Recombinant cytokine receptors and methods of use thereof

Abstract

Provided are recombinant cytokine receptors comprising an IL-2 intracellular domain and an extracellular domain that binds to a cytokine other than IL-2. Also provided herein are Treg cells comprising recombinant cytokine receptors and methods of use thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 368,085 filed on July 11, 2022, U.S. Provisional Application No. 63 / 498,802 filed on April 27, 2023, and U.S. Provisional Application No. 63 / 499,929 filed on May 3, 2023, the contents of each of which are hereby incorporated by reference in their entirety.

[0003] Sequence Listing Incorporated by Reference

[0004] The contents of the electronic sequence listing (237752000241SEQLIST.xml; size: 36,642 bytes; creation date: July 7, 2023) are incorporated herein by reference in their entirety. Background Art

[0005] The immune system plays a key role in maintaining the body's homeostasis, maintaining a balance between the elimination of foreign antigens and the self-tolerance of self-antigens. Specifically, overactive immune dysregulation may lead to various autoimmune disorders (e.g., irritable bowel syndrome, systemic lupus erythematosus, alopecia areata, multiple sclerosis), which is usually the result of overactive effector T lymphocytes or insufficient activity of regulatory T lymphocytes (i.e., Treg). Current therapies for autoimmune diseases include the administration of steroids, which may cause serious side effects in patients and generally provide little relief.

[0006] Regulatory T cells play a key role in maintaining body homeostasis to prevent other healthy tissues from being destroyed. Treg is a unique subset of T cells that inhibits the cytotoxic or pro-inflammatory activity of effector CD4+ or effector CD8+ T cells. After key Treg genes, especially CD25 and FOXP3 are upregulated, Treg differentiates from the parental T lymphocyte lineage (see, e.g., Chen, ML et al. (2005), Proc Natl Acad Sci USA 102(2):419-424; and Liu, VC et al. (2007), J Immunol 178(5):2883-2892, hereby incorporated by reference in their entirety). After T cell receptor (TCR) activation, these Tregs are responsible for directly inhibiting 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, hereby incorporated by reference in its entirety) and engagement of immune checkpoint receptors (e.g., TIGIT or CTLA-4 engagement) (see, e.g., Knochelmann, HM et al. (2018), Cell Mol Immunol; 15(5): 458-469, hereby incorporated by reference in its entirety). Although effector T cells are able to produce cytokine IL-2 to support self-expansion after TCR activation, Treg cells rely on exogenous IL-2 to promote Treg survival and maintenance because Treg cannot produce their own IL-2. However, like all T lymphocyte lineages, Treg cells require IL-2 signaling for survival and proliferation. This biological mechanism ensures that Tregs are maintained in a tissue niche rich in active effector T cells, thereby creating a cellular negative feedback mechanism, thereby promoting Treg expansion through increased IL-2 production by target effector T cells, which is then used to inhibit effector T cell activity, thereby inhibiting its own expansion and survival downstream (see, e.g., Shevyrev, D & Tereshchenko, V (2020), Front Immunol; 10: 3100, 1-13). Therefore, targeting the IL-2 receptor signaling pathway in Tregs is one of the target areas recently devoted to identifying new therapies for autoimmune disorders. Summary of the invention

[0007] In one aspect, the present application relates to a recombinant cytokine receptor comprising an intracellular domain of IL-2Rβ and an extracellular domain from a cytokine other than IL-2. Also provided herein are cells expressing the recombinant cytokine receptor and methods of using the same. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1AThe organization of the DNA cassette encoding the untethered cytokine receptor is shown. The black arrow indicates the direction of transcription. MND represents the promoter region. The recombinant cytokine receptor comprises the extracellular domain and transmembrane domain of the IL receptor (represented by ILXR, where X is 2, 4, 7, 9 or 21; thus, the IL extracellular domain and transmembrane domain can be from any one of IL-2, IL-4, IL-7, IL-9 and IL-21) and the IL-2Rβ intracellular domain. The construct also comprises the P2A self-cleaving peptide. EGFR is an epidermal growth factor receptor transduction marker. Figure 1B Untethered recombinant receptor protein constructs are shown. Figure 1C The organization of the DNA cassette encoding the tethered cytokine receptor is shown. The black arrow indicates the direction of transcription. MND represents the promoter region. Figure 1D Tethered protein constructs are shown. Figure 1E Schematic representations of exemplary recombinant cytokine receptors are shown, which can be untethered or tethered to a cytokine.

[0009] Figure 2A The extent of secretory embryonic alkaline phosphatase (SEAP) protein production and secretion by HEKBlue cells expressing untethered recombinant cytokine receptors is shown. HEKBlue cells express an IL-2 signaling reporter construct. Cells were cultured in the presence or absence of IL-2 or the following doses of the indicated cytokines: IL-4: 0.4 ng / mL and 4 ng / mL ("10X"); IL-7: 1 ng / mL and 10 ng / mL ("10X"); IL-9: 2 ng / mL and 20 ng / mL ("10X"); and IL-21: 5 ng / mL and 50 ng / mL ("10X"). Figure 2B Shown are the extent of SEAP production and secretion by HEKBlue cells expressing tethered recombinant cytokine receptors. Figure 2C Shown is a dose response curve based on the absorbance values ​​of SEAP produced by HEKBlue cells carrying an IL-2 reporter construct when cultured under increasing doses of IL-2. Figure 2D Schematic diagram of the tethered recombinant cytokine receptor transactivation assay is shown. Jurkat T cells expressing tethered recombinant cytokine receptors were co-cultured overnight with IL-2 reporter cells expressing an untethered form of the recombinant cytokine receptor. Figure 2EShown is the absorbance readout of SEAP production by IL-2 reporter cells expressing untethered recombinant cytokine receptors (IL-4Rα / IL-2Rβ, IL-7Rα / IL-2Rβ, IL-9Rα / IL-2Rβ, or IL-21Rα / IL-2Rβ) co-cultured with Jurkat T cells expressing the same tethered recombinant cytokine receptor construct as the untethered IL-XRα / IL-2Rβ in the IL-2 reporter cells, as Figure 2D Painted. Figure 2F Shown are representative FACS plots of IL-9Rα levels on control Tregs compared to Tregs expressing IL-9Rα / IL-2Rβ recombinant cytokines (“IL9 / 2”).

[0010] Figure 3A Representative fluorescence activated cell sorting (FACS) images of regulatory T cells (Treg cells) that successfully expressed one of the tethered or untethered IL-4Rα / IL-2Rβ at day 7 and day 12 after transduction are shown. Cells were stained with EGFR antibodies to identify EGFR+ cells and then sorted. The flow cytometry standard (FCS) is forward scatter, and EGFR is gated on side scatter. The boxes mark the cell populations of EGFR+ or EGFR-. The percentage of the cell populations of EGFR+ or EGFR- is shown. Figure 3B Shown are representative FACS plots of Treg cells successfully expressing either tethered or untethered IL-7Rα / IL-2Rβ at days 7 and 12 post-transduction. Figure 3C Representative FACS plots of Treg cells successfully expressing tethered or untethered IL-21Rα / IL-2Rβ or IL-9Rα / IL-2Rβ at days 7 and 12 after transduction are shown. Figure 3D Shown are representative FACS plots of Treg cells successfully expressing tethered or untethered IL-4Rα / IL-2Rβ when cultured in the presence of IL-4 cytokine at days 7 and 12 post-transduction. Figure 3E Shown are FACS plots of Treg cells successfully expressing tethered or untethered IL-7Rα / IL-2Rβ when cultured in the presence of IL-7 cytokine at days 7 and 12 post-transduction. Figure 3F Shown are representative FACS plots of regulatory T cells (Treg cells) that successfully expressed tethered or untethered IL-9Rα / IL-2Rβ when cultured in the presence of IL-9 cytokine at days 7 and 12 post-transduction. Figure 3GShown are representative FACS plots of Treg cells that successfully expressed tethered or untethered IL-21Rα / IL-2Rβ when cultured in the presence of IL-21 cytokine at days 7 and 12 post-transduction. Figure 3H Shown are representative FACS plots of Treg cells successfully expressing tethered or untethered IL-7Rα / IL-2Rβ when cultured in the presence of IL-2 cytokine at days 7 and 12 post-transduction. Fig. 3I Shown are representative FACS plots of Treg cells successfully expressing untethered IL-4Rα / IL-2Rβ, IL-9Rα / IL-2Rβ, or IL-21Rα / IL-2Rβ when cultured in the presence of IL-2 cytokine at days 7 and 12 post-transduction.

