliferation or differentiation. The cells are generally maintained in culture for about 3 to about 5 days, about 4 to about 10 days, about 5 to about 20 days, about 10 to about 23 days, about 15 to about 30 days, or about 23 to about 30 days. It will be appreciated that the cells may be maintained for an appropriate amount of time required to achieve the desired result (i.e., the desired cell composition or level of proliferation). For example, to generate a cell composition comprising primarily Tregs, the cells may be maintained in culture for about 30 days.

[0133] In some embodiments, the method further comprises detecting one or more Treg markers provided herein. In some embodiments, the method further comprises detecting IL-2 signaling. In some embodiments, the method further comprises detecting phosphorylated STAT-5.

[0134] In some embodiments, the suppressive activity of transduced Tregs is increased upon transduction with a recombinant cytokine receptor provided herein. In some embodiments, the rate of cytotoxic T cell division is decreased by transduced Treg cells provided herein. In some embodiments, the rate of CD4+ T cell division is decreased. In some embodiments, the rate of CD8+ T cell division is decreased. In some embodiments, the activity of cytotoxic T cells is decreased.

[0135] In some embodiments, provided herein are methods for expanding transduced Treg cells in the absence of exogenous IL-2, comprising culturing cells expressing a recombinant cytokine receptor provided herein. In some embodiments, the cultured cells express a recombinant cytokine receptor in which IL-2Rβ is tethered to an IL-2 cytokine. In some embodiments, the cultured cells comprising a recombinant cytokine receptor provided herein receive IL-2 signaling stimulation from the tethered IL-2 cytokine. In some embodiments, the cells are cultured in a culture medium containing a cytokine other than IL-2. In some embodiments, the cells are cultured in a medium containing two, three, four, or five or more cytokines.

[0136] V. Pharmaceutical Compositions, Articles of Manufacture, and Kits Further provided by the present application is a pharmaceutical composition comprising a cell (e.g., a T cell, such as a Treg cell) comprising a recombinant cytokine receptor described herein.

[0137] The pharmaceutical compositions may be suitable for various modes of administration described herein, including, for example, systemic or local administration.

[0138] In some embodiments, the pharmaceutical composition is formulated for intravenous administration. Pharmaceutical compositions used for in vivo administration are generally formulated as sterile, substantially isotonic, and in full compliance with all US Food and Drug Administration Good Manufacturing Practice (GMP) regulations. Sterilization is easily achieved by filtration through a sterile filtration membrane. In some embodiments, the composition is pathogen-free. For injection, the pharmaceutical composition can be in the form of a solution, for example, in a physiologically compatible buffer (such as Hank's solution or Ringer's solution).

[0139] In some embodiments, the pharmaceutical composition is suitable for administration to a human. In some embodiments, the pharmaceutical composition is suitable for administration to a rodent (e.g., a mouse, a rat) or a non-human primate (e.g., a cynomolgus monkey). In some embodiments, the pharmaceutical composition is cryopreserved.

[0140] The present application also provides kits that include the compositions (such as pharmaceutical compositions) described herein, and may further include instruction(s) for how to use the compositions (such as the uses described herein). The kits described herein may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any of the methods described herein.

[0141] VI. Treatment Methods In some embodiments, provided herein are methods of treating an immune-related disorder, comprising administering cells (e.g., Treg cells) comprising a recombinant cytokine receptor provided herein. In some embodiments, the recombinant cytokine receptor comprises an interleukin-2 receptor beta (IL-2Rβ or IL-2RB) polypeptide, N-terminally tethered to an IL-2 cytokine. In some embodiments, the recombinant cytokine receptor used in the methods of treating an immune-related disorder is capable of signaling in the absence of exogenous IL-2. In some embodiments, the recombinant cytokine receptor used in the methods of treating an immune-related disorder comprises an IL-2 cytokine, a polypeptide linker, and an IL-2Rβ polypeptide. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F. Thus, in some embodiments, methods are provided for treating immune-related disorders, comprising administering cells (e.g., Treg cells) comprising a recombinant cytokine receptor comprising: (I) an IL-2 cytokine; (II) a polypeptide linker; and (III) an IL-2Rβ polypeptide. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 11.In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 11. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiments, the cell is a Treg.

[0142] In some embodiments, a method of treating an immune-related disorder comprises administering cells (e.g., Treg cells) comprising a recombinant cytokine receptor comprising IL-2Rβ tethered to a wild-type IL-2 cytokine. In some embodiments, the method comprises transducing cells (e.g., Treg cells) with a vector encoding the recombinant cytokine receptor. In some embodiments, the cells are autologous to the individual being treated. In some embodiments, the wild-type IL-2 cytokine comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the wild-type IL-2 cytokine comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, a method of treating an immune disorder comprises transducing cells (e.g., Treg cells) with a recombinant cytokine receptor comprising, from N- to C-terminus, (I) wild-type IL-2 cytokine; (II) a polypeptide linker; and (III) IL-2Rβ. In some embodiments, the recombinant cytokine receptor does not comprise a TCR activation domain or costimulatory domain (e.g., an activation or costimulatory domain of CD3 or CD28 (such as a CD28 signaling domain)). In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134.For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in an IL-2 cytokine and an IL-2Rβ polypeptide, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiments, the cell is a Treg.

[0143] In some embodiments, a method for treating an immune-related disorder comprises administering cells (e.g., Treg cells) comprising a recombinant cytokine receptor comprising an IL-2 cytokine, wherein the IL-2 cytokine comprises one or more amino acid substitutions at amino acid positions selected from 18, 22, 126, 38, 43, and 61. In some embodiments, a method for an immune-related disorder comprises administering cells (e.g., Treg cells) comprising a recombinant cytokine receptor comprising an IL-2 cytokine, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of R38D, K43E, and E61R. In some embodiments, a method for treating an immune-related disorder comprises administering cells (e.g., Treg cells) comprising a recombinant cytokine receptor comprising an IL-2 cytokine, wherein the IL-2 cytokine comprises the amino acid substitutions R38D, K43E, and E61R ("3x IL-2"). In some embodiments, a method of treating an immune-related disorder comprises administering a cell (e.g., a Treg cell) comprising a recombinant cytokine receptor comprising an IL-2 cytokine, wherein the IL-2 cytokine comprises the amino acid substitutions R38D, K43E, and E61R, and one or more additional amino acid substitutions. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F.In some embodiments, the method comprises transducing cells (e.g., Treg cells) with a vector encoding a recombinant cytokine receptor. In some embodiments, the cells are autologous to the individual being treated. In some embodiments, the 3x IL-2 cytokine comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the 3x IL-2 cytokine comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the method of treating an immune-related disorder comprises administering cells (e.g., Treg cells) comprising a recombinant cytokine receptor comprising, from N-terminus to C-terminus, (I) 3x IL-2 cytokine; (II) a polypeptide linker; and (III) an IL-2Rβ polypeptide. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the recombinant cytokine receptor comprises 3x IL-2 cytokine having an amino acid sequence comprising at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiments, the cell is a Treg.

[0144] In some embodiments, a method of treating an immune-related disorder comprises administering cells (e.g., Treg cells) comprising a recombinant cytokine receptor comprising an IL-2 cytokine, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of L18R, Q22E, and Q126H. In some embodiments, a method of treating an immune-related disorder comprises administering cells (e.g., Treg cells) comprising a recombinant cytokine receptor comprising an IL-2 cytokine, wherein the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126H ("REH IL-2"). In some embodiments, a method of treating an immune-related disorder comprises administering cells (e.g., Treg cells) comprising a recombinant cytokine receptor comprising an IL-2 cytokine, wherein the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126H, and one or more additional amino acid substitutions. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F. In some embodiments, the method comprises transducing a cell (e.g., a Treg cell) with a vector encoding the recombinant cytokine receptor. In some embodiments, the cells are autologous to the individual being treated. In some embodiments, the REH IL-2 cytokine comprises the amino acid sequence set forth in SEQ ID NO:6.In some embodiments, the REH IL-2 cytokine comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:6. In some embodiments, a method of treating an immune-related disorder comprises administering a cell (e.g., a Treg cell) comprising a recombinant cytokine receptor comprising, from N- to C-terminus, (I) a REH IL-2 cytokine; (II) a polypeptide linker; and (III) an IL-2Rβ polypeptide. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiments, the cell is a Treg.

[0145] In some embodiments, a method of treating an immune-related disorder comprises administering cells (e.g., Treg cells) comprising a recombinant cytokine receptor comprising an IL-2 cytokine, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of L18R, Q22E, Q126H, R38D, K43E, and E61R. In some embodiments, a method of treating an immune-related disorder comprises administering cells (e.g., Treg cells) comprising a recombinant cytokine receptor comprising an IL-2 cytokine, wherein the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, Q126H, R38D, K43E, and E61R ("3x REH IL-2"). In some embodiments, a method of treating an immune-related disorder comprises administering cells (e.g., Treg cells) comprising a recombinant cytokine receptor comprising an IL-2 cytokine, wherein the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, Q126H, R38D, K43E, and E61R, and one or more additional amino acid substitutions. In some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions at position(s) 15, 16, 19, 20, 22, 23, and 81, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions at position(s) 133 and / or 134. For example, in some embodiments, the recombinant cytokine further comprises one or more amino acid substitutions in the IL-2 cytokine and IL-2Rβ, where the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D, and the IL-2Rβ polypeptide comprises one or both amino acid substitutions H133D and / or Y134F. In some embodiments, the method comprises transducing a cell (e.g., a Treg cell) with a vector encoding the recombinant cytokine receptor. In some embodiments, the cells are autologous from the individual being treated.In some embodiments, the 3x REH IL-2 cytokine comprises the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the 3x REH IL-2 cytokine comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12. In some embodiments, a method of treating an immune-related disorder comprises administering a cell (e.g., a Treg cell) comprising a recombinant cytokine receptor comprising, from N- to C-terminus, (I) a 3x REH IL-2 cytokine; (II) a polypeptide linker; and (III) an IL-2Rβ polypeptide. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiments, the cell is a Treg.