[0011] Figure 4A Line graphs representing the percentage of EGFR+Treg cells expressing tethered or untethered IL-7Rα / IL-2Rβ recombinant cytokine receptors at days 7 and 13 when cultured in the absence of cytokines are shown. A construct transfected with EGFR was used as a positive control (EGFR). UT stands for untransduced Tregs. Figure 4B Shown are line graphs representing the percentage of EGFR+ Treg cells expressing tethered or untethered IL-7Rα / IL-2Rβ recombinant cytokine receptors at days 7 and 13 when cultured in the presence of IL-7 cytokine. Figure 4C Shown are line graphs representing the percentage of EGFR+ Treg cells expressing tethered or untethered IL-7Rα / IL-2Rβ recombinant cytokine receptors at days 7 and 13 when cultured in the presence of IL-2 cytokine.

[0012] Figure 5A Line graphs representing the percentage of live Treg cells expressing tethered or untethered IL-7Rα / IL-2Rβ recombinant cytokine receptors when cultured in the absence of cytokines at days 2, 5, 7, 9, 12, and 14 are shown. A construct transfected with EGFR was used as a positive control (EGFR). UT represents untransduced Treg cells. Figure 5B Line graphs representing the percentage of live Treg cells expressing tethered or untethered IL-7Rα / IL-2Rβ recombinant cytokine receptors when cultured in the presence of IL-7 cytokine are shown at days 2, 5, 7, 9, 12, and 14. A construct transfected with EGFR was used as a positive control (EGFR). UT represents untransduced Treg cells. Figure 5CShown are line graphs representing the percentage of live Treg cells expressing tethered or untethered IL-7Rα / IL-2Rβ recombinant cytokine receptors on days 2, 5, 7, 9, 12, and 14 when cultured in the presence of IL-2 cytokine. Figure 5D Shown are line graphs representing the percentage of live Treg cells expressing tethered or untethered IL-4Rα / IL-2Rβ, IL-9Rα / IL-2Rβ, or IL-21Rα / IL-2Rβ recombinant cytokine receptors on days 5, 7, 9, 12, and 14 when cultured in the absence of cytokines. Figure 5E Shown are line graphs representing the percentage of live Treg cells expressing tethered or untethered IL-4Rα / IL-2Rβ, IL-9Rα / IL-2Rβ, or IL-21Rα / IL-2Rβ recombinant cytokine receptors on days 5, 7, 9, 12, and 14 when cultured in the presence of IL-2 cytokine. Fig. 5F Shown are line graphs representing the percentage of live Treg cells expressing tethered or untethered IL-4Rα / IL-2Rβ recombinant cytokine receptors on days 5, 7, 9, 12, and 14 when cultured in the presence of IL-4 cytokine. Figure 5G Shown are line graphs representing the percentage of live Treg cells expressing tethered or untethered IL-9Rα / IL-2Rβ recombinant cytokine receptors on days 5, 7, 9, 12, and 14 when cultured in the presence of IL-9 cytokine. Figure 5H Shown are line graphs representing the percentage of live Treg cells expressing tethered or untethered IL-21Rα / IL-2Rβ recombinant cytokine receptors on days 5, 7, 9, 12, and 14 when cultured in the presence of IL-21 cytokine.

[0013] Fig. 6A Line graphs representing the number of Treg cells expressing tethered or untethered IL-7Rα / IL-2Rβ recombinant cytokine receptors when cultured in the absence of cytokines at days 2, 5, 7, 9, 12, and 14 are shown. A construct transfected with EGFR was used as a positive control (EGFR). UT represents untransduced Treg cells. Figure 6B Shown are line graphs representing the number of Treg cells expressing tethered or untethered IL-7Rα / IL-2Rβ recombinant cytokine receptors on days 2, 5, 7, 9, 12, and 14 when cultured in the presence of IL-7 cytokine. Figure 6CShown are line graphs representing the number of Treg cells expressing tethered or untethered IL-7Rα / IL-2Rβ recombinant cytokine receptors on days 2, 5, 7, 9, 12, and 14 when cultured in the presence of IL-2 cytokine. Fig.6D Shown are line graphs representing the number of Treg cells expressing tethered or untethered IL-4Rα / IL-2Rβ, IL-9Rα / IL-2Rβ, or IL-21Rα / IL-2Rβ recombinant cytokine receptors on days 2, 5, 7, 9, 12, and 14 when cultured in the absence of cytokines. Fig. 6E Shown are line graphs representing the number of Treg cells expressing tethered or untethered IL-4Rα / IL-2Rβ, IL-9Rα / IL-2Rβ, or IL-21Rα / IL-2Rβ recombinant cytokine receptors on days 2, 5, 7, 9, 12, and 14 when cultured in the presence of IL-2 cytokine. Fig. 6F Shown are line graphs representing the number of Treg cells expressing tethered or untethered IL-4Rα / IL-2Rβ recombinant cytokine receptors on days 2, 5, 7, 9, 12, and 14 when cultured in the presence of IL-4 cytokine. Figure 6G Shown are line graphs representing the number of Treg cells expressing tethered or untethered IL-9Rα / IL-2Rβ recombinant cytokine receptors on days 2, 5, 7, 9, 12, and 14 when cultured in the presence of IL-9 cytokine. Figure 6H Shown are line graphs representing the number of Treg cells expressing tethered or untethered IL-21Rα / IL-2Rβ recombinant cytokine receptors on days 2, 5, 7, 9, 12, and 14 when cultured in the presence of IL-21 cytokine. Fig.6I Shown are bar graphs representing the in vitro cell expansion fold of control Tregs cultured in the presence of IL-2 cytokine, Tregs expressing tethered IL-9Rα / IL-2Rβ recombinant cytokine receptors cultured in the presence of IL-2 (“IL-9SR”), and Tregs expressing tethered IL-9Rα / IL-2Rβ recombinant cytokine receptors cultured in the absence of IL-2. Figure 6J Provided are line graphs showing the fold expansion over time of control Tregs and Tregs expressing tethered IL-9Rα / IL-2Rβ recombinant cytokine receptors (“IL-9STRs”) cultured in the presence (left panel) or in the absence (right panel) of IL-2.

[0014] Figures 7A-7R Shown are the expression levels of Treg markers, particularly as identified by flow cytometry. Figures 7A-7C Figure 2 shows FOXP3 expression in Tregs expressing IL-7Rα / IL-2Rβ recombinant cytokine receptors, where FOXP3 expression was detected by FACS analysis in the absence of cytokines ( Fig. 7A ) and IL-7 ( Figure 7B ) or with IL-2( Figure 7C ) Average FOXP3 expression in control Tregs and recombinant Tregs cultured together. Figure 7D-7F CD25 expression in Tregs expressing IL-7Rα / IL-2Rβ recombinant cytokine receptors is shown, where CD25 expression in Tregs expressing IL-7Rα / IL-2Rβ recombinant cytokine receptors was detected by FACS analysis in the absence of cytokines ( Fig.7D ) and IL-7 ( Fig. 7E ) or with IL-2( Figure 7F ) Mean CD25 expression in control Tregs and recombinant Tregs cultured with IL-2. Dashed lines indicate baseline mean FOXP3 or CD25 expression levels in control Tregs cultured with IL-2. Figure 7G Shown is CD71 expression in Tregs expressing tethered or untethered IL-7Rα / IL-2Rβ recombinant cytokine receptors cultured in the absence of cytokines, with IL-7, or with IL-2. Figure 7H Shown is the co-expression of HELIOS and FOXP3 in Tregs expressing tethered IL-7Rα / IL-2Rβ recombinant cytokine receptors at days 7 and 12 when Tregs were cultured in the absence of cytokines. Fig.7I Shown is the co-expression of HELIOS and FOXP3 in Tregs expressing tethered IL-7Rα / IL-2Rβ recombinant cytokine receptors when Tregs were cultured with IL-2 at days 7 and 12. Figures 7J-7K Figure 2 shows the expression of tethered or untethered IL-4Rα / IL-2Rβ ( Figure 7J ) or expressing tethered or untethered IL-9Rα / IL-2Rβ or tethered or untethered IL-21Rα / IL-2Rβ ( Figure 7K ) HELIOS and FOXP3 co-expression in Tregs expressing recombinant cytokine receptors. Figure 7L-7M Figure 2 shows the expression of tethered or untethered IL-4Rα / IL-2Rβ ( Figure 7L ) or expressing tethered or untethered IL-9Rα / IL-2Rβ or tethered or untethered IL-21Rα / IL-2Rβ ( Figure 7M) HELIOS and FOXP3 co-expression in Tregs expressing recombinant cytokine receptors. Figure 7N Shown is the co-expression of HELIOS and FOXP3 in Tregs expressing tethered or untethered IL-4Rα / IL-2Rβ recombinant cytokine receptors cultured with IL-4 at days 7 and 12. Fig.7O Shown is the co-expression of HELIOS and FOXP3 in Tregs expressing tethered or untethered IL-9Rα / IL-2Rβ recombinant cytokine receptors cultured with IL-9 at days 7 and 12. Figure 7P Shown is the co-expression of HELIOS and FOXP3 in Tregs expressing tethered or untethered IL-21Rα / IL-2Rβ recombinant cytokine receptors cultured with IL-21 at days 7 and 12. Figure 7Q Shown is a bar graph representation of FOXP3 mean fluorescence intensity (MFI) as measured by flow cytometry in EGFR-negative and EGFR-positive cells from control Tregs and Tregs expressing tethered IL-9Rα / IL-2Rβ recombinant cytokine receptors (“IL-9TSR”) cultured in the presence or absence of IL-2. Figure 7R Provided are bar graphs representing the quantification of percent methylation at the TSDR FOXP3 locus in conventional CD4+ T cells, control Tregs, and Tregs expressing tethered IL-9Rα / IL-2Rβ recombinant cytokine receptors ("tethered SRs").