[0146] In some embodiments, the recombinant cytokine receptor of the present disclosure is expressed in a T cell. In some embodiments, one or more recombinant cytokine receptors are expressed in a T cell. In some embodiments, the recombinant cytokine receptor is co-expressed with a chimeric antigen receptor (CAR). In some embodiments, the T cell is a regulatory T cell (Treg). In some embodiments, the Treg is CD4+, IL-2Rα+, CD127lo. In some embodiments, the Treg expresses FOXP3 and / or HELIOS. In some embodiments, the recombinant cytokine receptor is expressed in a Treg cell.

[0147] The immune conditions, diseases, disorders, and reactions or responses treated according to the methods and compositions of the present invention refer to diseases in which the immune system contributes to the pathogenesis or may be part of the treatment. These reactions include, but are not limited to, cancer, inflammation, autoimmune conditions, disorders, or diseases, and persistent and ongoing immune responses to infectious non-self antigens from bacterial, viral (e.g., HCV), fungal, or parasitic organisms that invade and persist in mammals and humans. Such conditions and disorders include allergies and / or asthma. Allergies and asthma can result from sensitization by foreign or non-self antigens such as pollen, animal dander, and food proteins. The source of the inducing foreign antigen can be plants, fungi, mold, or other environmental pollutants.

[0148] Autoimmunity is defined as a persistent and progressive immune response to non-infectious self-antigens, distinct from infectious non-self-antigens from bacterial, viral, fungal, or parasitic organisms that invade and persist in mammals and humans. Autoimmune conditions include graft-versus-host disease, autoimmune polyendocrine syndrome, type 1 diabetes mellitus (TIDM), autoimmune gastritis, autoimmune uveoretinitis, autoimmune vasculitis, colitis, thyroiditis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune hepatitis, autoimmune inner ear disease, axonal and neuropathy, Behçet's disease, bullous pemphigoid, Castleman's disease, celiac disease, Chagas disease, and chronic inflammation. CIDP, chronic relapsing multifocal osteomyelitis, Churg-Strauss cicatricial pemphigoid / benign mucous membrane pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, Devic's disease, discoid lupus, Dressler's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans' syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant Myocarditis, glomerulonephritis, Goodpasture's syndrome, polyangiogranulomatosis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes gestationis or pemphigoid of pregnancy, hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis, Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosis, lignified conjunctivitis, linear IgA disease, lupus , Lyme disease, chronic Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, Mooren's ulcer, Much-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcal disease), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry-Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy,Perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progestational dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia, pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, arthritis These include horse arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome, subacute bacterial endocarditis, Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis (polyangiopathy).

[0149] "Autoantigen" or "self-antigen," as used herein, refers to an antigen or epitope that is native to a mammal and immunogenic in said mammalian disease. One aspect of the present application provides a method of treating an immune-related disorder. The cells can be "allogeneic cells" (allogeneic), which are isolated from one individual (donor) and infused into another, while "autologous cells" (autologous) refer to cells that are isolated and infused back into the same individual. In some embodiments, the cells are autologous to the individual. In some embodiments, the individual is human. In some embodiments, the cells are T cells. In some embodiments, the cells are Tregs. In some embodiments, the Treg cells are isolated from human peripheral blood mononuclear cells (PBMCs). In some embodiments, the Treg cells are modified ex vivo with vectors encoding one or more recombinant cytokine receptors. In some embodiments, the Treg cells are expanded ex vivo in the absence of exogenous IL-2. In some embodiments, Treg cells are modified with a nucleic acid or vector encoding a recombinant cytokine receptor. In some embodiments, Treg cells are modified to proliferate in the absence of exogenous IL-2. In some embodiments, Treg cells have one or more markers of IL-2 signaling detected. In some embodiments, the marker of IL-2 signaling detected is phosphorylated STAT5. In some embodiments, Tregs are CD4+, IL-2Rα+, CD1271. In some embodiments, Tregs express FOXP3 and / or HELIOS. In some embodiments, Treg cells are administered to the same individual to treat an immune-related disorder.

[0150] In some embodiments, a method for treating an immune-related disorder comprises administering Treg cells to an individual in need of such treatment. In some embodiments, the recombinant cytokine receptor is co-expressed with a chimeric antigen receptor (CAR). In some embodiments, the T cells are regulatory T cells (Tregs). In some embodiments, the Tregs are CD4+, IL-2Rα+, CD127lo. In some embodiments, the Tregs express FOXP3 and / or HELIOS.

[0151] In some embodiments, the method of treating an immune-related disorder comprises administering autologous Treg cells to an individual. In some embodiments, the individual is human. In some embodiments, the method of treating an immune-related disorder comprises administering Treg cells isolated from human peripheral blood mononuclear cells (PBMCs). In some embodiments, the method of treating an immune-related disorder comprises administering Treg cells modified ex vivo with a vector encoding one or more recombinant cytokine receptors. In some embodiments, the method of treating an immune-related disorder comprises administering Treg cells expanded ex vivo in the absence of exogenous IL-2. In some embodiments, the method of treating an immune-related disorder comprises administering Treg cells modified with a nucleic acid or vector encoding a recombinant cytokine receptor. In some embodiments, the method of treating an immune-related disorder comprises administering Treg cells modified to proliferate in the absence of exogenous IL-2. In some embodiments, the method of treating an immune-related disorder comprises administering Treg cells in which one or more markers of IL-2 signaling are detected. In some embodiments, a method of treating an immune-related disorder comprises administering Treg cells in which a detected marker of IL-2 signaling is phosphorylated STAT5. In some embodiments, a method of treating an immune-related disorder comprises administering Treg cells that are CD4+, IL-2Rα+, CD127lo. In some embodiments, a method of treating an immune-related disorder comprises administering Treg cells that express FOXP3 and / or HELIOS. In some embodiments, a method of treating an immune-related disorder comprises administering Treg cells.

[0152] In some embodiments, a method of treating an immune-related disorder comprises administering Treg cells comprising a recombinant cytokine receptor of the present disclosure. In some embodiments, the suppressive activity of Tregs against CD8+ T cells and / or CD4+ T cells is increased. In some embodiments, the suppressive activity of Tregs against CD8+ T cells and / or CD4+ T cells when cultured without exogenous IL-2 is increased. In some embodiments, the suppressive activity of Tregs against CD8+ T cells and / or CD4+ T cells is increased compared to unmodified Tregs cultured without exogenous IL-2. In some embodiments, the suppressive activity of transduced Tregs against CD8+ T cells and / or CD4+ T cells cultured without exogenous IL-2 is approximately the same as or approximately the same as unmodified Treg cells cultured with IL-2. In some embodiments, the suppressive activity of transduced Tregs reduces the rate of CD4+ T cell and / or CD8+ T cell division.

[0153] In some embodiments, a method of treating an immune-related disorder comprises transducing a population of cells with a recombinant cytokine receptor. In some embodiments, a method of treating an immune-related disorder comprises transducing a population of T cells with a recombinant cytokine receptor. In some embodiments, a method of treating an immune-related disorder comprises transducing a population of Treg cells with a recombinant cytokine receptor. In some embodiments, cell viability is increased. In some embodiments, at least about 60-99% of the cells in a population of Treg cells transduced with a recombinant cytokine remain viable in vitro for approximately 7-20 days after transduction. In some embodiments, at least about 60-99% of the cells in a population of Treg cells transduced with a recombinant cytokine remain viable in vitro for approximately 7-14 days after transduction. In some embodiments, at least about 60% of the cells in a population of Treg cells transduced with a recombinant cytokine remain viable in vitro for approximately 7 days after transduction.

[0154] In some embodiments, a method of treating an immune-related disorder comprises transducing a population of cells with a recombinant cytokine receptor. In some embodiments, a method of treating an immune-related disorder comprises transducing a population of T cells with a recombinant cytokine receptor. In some embodiments, a method of treating an immune-related disorder comprises transducing a population of Treg cells with a recombinant cytokine receptor. In some embodiments, cell viability is increased. In some embodiments, at least about 60-99% of the cells in a population of Treg cells transduced with the recombinant cytokine persist in vivo for at least about 3 days or more. In some embodiments, at least about 60-90% of the cells in a population of Treg cells transduced with the recombinant cytokine persist in vivo for at least about 3 days or more. In some embodiments, at least about 60% of the cells in a population of Treg cells transduced with the recombinant cytokine persist in vivo for at least about 3 days or more. In some embodiments, the population of recombinant cytokine-transduced Treg cells persists in vivo for at least about any of 1, 2, 3, 5, 6, 8, 10, or 11 months, or for at least about 1 year or more.

[0155] In some embodiments, a method of treating an immune-related disorder comprises transducing a cell population with a recombinant cytokine receptor. In some embodiments, a method of treating an immune-related disorder comprises transducing a population of T cells with a recombinant cytokine receptor. In some embodiments, a method of treating an immune-related disorder comprises transducing a population of Treg cells with a recombinant cytokine receptor, resulting in a cell population containing more viable cells compared to a composition comprising the same population of Treg cells not transduced with the recombinant cytokine receptor. In some embodiments, at least about 60-99% of the cells in a population of Treg cells transduced with the recombinant cytokine remain viable in vitro for approximately 14-23 days after transduction. In some embodiments, at least about 60-90% of the cells in a population of Treg cells transduced with the recombinant cytokine remain viable in vitro for approximately 14-23 days after transduction. In some embodiments, at least about 60% of the cells in a population of Treg cells transduced with the recombinant cytokine remain viable in vitro for approximately 14 days after transduction.

[0156] In some embodiments, a method for treating an immune-related disorder comprises transducing a cell population with a recombinant cytokine receptor. In some embodiments, a method for treating an immune-related disorder comprises transducing a population of T cells with a recombinant cytokine receptor. In some embodiments, a method for treating an immune-related disorder comprises transducing a population of Treg cells with a recombinant cytokine receptor, resulting in a cell population containing more viable cells compared to a composition comprising the same population of Treg cells not transduced with the recombinant cytokine receptor. In some embodiments, at least about 60-99% of the cells in a population of Treg cells transduced with the recombinant cytokine persist in vivo for at least about 3 days or more. In some embodiments, at least about 60-90% of the cells in a population of Treg cells transduced with the recombinant cytokine persist in vivo for at least about 3 days or more. In some embodiments, at least about 60% of the cells in a population of Treg cells transduced with the recombinant cytokine persist in vivo for at least about 3 days or more. In some embodiments, the population of recombinant cytokine-transduced Treg cells persists in vivo for at least about any of 1, 2, 3, 5, 6, 8, 10, or 11 months, or for at least 1 year or more.