[0015] Figures 8A-8M Representative histograms and bar graphs showing the extent of STAT5 phosphorylation (pSTAT5) in Tregs expressing recombinant cytokine receptors are shown. Phosphorylation in the absence of cytokines ( Fig. 8A ) and IL-7 ( Figure 8B ) or with IL-2( Figure 8C ) and Tregs expressing tethered or untethered IL-7Rα / IL-2Rβ recombinant cytokine receptors, as well as in control Tregs ( Fig.8D ), in Tregs expressing IL-7-tethered IL-7Rα / IL-2Rβ recombinant cytokine receptors in the absence of cytokines ( Fig. 8E ) or in Tregs expressing IL-7Rα / IL-2Rβ recombinant cytokine receptors in the presence of IL-7 ( Figure 8F ) are presented as pSTAT5 levels. Figure 8GShown are representative histograms of pSTAT5 levels in response to cognate cytokine stimulation of control Tregs compared to Tregs expressing untethered IL-4Rα / IL-2Rβ, IL-9Rα / IL-2Rβ, or IL-21Rα / IL-2Rβ cytokine receptors. Figure 8H Shown are representative histograms of pSTAT5 levels in the absence of cytokine stimulation of control Tregs compared to Tregs expressing tethered IL-4Rα / IL-2Rβ, IL-9Rα / IL-2Rβ, or IL-21Rα / IL-2Rβ cytokine receptors. Figures 8I-8K Representative histograms of pSTAT5 levels from EGFR-positive cells of control Tregs and Tregs expressing tethered IL-9Rα / IL-2Rβ cytokine receptors (“IL-9SR”) in the presence or absence of IL-2 are provided ( Fig.8I and 8K ) and bar charts ( Figure 8J ). Figure 8L-8M Representative histograms of pSTAT5 levels from EGFR-negative cells of control Tregs and Tregs expressing tethered IL-9Rα / IL-2Rβ cytokine receptors (“IL-9SR”) in the presence or absence of IL-2 are provided ( Figure 8L ) and bar charts ( Figure 8M ).

[0016] Figures 9A-9D The results show that the expression of IL-4Rα / IL-2Rβ ( Fig.9A )、IL-7Rα / IL-2Rβ( Fig. 9B )、IL-9Rα / IL-2Rβ( Fig. 9C ) and IL-21Rα / IL-2Rβ( Fig.9D ) Cognate cytokine dose dependence of pSTAT5 levels in Tregs expressing recombinant cytokine receptors.

[0017] Figures 10A-10D It is shown that in the absence of cytokines ( Fig. 10A )、0.156U IL-2( Fig. 10B )、2.5U IL-2( Fig. 10C ) or 10U IL-2 ( Fig. 10DFigure IL-2 dose-dependency of pSTAT5 levels in control Tregs (EGFR-Tregs) and Tregs expressing IL-7-tethered IL-7Rα / IL-2Rβ recombinant cytokine receptors (EGFR+Tregs) after 40 min of stimulation with EGFR.

[0018] Fig.11A and 11B Shown are the suppressive activities of Tregs expressing IL-7-tethered IL-7Rα / IL-2Rβ recombinant cytokine receptors on effector CD4+ T cells and effector CD8+ T cells, respectively. Fig. 11C and 11D Shown are the suppressive activities of Tregs expressing IL-9 tethered IL-9Rα / IL-2Rβ recombinant cytokine receptors (“IL-9TSR”) on effector CD4+ T cells and effector CD8+ T cells, respectively. Fig.11E and 11F Comparison of the suppressive activity of Tregs expressing (i) IL-4 tethered IL-4Rα / IL-2Rβ recombinant cytokine receptor ("tethered 4 / 2"), (ii) IL-7 tethered IL-7Rα / IL-2Rβ recombinant cytokine receptor ("tethered 7 / 2"), or (iii) IL-9 tethered IL-9Rα / IL-2Rβ recombinant cytokine receptor ("tethered 9 / 2") on effector CD4+ T cells and effector CD8+ T cells, respectively, is shown. Suppressive activity was calculated as the percentage of non-dividing effector T cells at the specified cell ratio in PBMC / Treg co-cultures. Control Tregs were incubated with IL-2; Tregs expressing tethered IL-7Rα / IL-2Rβ or IL-9Rα / IL-2Rβ recombinant cytokine receptors were incubated without cytokines.

[0019] Figures 12A-12D Shown are the levels of Treg lineage and activation markers, including FOXP3 ( Fig. 12A )、CD25( Fig. 12B )、CTLA4( Fig. 12C ) and GARP( Fig.12D ).

[0020] Fig.13A A schematic diagram of a Treg (re)stimulation experiment testing Treg responses to anti-CD3 / anti-CD28 antibodies versus CD19 antigen stimulation in vitro is provided. Fig. 13BShown is the proliferation of control Tregs and Tregs expressing tethered IL-9Rα / IL-2Rβ recombinant cytokine receptors (“IL-9SR Tregs”) cultured in vitro in the presence or absence of IL-2 over time.

[0021] Figures 14A-14C Shows the use of Fig.13A Mean fluorescence intensity (MFI) over time of markers of Treg proliferation, activation or exhaustion, such as CD71 ( Fig.14A )、ICOS( Fig. 14B ) and PD-1( Fig. 14C ).

[0022] Figures 15A-15C The percentage of Tregs producing cytokines, such as IL-10 (IL-10) and IL-2Rβ (IL-2Rβ) on days 2 and 19 is shown for control Tregs and Tregs expressing tethered IL-9Rα / IL-2Rβ recombinant cytokine receptors (“IL9 SR”) cultured in vitro with IL-2. Fig.15A ), IFN-γ( Fig. 15B ) and GrB( Fig. 15C ).

[0023] Fig.16A Shown are the numbers of Tregs engrafted and expanded in vivo in the spleen, lung, and liver in an immunocompromised NSG mouse model of (i) control Tregs, (ii) Tregs expressing tethered IL-4Rα / IL-2Rβ recombinant cytokine receptors ("Tethered 4 / 2"), (iii) Tregs expressing tethered IL-7Rα / IL-2Rβ recombinant cytokine receptors ("Tethered 7 / 2"), or (iv) Tregs expressing tethered IL-9Rα / IL-2Rβ recombinant cytokine receptors ("Tethered 9 / 2"). Fig. 16B Shown are the numbers of Tregs engrafted and expanded in vivo in the blood, spleen, lung, and liver of control Tregs and Tregs expressing tethered IL-9Rα / IL-2Rβ recombinant cytokine receptors (“IL-9TSR”) in an immunocompromised NSG mouse model. Fig. 16C Shown are cell counts over time for control Tregs and Tregs expressing tethered IL-9Rα / IL-2Rβ recombinant cytokine receptors (IL-9TSR") cultured in the presence (left) or absence (right) of IL-2. Fig.13A Cells were stimulated on days 0, 7, and 14 as indicated in FIG. Fig.16D (picture) and Fig.16E(FACS graph) shows the number of human immune cells circulating in the blood 15 days after adoptive cell transfer in the presence versus absence of exogenous IL-2 and the percentage of those immune cells that are Treg cells (e.g., "CD19 CAR+IL9SR" or "CAR+IL-9SR").

[0024] Fig.17 The in vivo suppressive activity of Tregs expressing IL-9 tethered IL-9Rα / IL-2Rβ recombinant cytokine receptors (“IL-9SR Tregs”) in a humanized mouse model of graft-versus-host disease (GVHD) is shown. PBMC, peripheral blood mononuclear cells; 5M, 5 million (5×10 6 ).

[0025] Fig.18 Shown is the progression of EAE scores over time in control mice and mice with constitutively active STAT5b (CA-STAT5b) in FOXP3-expressing cells (ie, Tregs) as an indicator of the severity of neurological autoimmunity (ie, multiple sclerosis).

[0026] Figures 19A-19B The fold expansion of wild-type (WT) Tregs with or without IL-2 and CA-STAT5b Tregs without IL-2 are shown as bar graphs ( Fig.19A ) and FACS plots ( Fig.19B ). Figures 19C-19D It was shown that in vitro ( Fig.19C ) and in vivo graft-versus-host disease (GVHD) mouse models ( Fig.19D ) The suppressive activity of WT Treg and CA-STAT5b Treg.

[0027] Figures 20A-20B The persistence and expansion of IL-2 secreting Tregs are shown. Fig. 20A Shown are the percentages of IL-2 secreting Tregs that persist over time when cultured with or without IL-2. Fig. 20B Shown are the percentages of IL-2 secreting Tregs that expanded when cultured with (lower panel) or without (upper panel) IL-2.

[0028] Fig.21A Shown are cell counts over time for control Tregs and Tregs expressing an IL-2 tethered protein tag cultured with or without IL-2. Fig. 21B Shown is FACS analysis of IL-2 and EGFR protein signature levels over time in Tregs expressing IL-2-tethered EGFR grown in vitro in the absence of IL-2. Fig. 21CProvided is a bar graph showing the amount of soluble IL-2 accumulated over time in the culture medium of Tregs expressing an IL-2 tethered protein tag grown in vitro in the absence of IL-2.

[0029] Fig. 22 Heatmaps of RNA sequencing analysis performed on wild-type Tregs and CA-STAT5b Tregs are shown. 278 differentially expressed genes (DEGs) were identified, 99 of which were downregulated and 179 were upregulated in CA-STAT5b Tregs compared to wild-type Tregs.

[0030] Fig.23A Schematic representation of an additional exemplary recombinant cytokine receptor is shown, wherein the recombinant cytokine receptor comprises an IL-9 cytokine tethered to a receptor having an IL-9Rα extracellular domain (ED) and an IL-2Rβ transmembrane (TM) and intracellular (ID) domain ("IL9TSR(IL2RTM)"; right), which is tethered to an exemplary recombinant cytokine receptor having an IL-9Rα ED and TD and an IL-2Rβ ID ("IL9TSR(IL9RTM)", left, previously described in Figure 1E ) for comparison. Fig. 23B Line graphs representing the fold expansion of Treg cells expressing only EGFR (control Treg), IL9TSR (IL9RTM) construct (also referred to as "T 9 / 2 (OG)" or "IL9TSR") and IL9TSR (IL2RTM) (also referred to as "IL-2TM", "IL9TSR (IL2TM)" or "T9 / 2TSR (IL2RTM)") over time are shown. The fold expansion of cells cultured with (upper graph) or without (lower graph) exogenous IL-2 was measured over time. Fig.23C Shown are FACS plots comparing cell size (i.e., SCC-H, side scatter height) and EGFR to assess the survival of Treg cells expressing EGFR at days 7 and 14 post-transduction as readout for recombinant cytokine receptor constructs for Treg cells expressing EGFR only, IL9TSR(IL9RTM) construct (i.e., “IL9TSR”), and IL9TSR(IL2RTM) construct (i.e., “IL9TSR(IL2TM)”). Fig.23DShown are bar graphs representing the mean fluorescence intensity (MFI) of the Treg marker FoxP3 as calculated and analyzed by FACS and flow cytometry of Treg cells expressing EGFR only (cultured with and without exogenous IL-2), IL9TSR (IL9RTM) construct (i.e., "OG"; cultured without exogenous IL-2), and IL9TSR (IL2RTM) construct (i.e., "IL-2TM"; cultured without exogenous IL-2) at day 7 (upper panel) and day 14 (lower panel) post-transduction. Fig.23E Shown are the percentage and distribution of Treg cells expressing phosphorylated STAT5 on day 15 in cultures of Treg cells expressing EGFR alone (pulsed with and without exogenous IL-2 as pSTAT5 positive and negative controls, respectively), IL9TSR (IL9RTM) construct (pulsed without exogenous IL-2), and IL9TSR (IL2RTM) construct (pulsed without exogenous IL-2). DETAILED DESCRIPTION

[0031] Successful adoptive cell therapy requires robust expansion and persistence of the cells administered, and the environmental signals received by the cells greatly contribute to these behaviors. In some embodiments, a system is provided herein, wherein the proliferation of immune cells (e.g., T cells) and their persistence in vivo, the production of immune activation cytokines and immune function can be enhanced by introducing recombinant cytokine receptors. These recombinant cells can be used as cell therapies, such as autoimmune disorders. The present invention generally relates to the field of immunology, and partly to compositions and methods for growing, modifying and amplifying cells, wherein the cells include, for example, recombinant cytokine receptors that allow immune cells (such as Treg cells) to proliferate in the absence of IL-2. Recombinant cytokine receptors for activating IL-2Rβ signaling using alternative cytokines (e.g., in addition to IL-2) are provided herein. Recombinant cytokine receptors that are not dependent on exogenous cytokines for activation are also provided herein. Therefore, cells transduced with recombinant cytokine receptors described herein have certain advantages compared to cells that have not been transduced, including IL-2 independence. In some embodiments of the methods and compositions provided herein, cells are engineered to include recombinant cytokine receptors as described herein. The recombinant cytokine receptors described herein provide stimulatory cytokine signals to cells and improve the efficacy of cell therapy.In some embodiments, the cell is a T cell, such as a Treg.

[0032] Cells (e.g., Tregs) containing these recombinant cytokine receptors can still be activated, for example, by canonical TCR activation in the presence of a target antigen to inhibit conventional T cells. Therefore, Tregs cannot inhibit off-target T cells or produce inhibitory cytokines in the absence of their target T cells. Due to the continued suppression of conventional T cells, this off-target activity may lead to an increased risk of chronic infection or cancer progression. Therefore, in some embodiments, engineered Treg cells have fewer off-target effects and side effects compared to alternatives.

[0033] Each recombinant cytokine receptor provided herein shows a similar trend, although to different degrees. For example, exemplary recombinant cytokine receptors tested herein include: untethered IL-4Rα / IL-2Rβ, IL-7Rα / IL-2Rβ, IL-9Rα / IL-2Rβ and IL-21Rα / IL-2Rβ recombinant cytokine receptors, each of which opens intracellular IL-2 signaling (e.g., when transduced into Treg) after binding to the corresponding cognate ligand (i.e., IL-4, IL-7, IL-9 or IL-21, respectively); and cytokine-tethered IL-4Rα / IL-2Rβ, IL-7Rα / IL-2Rβ, IL-9Rα / IL-2Rβ and IL-21Rα / IL-2Rβ recombinant cytokine receptors, wherein intracellular IL-2 signaling is constitutively activated. In vitro analyses demonstrated varying degrees of Treg survival, proliferation, or function (e.g., both tethered and untethered IL-21Rα / IL-2Rβ recombinant cytokine receptors showed the lowest proliferation rate and earliest decline in Treg in vitro survival of the eight receptors tested). Further in vivo experiments demonstrated that tethered IL-9Rα / IL-2Rβ recombinant cytokine receptors conferred the highest degree of Treg engraftment, survival, and proliferation, even though both tethered IL-4Rα / IL-2Rβ and IL-7Rα / IL-2Rβ recombinant cytokine receptors showed varying degrees of in vivo engraftment, survival, and proliferation in the receptors tested and in the tissues tested (see Fig.16A ). Therefore, the in vitro and in vivo functional characteristics of recombinant cytokine receptors are surprising, and these differences can be exploited to address the biological constraints of different diseases where Treg targeting may prove beneficial for therapeutic purposes.