[0157] In some embodiments, a method for treating an immune-related disorder comprises transducing a cell population with a recombinant cytokine receptor. In some embodiments, a method for treating an immune-related disorder comprises transducing a population of T cells with a recombinant cytokine receptor. In some embodiments, a method for treating an immune-related disorder comprises transducing a population of Treg cells with a recombinant cytokine receptor, resulting in a cell population containing more viable cells compared to a composition comprising the same population of Treg cells not transduced with the recombinant cytokine receptor. In some embodiments, at least about 60-99% of the cells in a population of Treg cells transduced with the recombinant cytokine persist in vivo for at least about 14 days or more. In some embodiments, at least about 60-90% of the cells in a population of Treg cells transduced with the recombinant cytokine persist in vivo for at least about 14 days or more. In some embodiments, at least about 60% of the cells in a population of Treg cells transduced with the recombinant cytokine persist in vivo for at least about 14 days or more. In some embodiments, the population of recombinant cytokine-transduced Treg cells persists in vivo for at least about any of 1, 2, 3, 5, 6, 8, 10, or 11 months, or for at least 1 year or more.

[0158] In some embodiments, a method for treating an immune-related disorder comprises transducing a cell population with a recombinant cytokine receptor. In some embodiments, a method for treating an immune-related disorder comprises transducing a population of T cells with a recombinant cytokine receptor. In some embodiments, a method for treating an immune-related disorder comprises transducing a population of Treg cells with a recombinant cytokine receptor, resulting in a cell population containing more viable cells compared to a composition comprising the same population of Treg cells not transduced with the recombinant cytokine receptor. In some embodiments, at least about 60-99% of the cells in a population of Treg cells transduced with the recombinant cytokine persist in vivo for at least about 23 days or more. In some embodiments, at least about 60-90% of the cells in a population of Treg cells transduced with the recombinant cytokine persist in vivo for at least about 23 days or more. In some embodiments, at least about 60% of the cells in a population of Treg cells transduced with the recombinant cytokine persist in vivo for at least about 23 days or more. In some embodiments, the population of recombinant cytokine-transduced Treg cells persists in vivo for at least about any of 1, 2, 3, 5, 6, 8, 10, or 11 months, or for at least 1 year or more.

[0159] In some embodiments, a method of treating an immune-related disorder comprises transducing a population of Treg cells transduced with a recombinant cytokine receptor, whereby the population of transduced Treg cells expands at least two-fold over a population of the same Treg cells not transduced with the recombinant cytokine receptor. In some embodiments, the method of treating an immune-related disorder comprises a composition whereby the population of Treg cells transduced with the recombinant cytokine receptor expands at least two-fold, and whereby the population of Treg cells transduced with the recombinant cytokine receptor maintains expression of at least one Treg marker selected from the group consisting of CD4+, IL-2Rα+, and CD127lo. In some embodiments, the Treg marker may also comprise FOXP3 and / or HELIOS.

[0160] In some embodiments, the recombinant cytokine receptors of the present disclosure may also be used in combination with chimeric antigen receptors (CARs). In some embodiments, the modified Tregs comprise a recombinant cytokine receptor and a chimeric antigen.

[0161] VII. Methods for Expanding Transduced Treg Cells In some embodiments, the method of expanding transduced T cells comprising a recombinant cytokine receptor does not activate IL-2 signaling in T cells that do not comprise a recombinant cytokine receptor. In some embodiments, the method of expanding transduced T cells comprising a recombinant cytokine receptor does not activate IL-2 signaling in Treg cell(s) that do not comprise a recombinant cytokine receptor.

[0162] In some embodiments, the method of expanding transduced T cells comprising a recombinant cytokine receptor does not activate signaling of another distinct IL-2 receptor. In some embodiments, the method of expanding transduced T cells comprising a recombinant cytokine receptor does not cause the transduced Treg cells to secrete one or more cytokines at a level greater than that of Treg cells cultured with exogenous IL-2 cytokine. In some embodiments, the method of expanding transduced T cells comprising a recombinant cytokine receptor does not cause the transduced Treg cells to secrete one or more cytokines at a level greater than that of Treg cells cultured with exogenous IL-2 cytokine.