[0034] In addition, various proposed methods for activating the IL-2 signaling pathway in Treg have failed to show functional success. See Example 9, in which three separate methods all failed to support Treg function: constitutively active STAT-5Treg model, IL-2 secretion Treg model, and IL-2 tethered tag protein Treg model all failed to promote the activity of Treg against conventional T cells, thereby proving that not every method for promoting IL-2 independence can succeed when applied to Treg cells. In addition, compared with conventional T cells, it is known that Treg cells are very different in function, molecular characteristics, and genetic characteristics (for example, see 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 hereby incorporated by reference as a whole), so Treg-specific customization methods are needed, as described herein.

[0035] All publications, including patent documents, scientific articles, and databases, mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. If definitions set forth herein are contrary to or inconsistent with definitions set forth in patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over those set forth herein by reference.

[0036] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0037] I. Recombinant Cytokine Receptors

[0038] 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 an extracellular cytokine binding domain (ED; also referred to as an "extracellular cytokine receptor domain"), a transmembrane domain (TD), and an intracellular cytokine signaling domain (ID; also referred to as an "intracellular domain"). In some embodiments, provided herein is a receptor comprising an ED tethered to its cognate cytokine, so that the receptor can be activated in the absence of any exogenous cytokine. The recombinant cytokine receptor described herein is derived from an interleukin cytokine receptor, wherein the ID includes the ID of the interleukin-2 receptor β (IL-2Rβ) chain of IL-2R. Canonical IL-2 signaling causes STAT5 phosphorylation, thereby activating STAT5 nuclear translocation and initiating target gene transcription. In some cells, such as T cells, including Treg cells, IL-2 signaling is essential for cell survival and cell proliferation. Thus, a recombinant cytokine receptor comprising the IL-2Rβ intracellular domain that binds to a cytokine other than IL-2 is able to activate cell survival and proliferation in the absence of IL-2 by binding to another cytokine.

[0039] In some embodiments, the recombinant cytokine receptors described herein are described for an extracellular domain and an intracellular domain. Thus, for example, an "IL-4Rα / IL-2Rβ" receptor comprises an IL-4 receptor extracellular domain connected to an IL-2Rβ intracellular domain. In some embodiments, the receptor comprises a transmembrane domain from a cytokine receptor that is the same as the extracellular domain. In some embodiments, the receptor comprises a transmembrane domain from a cytokine that is different from the extracellular domain.

[0040] In some embodiments, the recombinant cytokine receptor is more effective for maintaining immune cell persistence. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a Treg.

[0041] In some embodiments, compared with other strategies for producing IL-2 independent Treg (e.g., such as engineered to secrete IL-2 Treg), recombinant cytokine receptors are more effective for maintaining Treg persistence. In some embodiments, compared with IL-2 secreting Treg cells, cells transduced with recombinant cytokine receptors provided herein can maintain Treg phenotypes and / or have increased persistence.

[0042] 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 Treg survival and proliferation in the absence of IL-2; (3) increased IL-2 receptor signaling through STAT5 phosphorylation; and (4) the ability to suppress effector T cells in the absence of IL-2 to about or approximately the same extent as wild-type Treg cells grown in the presence of IL-2.

[0043] Table 1. Untethered recombinant cytokine receptor sequences

[0044]

[0045] Table 2. Tethered recombinant cytokine receptor sequences

[0046]

[0047] Provided herein is a recombinant cytokine receptor comprising a cytokine receptor ED and TD, an ID of interleukin-2 receptor β (IL-2Rβ), wherein the ED does not bind to 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 interleukin ED, TD, and ID. In some embodiments, the ED binds to a cytokine other than IL-2. Therefore, in some embodiments, a recombinant cytokine receptor is provided, comprising: (I) ED of IL-4Rα, IL-7Rα, IL-9Rα, or IL-21Rα; (II) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα, or IL-21Rα; (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence shown in SEQ ID NO: 1-5. 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 shown in SEQ ID NOs: 1-5. In some embodiments, the recombinant cytokine receptor comprises an ED 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 shown in SEQ ID NOs: 11-14.

[0048] In some embodiments, the recombinant cytokine receptor comprises (I) the ED of IL-4Rα; (II) the TD of IL-4Rα; and (III) the ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the recombinant cytokine receptor comprises an ED 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 with the amino acid sequence shown in SEQ ID NO:11. In some embodiments, the recombinant cytokine receptor comprises: (I) an ED 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 of SEQ ID NO: 11; (II) a TD 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 of SEQ ID NO: 16; and (III) an ID 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 of SEQ ID NO: 20. In some embodiments, the recombinant cytokine receptor comprises (I) an ED comprising the amino acid sequence of SEQ ID NO:11; (II) a TD comprising the amino acid sequence of SEQ ID NO:16; and (III) an ID comprising the amino acid sequence of SEQ ID NO:20.

[0049] In some embodiments, the recombinant cytokine receptor comprises (I) ED of IL-4Rα; (II) TD of IL-2Rβ; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises an ED 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 shown in SEQ ID NO: 11. In some embodiments, the recombinant cytokine receptor comprises: (I) an ED 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 of SEQ ID NO: 11; (II) a TD 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 of SEQ ID NO: 21; and (III) an ID 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 of SEQ ID NO: 20. In some embodiments, the recombinant cytokine receptor comprises (I) an ED comprising the amino acid sequence of SEQ ID NO:11; (II) a TD comprising the amino acid sequence of SEQ ID NO:21; and (III) an ID comprising the amino acid sequence of SEQ ID NO:20.

[0050] In some embodiments, the recombinant cytokine receptor comprises (I) the ED of IL-7Rα; (II) the TD of IL-7Rα; and (III) the ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the recombinant cytokine receptor comprises an ED 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 with the amino acid sequence shown in SEQ ID NO:12. In some embodiments, the recombinant cytokine receptor comprises: (I) an ED 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 of SEQ ID NO: 12; (II) a TD 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 of SEQ ID NO: 17; and (III) an ID 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 of SEQ ID NO: 20. In some embodiments, the recombinant cytokine receptor comprises (I) an ED comprising the amino acid sequence of SEQ ID NO:12; (II) a TD comprising the amino acid sequence of SEQ ID NO:17; and (III) an ID comprising the amino acid sequence of SEQ ID NO:20.

[0051] In some embodiments, the recombinant cytokine receptor comprises (I) ED of IL-7Rα; (II) TD of IL-2Rβ; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises an ED 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 shown in SEQ ID NO: 12. In some embodiments, the recombinant cytokine receptor comprises: (I) an ED 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 of SEQ ID NO: 12; (II) a TD 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 of SEQ ID NO: 21; and (III) an ID 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 of SEQ ID NO: 20. In some embodiments, the recombinant cytokine receptor comprises (I) an ED comprising the amino acid sequence of SEQ ID NO:12; (II) a TD comprising the amino acid sequence of SEQ ID NO:21; and (III) an ID comprising the amino acid sequence of SEQ ID NO:20.

[0052] In some embodiments, the recombinant cytokine receptor comprises (I) ED of IL-9Rα; (II) TD of IL-9Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence shown in SEQ ID NO:3. 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 with the amino acid sequence shown in SEQ ID NO:3. In some embodiments, the recombinant cytokine receptor comprises an ED 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 with the amino acid sequence shown in SEQ ID NO:13. In some embodiments, the recombinant cytokine receptor comprises: (I) an ED 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 of SEQ ID NO: 13; (II) a TD 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 of SEQ ID NO: 18; and (III) an ID 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 of SEQ ID NO: 20. In some embodiments, the recombinant cytokine receptor comprises (I) an ED comprising the amino acid sequence of SEQ ID NO:13; (II) a TD comprising the amino acid sequence of SEQ ID NO:18; and (III) an ID comprising the amino acid sequence of SEQ ID NO:20.

[0053] In some embodiments, the recombinant cytokine receptor comprises (I) ED of IL-9Rα; (II) TD of IL-2Rβ; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises an ED 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 shown in SEQ ID NO: 13. In some embodiments, the recombinant cytokine receptor comprises: (I) an ED 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 of SEQ ID NO: 13; (II) a TD 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 of SEQ ID NO: 21; and (III) an ID 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 of SEQ ID NO: 20. In some embodiments, the recombinant cytokine receptor comprises (I) an ED comprising the amino acid sequence of SEQ ID NO: 13; (II) a TD comprising the amino acid sequence of SEQ ID NO: 21; and (III) an ID comprising the amino acid sequence of SEQ ID NO: 20. 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 shown in SEQ ID NO: 5. In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence shown in SEQ ID NO: 5.