[0163] In some embodiments, the method of expanding transduced T cells comprising a recombinant cytokine receptor allows the transduced Treg cells to divide and proliferate without exogenous IL-2. In some embodiments, the method of expanding transduced T cells comprises a method in which the relative amount of Treg cells in a composition comprising a population of Treg cells transduced with a recombinant cytokine receptor increases over time. In some embodiments, the method of expanding transduced T cells comprises a method in which a population of Treg cells transduced with a recombinant cytokine receptor expanded without IL-2 contains a similar number of viable cells compared to a composition comprising the same population of Treg cells not transduced with a recombinant cytokine receptor expanded with IL-2. In some embodiments, the method of expanding transduced T cells comprises a method in which at least about 80% of a population of Treg cells transduced with a recombinant cytokine maintain FOXP3 and / or HELIOS expression for about 14 days after transduction. In some embodiments, the method of expanding transduced T cells includes a method wherein at least about 80% of the cells in a population of recombinant cytokine-transduced Treg cells maintain FOXP3 and / or HELIOS expression for about 23 days after transduction.

[0164] In some embodiments, the method of expanding transduced T cells comprises a method in which a population of Treg cells transduced with a recombinant cytokine receptor is expanded at least two-fold over a population of the same Treg cells not transduced with the recombinant cytokine receptor. In some embodiments, the method of expanding transduced T cells comprises a method in which a population of Treg cells transduced with a recombinant cytokine receptor is expanded at least two-fold, and the population of Treg cells transduced with the recombinant cytokine receptor maintains expression of at least one Treg marker selected from the group consisting of CD25+, FOXP3, and HELIOS.

[0165] In some embodiments, the method of expanding transduced T cells includes a method in which the relative amount of Treg cells in a composition comprising a population of Treg cells transduced with a recombinant cytokine receptor increases over time when cultured without IL-2.

[0166] VIII.Definitions It is recognized that certain features of the disclosure that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features that are, for brevity, described in the c...

Claims

1. A regulatory T cell (Treg) comprising a recombinant cytokine receptor, the recombinant cytokine receptor comprising: IL-2 cytokine; IL-2 receptor beta extracellular domain; a transmembrane domain; and IL-2 receptor beta intracellular domain, the IL-2 receptor beta extracellular domain is tethered to the IL-2 cytokine by a polypeptide linker; The Treg.

2. The Treg of claim 1, wherein the recombinant cytokine receptor forms a protein complex with IL-2Rγ.

3. The Treg of claim 1, wherein the recombinant cytokine receptor participates in IL-2 signaling in the absence of exogenous IL-2.

4. 2. The Treg of claim 1, wherein the IL-2 cytokine comprises at least one amino acid substitution that reduces affinity for IL-2Rα and / or IL-2Rγ by at least about two-fold.

5. The Treg of claim 3, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from amino acid positions 18, 22, 126, 38, 43, 61, 15, 16, 19, 20, 22, 23, and 81.

6. 6. The Treg of claim 5, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of L18R, Q22E, Q126H, R38D, K43E, E61R, E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D.

7. 7. The Treg of claim 6, wherein the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126H; and / or R38D, K43E, and E61R; and / or E15S, H16Q, L19V, D20L, M23Q, and R81D; and / or E15S, H16Q, L19V, D20L, Q22K, and M23A.

8. The recombinant cytokine receptor a. WT IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain; b. 3x IL-2 cytokines, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain; c. REH IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain; d. 3x REH IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain; e. an IL-2 cytokine comprising substitutions at positions E15S, H16Q, L19V, D20L, M23Q, and R81D, a polypeptide linker, an IL-2Rβ extracellular domain comprising substitutions at positions H133D and Y134F, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain; or f. an IL-2 cytokine comprising substitutions at positions E15S, H16Q, L19V, D20L, Q22K, and M23A, a polypeptide linker, an IL-2Rβ extracellular domain comprising substitutions at positions H133D and Y134F, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain; The Treg of claim 1 , comprising:

9. The Treg of claim 1 , wherein the polypeptide linker comprises glycine and serine.

10. The recombinant cytokine receptor a. an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO:4; b. an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO:5; c. an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO:6; or d. The Treg of claim 8, comprising an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 12, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain.

11. The recombinant cytokine receptor a. an IL-2 cytokine comprising an amino acid sequence that comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12; b. a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21; c. an IL-2 receptor beta extracellular domain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13; d. an IL-2 receptor β transmembrane domain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14; and / or e. an IL-2 receptor beta intramembrane domain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15; The Treg of claim 1 , comprising:

12. 12. The Treg of claim 11, wherein the IL-2 cytokine comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

4.

13. 13. The Treg of claim 12, wherein the recombinant cytokine receptor does not comprise a T cell receptor activation domain or a T cell costimulatory domain, and optionally, the T cell receptor activation domain or the T cell costimulatory domain is a CD28 signaling domain.

14. a. an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO:4, a polypeptide linker comprising the amino acid sequence of SEQ ID NO:9, an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO:13, an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO:14, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO:15; b. an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO:5, a polypeptide linker comprising the amino acid sequence of SEQ ID NO:9, an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO:13, an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO:14, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO:15; c. an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO:6, a polypeptide linker comprising the amino acid sequence of SEQ ID NO:9, an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO:13, an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO:14, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO:15; or d. an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 12, a polypeptide linker comprising the amino acid sequence of SEQ ID NO: 9, an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15; The Treg of claim 11, comprising:

15. 15. The Treg of claim 14, wherein the recombinant cytokine receptor comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 11.