[0054] In some embodiments, the recombinant cytokine receptor comprises (I) ED of IL-21Rα; (II) TD of IL-21Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence shown in SEQ ID NO:4. 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 with the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the recombinant cytokine receptor comprises an ED 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 with the amino acid sequence shown in SEQ ID NO:14. In some embodiments, the recombinant cytokine receptor comprises: (I) an ED 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 of SEQ ID NO: 14; (II) a TD 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 of SEQ ID NO: 19; and (III) an ID 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 of SEQ ID NO: 20. In some embodiments, the recombinant cytokine receptor comprises (I) an ED comprising the amino acid sequence of SEQ ID NO:14; (II) a TD comprising the amino acid sequence of SEQ ID NO:19; and (III) an ID comprising the amino acid sequence of SEQ ID NO:20.

[0055] In some embodiments, the recombinant cytokine receptor comprises (I) ED of IL-21Rα; (II) TD of IL-2Rβ; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises an ED having an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the recombinant cytokine receptor comprises: (I) an ED 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 of SEQ ID NO: 14; (II) a TD 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 of SEQ ID NO: 21; and (III) an ID 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 of SEQ ID NO: 20. In some embodiments, the recombinant cytokine receptor comprises (I) an ED comprising the amino acid sequence of SEQ ID NO:14; (II) a TD comprising the amino acid sequence of SEQ ID NO:21; and (III) an ID comprising the amino acid sequence of SEQ ID NO:20.

[0056] In some embodiments, the recombinant cytokine receptor comprises: (I) ED of IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; (II) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; (III) ID of IL-2Rβ; and (IV) a homologous cytokine tethered to the ED of the recombinant cytokine receptor through a polypeptide linker. In some embodiments, the ED is from the same cytokine receptor as the TD (e.g., ED from IL-4Rα and TD from IL-4Rα). In some embodiments, the homologous cytokine is IL-4, IL-7, IL-9 or IL-21. In some embodiments, the homologous cytokine is IL-4. In some embodiments, the homologous cytokine is IL-7. In some embodiments, the homologous cytokine is IL-9. In some embodiments, the homologous cytokine is IL-21. In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence shown in SEQ ID NO:6-9 and 15.

[0057] In some embodiments, the recombinant cytokine receptor comprises (I) the ED of IL-4Rα; (II) the TD of IL-4Rα; (III) the ID of IL-2Rβ; and (IV) a homologous cytokine IL-4 tethered to the ED of the recombinant cytokine receptor by a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence shown in SEQ ID NO:6. 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 shown in SEQ ID NO:6. In some embodiments, the recombinant cytokine receptor comprises an ED 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 shown in SEQ ID NO:11. In some embodiments, the recombinant cytokine receptor comprises: (I) an ED 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 of SEQ ID NO: 11; (II) a TD 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 of SEQ ID NO: 16; (III) a TD 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 of SEQ ID NO: 17. NO:20 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; and (IV) a cytokine 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 of SEQ ID NO:22, which is tethered to the ED of a recombinant cytokine receptor via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises (I) an ED comprising the amino acid sequence of SEQ ID NO:11; (II) a TD comprising the amino acid sequence of SEQ ID NO:16; (III) an ID comprising the amino acid sequence of SEQ ID NO:20; and (IV) a cytokine comprising the amino acid sequence of SEQ ID NO:22, which is tethered to the ED of the recombinant cytokine receptor via a polypeptide linker.

[0058] In some embodiments, the recombinant cytokine receptor comprises (I) ED of IL-4Rα; (II) TD of IL-2Rβ; (III) ID of IL-2Rβ; and (IV) a homologous cytokine IL-4 tethered to the ED of the recombinant cytokine receptor via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises an ED 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 shown in SEQ ID NO: 11. In some embodiments, the recombinant cytokine receptor comprises: (I) an ED 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 of SEQ ID NO: 11; (II) a TD 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 of SEQ ID NO: 21; (III) a TD 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 of SEQ ID NO: 22. NO:20 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; and (IV) a cytokine 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 of SEQ ID NO:22, which is tethered to the ED of a recombinant cytokine receptor via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises (I) an ED comprising the amino acid sequence of SEQ ID NO: 11; (II) a TD comprising the amino acid sequence of SEQ ID NO: 21; (III) an ID comprising the amino acid sequence of SEQ ID NO: 20; and (IV) a cytokine comprising the amino acid sequence of SEQ ID NO: 22, which is tethered to the ED of the recombinant cytokine receptor via a polypeptide linker.

[0059] In some embodiments, the recombinant cytokine receptor comprises (I) the ED of IL-7Rα; (II) the TD of IL-7Rα; (III) the ID of ILR-2β; and (IV) a homologous cytokine IL-7 tethered to the ED of the recombinant cytokine receptor via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence shown in SEQ ID NO:7. 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 shown in SEQ ID NO:7. In some embodiments, the recombinant cytokine receptor comprises an ED 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 shown in SEQ ID NO:12. In some embodiments, the recombinant cytokine receptor comprises: (I) an ED 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 of SEQ ID NO: 12; (II) a TD 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 of SEQ ID NO: 17; (III) a TD 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 of SEQ ID NO: NO:20 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; and (IV) a cytokine 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 of SEQ ID NO:23, which is tethered to the ED of a recombinant cytokine receptor via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises (I) an ED comprising the amino acid sequence of SEQ ID NO: 12; (II) a TD comprising the amino acid sequence of SEQ ID NO: 17; (III) an ID comprising the amino acid sequence of SEQ ID NO: 20; and (IV) a cytokine comprising the amino acid sequence of SEQ ID NO: 23, which is tethered to the ED of the recombinant cytokine receptor via a polypeptide linker.

[0060] In some embodiments, the recombinant cytokine receptor comprises (I) ED of IL-7Rα; (II) TD of IL-2Rβ; (III) ID of IL-2Rβ; and (IV) a homologous cytokine IL-7 tethered to the ED of the recombinant cytokine receptor via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises an ED 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 shown in SEQ ID NO: 12. In some embodiments, the recombinant cytokine receptor comprises: (I) an ED 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 of SEQ ID NO: 12; (II) a TD 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 of SEQ ID NO: 21; (III) a TD 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 of SEQ ID NO: NO:20 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; and (IV) a cytokine 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 of SEQ ID NO:23, which is tethered to the ED of a recombinant cytokine receptor via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises (I) an ED comprising the amino acid sequence of SEQ ID NO: 12; (II) a TD comprising the amino acid sequence of SEQ ID NO: 21; (III) an ID comprising the amino acid sequence of SEQ ID NO: 20; and (IV) a cytokine comprising the amino acid sequence of SEQ ID NO: 23, which is tethered to the ED of the recombinant cytokine receptor via a polypeptide linker.

[0061] In some embodiments, the recombinant cytokine receptor comprises (I) the ED of IL-9Rα; (II) the TD of IL-9Rα; (III) the ID of IL-2Rβ; and (IV) a homologous cytokine IL-9 tethered to the ED of the recombinant cytokine receptor by a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence shown in SEQ ID NO:8. 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 shown in SEQ ID NO:8. In some embodiments, the recombinant cytokine receptor comprises an ED 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 shown in SEQ ID NO:13. In some embodiments, the recombinant cytokine receptor comprises: (I) an ED 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 of SEQ ID NO: 13; (II) a TD 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 of SEQ ID NO: 18; (III) a TD 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 of SEQ ID NO: 19. NO:20 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; and (IV) a cytokine 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 of SEQ ID NO:24, which is tethered to the ED of a recombinant cytokine receptor via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises (I) an ED comprising the amino acid sequence of SEQ ID NO: 13; (II) a TD comprising the amino acid sequence of SEQ ID NO: 18; (III) an ID comprising the amino acid sequence of SEQ ID NO: 20; and (IV) a cytokine comprising the amino acid sequence of SEQ ID NO: 24, which is tethered to the ED of the recombinant cytokine receptor via a polypeptide linker.