16. IL-2 cytokine; IL-2 receptor beta extracellular domain; a transmembrane domain; and IL-2 receptor β intracellular domain 2. The Treg of claim 1, comprising a recombinant cytokine receptor consisting of: the IL-2 receptor beta extracellular domain is tethered to the IL-2 cytokine by a polypeptide linker; The Treg.

17. The Treg of claim 1, wherein the Treg is CD25+ and expresses FOXP3 and / or HELIOS.

18. The Treg of claim 17, further comprising a chimeric antigen receptor (CAR).

19. A method of treating an immune-related disorder in an individual, comprising administering to the individual the Treg of claim 1.

20. 20. The method of claim 19, wherein the cells are autologous to the individual.

21. 20. The method of claim 19, wherein the individual is a human.

22. 20. The method of claim 19, wherein the Tregs prevent, ameliorate, or cure an immune-related disorder.

23. IL-2 cytokine; IL-2 receptor beta extracellular domain; a transmembrane domain; and IL-2 receptor β intracellular domain A recombinant cytokine receptor comprising: the IL-2 receptor beta extracellular domain is tethered to the IL-2 cytokine by a polypeptide linker; The recombinant cytokine receptor.

24. 24. The recombinant cytokine receptor of claim 23, wherein the recombinant cytokine receptor forms a protein complex with IL-2Rγ.

25. 24. The recombinant cytokine receptor of claim 23, wherein the recombinant cytokine receptor participates in IL-2 signaling in the absence of exogenous IL-2.

26. 24. The recombinant cytokine receptor of claim 23, wherein the IL-2 cytokine comprises at least one amino acid substitution that reduces its affinity for IL-2Rα and / or IL-2Rγ by at least about two-fold.

27. 27. The recombinant cytokine receptor of claim 26, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from amino acid positions 18, 22, 126, 38, 43, 61, 15, 16, 19, 20, 22, 23, and 81.

28. 28. The recombinant cytokine receptor of claim 27, wherein the IL-2 cytokine comprises one or more amino acid substitutions selected from the group consisting of L18R, Q22E, Q126H, R38D, K43E, E61R, E15S, H16Q, L19V, D20L, Q22K, M23Q, M23A, and R81D.

29. 29. The recombinant cytokine receptor of claim 28, wherein the IL-2 cytokine comprises the amino acid substitutions L18R, Q22E, and Q126H; and / or R38D, K43E, and E61R; and / or E15S, H16Q, L19V, D20L, M23Q, and R81D; and / or E15S, H16Q, L19V, D20L, Q22K, and M23A.

30. a. WT IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain; b. 3x IL-2 cytokines, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain; c. REH IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain; d. 3x REH IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain; e. an IL-2 cytokine comprising substitutions at positions E15S, H16Q, L19V, D20L, M23Q, and R81D, a polypeptide linker, an IL-2Rβ extracellular domain comprising substitutions at positions H133D and Y134F, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain; or f. an IL-2 cytokine comprising substitutions at positions E15S, H16Q, L19V, D20L, Q22K, and M23A, a polypeptide linker, an IL-2Rβ extracellular domain comprising substitutions at positions H133D and Y134F, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain; 24. The recombinant cytokine receptor of claim 23, comprising:

31. a. an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO:4; b. an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO:5; c. an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO:6; or d. an IL-2 cytokine, a polypeptide linker, an IL-2Rβ extracellular domain, an IL-2Rβ transmembrane domain, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 12; 31. The recombinant cytokine receptor of claim 30, comprising:

32. 24. The recombinant cytokine receptor of claim 23, wherein the polypeptide linker comprises glycine and serine.

33. a. an IL-2 cytokine comprising an amino acid sequence that comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 12; b. a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 16-21; c. an IL-2 receptor beta extracellular domain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13; d. an IL-2 receptor β transmembrane domain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14; and / or e. an IL-2 receptor beta intramembrane domain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15; 24. The recombinant cytokine receptor of claim 23, comprising:

34. 34. The recombinant cytokine receptor of claim 33, wherein the IL-2 cytokine comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

4.

35. 35. The recombinant cytokine receptor of claim 34, wherein the recombinant cytokine receptor does not comprise a T cell receptor activation domain or a T cell costimulatory domain, and optionally, the T cell receptor activation domain or the T cell costimulatory domain is a CD28 signaling domain.

36. a. an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO:4, a polypeptide linker comprising the amino acid sequence of SEQ ID NO:9, an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO:13, an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO:14, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO:15; b. an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO:5, a polypeptide linker comprising the amino acid sequence of SEQ ID NO:9, an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO:13, an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO:14, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO:15; c. an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO:6, a polypeptide linker comprising the amino acid sequence of SEQ ID NO:9, an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO:13, an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO:14, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO:15; or d. an IL-2 cytokine comprising the amino acid sequence of SEQ ID NO: 12, a polypeptide linker comprising the amino acid sequence of SEQ ID NO: 9, an IL-2Rβ extracellular domain comprising the amino acid sequence of SEQ ID NO: 13, an IL-2Rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and an IL-2Rβ intracellular domain comprising the amino acid sequence of SEQ ID NO: 15; 34. The recombinant cytokine receptor of claim 33, comprising:

37. 37. The recombinant cytokine receptor of claim 36, wherein the recombinant cytokine receptor comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 11.