[0062] In some embodiments, the recombinant cytokine receptor comprises (I) ED of IL-9Rα; (II) TD of IL-2Rβ; (III) ID of IL-2Rβ; and (IV) a homologous cytokine IL-9 tethered to the ED of the recombinant cytokine receptor by a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises an ED 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 shown in SEQ ID NO: 13. In some embodiments, the recombinant cytokine receptor comprises: (I) an ED 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 of SEQ ID NO: 13; (II) a TD 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 of SEQ ID NO: 21; (III) a TD 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 of SEQ ID NO: 22; NO:20 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; and (IV) a cytokine 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 of SEQ ID NO:24, which is tethered to the ED of a recombinant cytokine receptor via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises (I) an ED comprising the amino acid sequence of SEQ ID NO: 13; (II) a TD comprising the amino acid sequence of SEQ ID NO: 21; (III) an ID comprising the amino acid sequence of SEQ ID NO: 20; and (IV) a cytokine comprising the amino acid sequence of SEQ ID NO: 24, tethered to the ED of the recombinant cytokine receptor via a polypeptide linker. 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 shown in SEQ ID NO: 15.In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence shown in SEQ ID NO:15.

[0063] In some embodiments, the recombinant cytokine receptor comprises (I) ED of IL-21Rα; (II) TD of IL-21Rα; (III) ID of IL-2Rβ; and (IV) a homologous cytokine IL-21 tethered to the ED of the recombinant cytokine receptor via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence shown in SEQ ID NO:9. 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 shown in SEQ ID NO:9. In some embodiments, the recombinant cytokine receptor comprises an ED 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 shown in SEQ ID NO:14. In some embodiments, the recombinant cytokine receptor comprises: (I) an ED 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 of SEQ ID NO: 14; (II) a TD 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 of SEQ ID NO: 19; (III) a TD 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 of SEQ ID NO: 19. NO:20 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; and (IV) a cytokine 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 of SEQ ID NO:25, which is tethered to the ED of a recombinant cytokine receptor via a polypeptide linker.In some embodiments, the recombinant cytokine receptor comprises (I) an ED comprising the amino acid sequence of SEQ ID NO: 14; (II) a TD comprising the amino acid sequence of SEQ ID NO: 19; (III) an ID comprising the amino acid sequence of SEQ ID NO: 20; and (IV) a cytokine comprising the amino acid sequence of SEQ ID NO: 25, which is tethered to the ED of the recombinant cytokine receptor via a polypeptide linker.

[0064] In some embodiments, the recombinant cytokine receptor comprises (I) ED of IL-21Rα; (II) TD of IL-2Rβ; (III) ID of IL-2Rβ; and (IV) a homologous cytokine IL-21 tethered to the ED of the recombinant cytokine receptor via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises an ED 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 with the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the recombinant cytokine receptor comprises: (I) an ED 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 of SEQ ID NO: 14; (II) a TD 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 of SEQ ID NO: 21; (III) a TD 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 of SEQ ID NO: NO:20 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; and (IV) a cytokine 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 of SEQ ID NO:25, which is tethered to the ED of a recombinant cytokine receptor via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises (I) an ED comprising the amino acid sequence of SEQ ID NO: 14; (II) a TD comprising the amino acid sequence of SEQ ID NO: 21; (III) an ID comprising the amino acid sequence of SEQ ID NO: 20; and (IV) a cytokine comprising the amino acid sequence of SEQ ID NO: 25, which is tethered to the ED of the recombinant cytokine receptor via a polypeptide linker.

[0065] In some embodiments, the cells expressing recombinant cytokine receptors can survive and proliferate in the absence of IL-2. In some embodiments, at least about 60% (such as at least about 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 recombinant cytokine receptors remain alive in the absence of IL-2. In some embodiments, cells are T cells. In some embodiments, cells are Treg cells.

[0066] In some embodiments, the cells expressing recombinant cytokine receptors can survive and proliferate in vitro in a suitable culture medium that can maintain Treg in the absence of IL-2. In some embodiments, at least about 60% (such as at least about 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 Treg transduced with recombinant cytokine receptors are transduced for about 2 days to about 30 days, such as about 10 to about 30 days, about 20 to about 30 days or about 23 to about 30 days in vitro. In some embodiments, at least about 60%-99% of Treg transduced with recombinant cytokine receptors are transduced for about 7 to about 20 days in vitro after transduction. In some embodiments, at least about 60%-90% of the Tregs transduced with recombinant cytokine receptors remain viable in vitro for about 7 to about 20 days after transduction. In some embodiments, at least about 60%-80% of the Tregs transduced with recombinant cytokine receptors remain viable in vitro for about 7 to about 20 days after transduction. In some embodiments, at least about 60%-99% of the Tregs transduced with recombinant cytokine receptors remain viable in vitro for about 7 days after transduction. In some embodiments, at least about 60%-90% of the Tregs transduced with recombinant cytokine receptors remain viable in vitro for about 7 days after transduction. In some embodiments, at least about 60%-80% of the Tregs transduced with recombinant cytokine receptors remain viable in vitro for about 7 days after transduction. In some embodiments, at least about 60%-99% of the Tregs transduced with recombinant cytokine receptors remain viable in vitro for about 12 days after transduction. In some embodiments, at least about 60%-90% of the Tregs transduced with recombinant cytokine receptors remain viable in vitro for about 12 days after transduction. In some embodiments, at least about 60%-80% of the Tregs transduced with recombinant cytokine receptors remain viable in vitro for about 12 days after transduction. In some embodiments, at least about 60%-99% of the Tregs transduced with recombinant cytokine receptors remain viable in vitro for about 14 days after transduction. In some embodiments, at least about 60%-90% of the Tregs transduced with recombinant cytokine receptors remain viable in vitro for about 14 days after transduction. In some embodiments, at least about 60%-80% of the Tregs transduced with recombinant cytokine receptors remain viable in vitro for about 14 days after transduction. In some embodiments, the viability of Tregs transduced with recombinant cytokine receptors is increased compared to Treg cells not transduced with recombinant cytokine receptors.In some embodiments, when cultured in the absence of a cognate receptor cytokine, the viability of Tregs transduced with a recombinant cytokine receptor is increased compared to Treg cells not transduced with a recombinant cytokine receptor. In some embodiments, the viability of Tregs is increased by at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 3.2 times, at least about 4 times, at least about 10 times, about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 45 times, at least about 50 times, or more compared to untransduced T cells.

[0067] In some embodiments, Treg expressing recombinant cytokine receptors can survive and proliferate in vivo in a host organism in the absence of IL-2. In some embodiments, at least about 60% (such as at least about 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 Treg transduced with recombinant cytokine receptors can persist in vivo for at least about 3 days or longer. In some embodiments, at least about 60%-99% of Treg transduced with recombinant cytokine receptors can persist in vivo for at least about 3 days or longer. In some embodiments, at least about 60%-90% of Treg transduced with recombinant cytokine receptors can persist in vivo for at least about 3 days or longer. In some embodiments, at least about 60%-80% of Tregs transduced with a recombinant cytokine receptor can persist in vivo for at least about 3 days or longer.

[0068] In some embodiments, cells transduced with a recombinant cytokine receptor have increased or sustained proliferation in vivo after transduction. In some embodiments, cells transduced with a recombinant cytokine receptor have more cells in an expanded 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.

[0069] In some embodiments, the Treg transduced with recombinant cytokine receptors has an increase or sustained proliferation after transduction. In some embodiments, compared with the number of cells before transduction, the Treg transduced with recombinant cytokine receptors has more cells after amplification. In some embodiments, compared with untransduced Treg cells, the Treg transduced with recombinant cytokine receptors has a higher proliferation rate. In some embodiments, compared with untransduced cells, the number of cells after transduction increases by at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 50 times, at least about 75 times, or more. In some embodiments, cells are cultured in the presence of cytokines after transduction. In some embodiments, cells are cultured in the absence of cytokines after transduction.

[0070] In some embodiments, cell viability, transduction of intracellular signaling, activation of intracellular signaling, or the ability to proliferate is increased following transduction with a recombinant cytokine receptor.