38. IL-2 cytokine; IL-2 receptor beta extracellular domain; a transmembrane domain; and IL-2 receptor β intracellular domain 24. The recombinant cytokine receptor of claim 23, comprising: the IL-2 receptor beta extracellular domain is tethered to the IL-2 cytokine by a polypeptide linker; The recombinant cytokine receptor.

39. A nucleic acid encoding the recombinant cytokine receptor of claim 23.

40. A vector comprising the nucleic acid of claim 39.

41. 41. The vector of claim 40, wherein the vector is a lentiviral vector.

42. 41. The vector of claim 40, further comprising a marker gene.

43. 43. The vector of claim 42, wherein the marker gene is a transmembrane protein.

44. 44. The vector of claim 43, wherein the transmembrane protein is EGFR.

45. 41. A composition comprising a Treg according to claim 1, a recombinant cytokine receptor according to claim 23, a nucleic acid according to claim 39, or a vector according to claim 40.

46. 41. A method of expanding transduced Treg cells in the absence of exogenous IL-2, comprising introducing the nucleic acid of claim 39 or the vector of claim 40 into Treg cells and culturing the cells.

47. The recombinant cytokine receptor i) does not activate IL-2 signaling in cells that do not contain the recombinant cytokine receptor; ii) does not activate IL-2 signaling in Tregs that do not contain said recombinant cytokine receptor; and / or iii) does not activate signaling of IL-2 receptors containing different amino acid sequences; 47. The method of claim 46.

48. 47. The method of claim 46, wherein the recombinant cytokine receptor does not cause the transduced Treg cells to secrete one or more cytokines at a higher level than Treg cells cultured with exogenous IL-2 cytokine.

49. 47. The method of claim 46, further comprising detecting at least one Treg marker selected from the group consisting of CD4+, CD25+, and CD127lo, and optionally further comprising detecting FOXP3 and / or HELIOS.

50. 47. The method of claim 46, further comprising detecting methylation at Treg-specific differentially regulated genes.

51. 51. The method of claim 50, wherein the Treg-specific differentially regulated gene is FOXP3.

52. The method of claim 46, wherein the Treg cells are capable of dividing and proliferating in the absence of IL-2.

53. 47. The method of claim 46, wherein one or more markers of endogenous IL-2 signaling are detected, and optionally, the one or more markers of endogenous IL-2 signaling comprises phosphorylated STAT-5.

54. 47. The method of claim 46, wherein the method generates Treg cells comprising a recombinant cytokine receptor, i. the in vitro and / or in vivo suppressive activity of the Tregs against CD8+ T cells and / or CD4+ T cells is increased compared to control Tregs not transduced with the recombinant cytokine receptor; ii. the rate of division of CD4+ T cells and / or CD8+ T cells when cultured in the presence of the Tregs is reduced compared to the rate of division of CD4+ T cells and / or CD8+ T cells when cultured without the Tregs; iii. the relative amount of Treg cells in a composition comprising a population of Treg cells transduced with a recombinant cytokine receptor increases over time; iv. a composition comprising a population of Treg cells transduced with said recombinant cytokine receptor expanded without IL-2 contains a similar number of viable cells compared to a composition comprising the same population of Treg cells not transduced with said recombinant cytokine receptor expanded with IL-2; v. at least 80% of the cells in the population of Treg cells transduced with the recombinant cytokine receptor maintain expression of FOXP3 and / or HELIOS for about 14 days post-transduction; vi. at least 80% of the cells in the population of Treg cells transduced with the recombinant cytokine receptor maintain expression of FOXP3 and / or HELIOS for about 23 days post-transduction; vii. the population of Treg cells transduced with the recombinant cytokine receptor expands at least two-fold over the population of the same Treg cells not transduced with the recombinant cytokine receptor; viii. the levels of IL-10 cytokine produced by said Tregs in vitro and / or in vivo are comparable to or increased compared to control Tregs not transduced with said recombinant cytokine receptor; ix. the IFN-γ cytokine levels produced by said Tregs in vitro and / or in vivo are comparable to or increased compared to control Tregs not transduced with said recombinant cytokine receptor; x. the levels of Gr-B cytokine produced by said Tregs in vitro and / or in vivo are comparable to or increased compared to control Tregs not transduced with said recombinant cytokine receptor; xi. the Tregs are functionally reactivated in vivo and / or in vitro to levels greater than control Tregs not transduced with the recombinant cytokine receptor; and / or xii. The level of Treg proliferation increases one or more times in vitro and / or in vivo following restimulation; The method.

55. 47. The method of claim 46, further comprising culturing the Treg cells in a composition comprising a cytokine other than IL-2.

56. 47. The method of claim 46, wherein said population of Treg cells transduced with said recombinant cytokine receptor is expanded at least two-fold, and wherein said population of Treg cells transduced with said recombinant cytokine receptor maintains expression of at least one Treg marker selected from the group consisting of CD25, FOXP3, and HELIOS.

57. 20. The method of claim 19, wherein the individual expresses IL-2.

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