[0071] In some embodiments, after transduction with a recombinant cytokine receptor, the cell viability, transduction of intracellular signaling, activation of intracellular signaling, or the ability to proliferate increases. In some embodiments, the recombinant cytokine receptor expressed in the cell transduces intracellular IL-2 signaling in the cell. In some embodiments, the recombinant cytokine receptor activates intracellular IL-2 signaling in the cell. In some embodiments, the recombinant cytokine receptor promotes intracellular IL-2 signaling in the cell. In some embodiments, the recombinant cytokine receptor increases intracellular IL-2 signaling in the cell compared to cells without recombinant cytokine receptors. In some embodiments, the intracellular domain of the recombinant cytokine receptor provided herein comprises an active IL-2Rβ intracellular domain. In some embodiments, the recombinant cytokine receptor comprises an active IL-2Rβ 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 recombinant cytokine receptor comprises an IL-2Rβ intracellular domain, which phosphorylates STAT5 by activating JAK1 kinase after activation of the recombinant cytokine receptor. In some embodiments, the IL-2Rβ intracellular domain phosphorylates STAT5 by activating JAK1 kinase after activation of the recombinant cytokine receptor. In some embodiments, the recombinant cytokine receptor phosphorylates Shc after activation of the recombinant cytokine receptor, thereby activating the downstream PI3K-AKT pathway. In some embodiments, the recombinant cytokine receptor phosphorylates Shc after activation of the recombinant cytokine receptor, thereby activating the downstream Ras / MAPK pathway. In some embodiments, the recombinant cytokine receptor comprises an IL-4Rα / IL-2Rβ, IL-7Rα / IL-2Rβ, IL-9Rα / IL-2Rβ or IL-21Rα / IL-2Rβ extracellular / intracellular receptor domain. In some embodiments, the cell is a T cell. In some embodiments, the cell is a Treg cell.

[0072] In some embodiments, the recombinant cytokine receptor phosphorylates STAT5 by activating JAK1 kinase after activation of the recombinant cytokine receptor. In some embodiments, the recombinant cytokine receptor phosphorylates Shc after activation of the recombinant cytokine receptor, thereby activating the downstream PI3K-AKT pathway. In some embodiments, the recombinant cytokine receptor phosphorylates Shc after activation of the recombinant cytokine receptor, thereby activating the downstream Ras / MAPK pathway. In some embodiments, the recombinant cytokine receptor comprises an IL-4Rα / IL-2Rβ extracellular / intracellular receptor domain. In some embodiments, the recombinant cytokine receptor is IL-4Rα / IL-2Rβ.

[0073] In some embodiments, recombinant cytokine receptors have the ability to stimulate STAT5 phosphorylation in cells (e.g., Treg).STAT5 signaling can be measured, for example, by using any suitable method known in the art to phosphorylate STAT5.For example, STAT5 phosphorylation can be measured using antibodies specific to the phosphorylated forms of these molecules in conjunction with flow cytometry analysis as described herein.

[0074] 1. Extracellular domain

[0075] IL-2 is a class I cytokine. Class I cytokine receptors typically have a large extracellular domain (ED) that contains multiple full βIg-like domains and Fn3 domains (see, e.g., Metcalfe, RD et al. (2020), Front Immunol; 11: 1424). These domains have a β-sandwich structure containing two antiparallel β folds. The two Fn3 domains form a cytokine binding homology region at the junction of the domains. The class I cytokine receptor ED contains a conserved WSXWS (Trp-Ser-X-Trp-Ser, where X is any amino acid) motif, which may be used to stabilize the receptor, undergo conformational changes after cytokine binding, and can be extensively glycosylated. Class I cytokine receptor chains, including IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα, and IL-21Rα, are most commonly found in heterodimers or heterotrimers. For example, IL-2Rβ can be present in conjunction with IL-2Rγ c Heterodimers or with IL-2Rα and IL-2Rγ c In the heterotrimer, the IL-2 binding affinity of IL-2Rβ alone is K d is about 100 nm, and the K d The K of the heterotrimer is about 1 nM. d is about 10 pM (see, e.g., Wang, X et al. (2009), Annu Rev Immunol; 27:29-60, hereby incorporated by reference in its entirety). Reconstitution studies have identified the homologous cytokine binding affinities of IL-4Rα, IL-7Rα, IL-9Rα, and IL-21Rα as follows: d About 266pM, K d About 250 pM, K d is about 100 pM (see, e.g., Lin, JX & Leonard, WJ (2018), Cold Spring Harb Perspect Biol; 10(9):a028449, hereby incorporated by reference in its entirety), and K dis about 70 pM (see, e.g., Kang, L et al. (2010), J Biol Chem; 285(16): 12223-12231, hereby incorporated by reference in its entirety). c When the chains are complexed, these binding affinities can be changed to higher affinities. The lengths of the EDs of IL-4Rα, IL-7Rα, IL-9Rα, and IL-21Rα are 232 residues, 239 residues, 270 residues, and 232 residues, respectively. The IL-2, IL-4, IL-7, IL-9, and IL-21 signaling pathways each have their own unique effects on T cell biology: T cell survival and proliferation; T helper 2 (T H 2) Differentiation from naive T cells and inhibition of T helper cells 1 (T H 1) Anti-inflammatory effects and induction of Treg differentiation; survival of naive and memory T cells; enhancement of Treg suppressive activity and T H 17 cell differentiation; and promoting CD8+ T cell clonal expansion and cytolytic activity.

[0076] 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 interleukin ED, TD and ID. Therefore, in some embodiments, a recombinant cytokine receptor is provided, which comprises: (I) ED of IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; (II) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; (III) ID of IL-2Rβ; and (IV) optionally, a homologous cytokine tethered to the ED of the recombinant cytokine receptor through a polypeptide linker. In some embodiments, ED comprises the amino acid sequence shown in SEQID NO: 11-14. In some embodiments, ED is from the same cytokine receptor as TD (e.g., ED from IL-4Rα and TD from IL-4Rα). In some embodiments, ED is from a cytokine receptor different from TD.

[0077] In some embodiments, the recombinant cytokine receptor comprises the ED of IL-4Rα. In some embodiments, the ED comprises the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the ED 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 with the amino acid sequence of SEQ ID NO: 11. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-4Rα; (II) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-4Rα; (II) TD of IL-4Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an ED 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 of SEQ ID NO: 11; (II) a TD 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 of SEQ ID NO: 16; and (III) a TD 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 of SEQ ID NO: 17. NO:20 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an ED comprising the amino acid sequence of SEQ ID NO:11; (II) a TD comprising the amino acid sequence of SEQ ID NO:16; and (III) an ID comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence shown in SEQ ID NO:1.

[0078] In some embodiments, the recombinant cytokine receptor comprises the ED of IL-4Rα. In some embodiments, the ED 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 with the amino acid sequence of SEQ ID NO: 11. In some embodiments, the ED comprises the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-4Rα; (II) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-4Rα; (II) TD of IL-2Rβ; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an ED 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 of SEQ ID NO: 11; (II) a TD 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 of SEQ ID NO: 21; and (III) a TD 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 of SEQ ID NO: 21. NO:20 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the ID of the amino acid sequence. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an ED comprising the amino acid sequence of SEQ ID NO: 11; (II) a TD comprising the amino acid sequence of SEQ ID NO:21; and (III) an ID comprising the amino acid sequence of SEQ ID NO:20.

[0079] In some embodiments, the recombinant cytokine receptor comprises the ED of IL-7Rα. In some embodiments, the IL-7Rα ED comprises the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the ED 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% with SEQ ID NO: 12. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-7Rα; (II) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-7Rα; (II) TD of IL-7Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an ED 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 of SEQ ID NO: 12; (II) a TD 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 of SEQ ID NO: 17; and (III) a TD 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 of SEQ ID NO: 18. NO:20 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an ED comprising the amino acid sequence of SEQ ID NO:12; (II) a TD comprising the amino acid sequence of SEQ ID NO:17; and (III) an ID comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence shown in SEQ ID NO:2.

[0080] In some embodiments, the recombinant cytokine receptor comprises the ED of IL-7Rα. In some embodiments, the ED 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 with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the ED comprises the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-7Rα; (II) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-7Rα; (II) TD of IL-2Rβ; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an ED 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 of SEQ ID NO: 12; (II) a TD 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 of SEQ ID NO: 21; and (III) a TD 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 of SEQ ID NO: 22. NO:20 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the ID of the amino acid sequence. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an ED comprising the amino acid sequence of SEQ ID NO:12; (II) a TD comprising the amino acid sequence of SEQ ID NO:21; and (III) an ID comprising the amino acid sequence of SEQ ID NO:20.

[0081] In some embodiments, the recombinant cytokine receptor comprises the ED of IL-9Rα. In some embodiments, the ED comprises the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the ED 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% with SEQ ID NO: 13. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-9Rα; (II) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-9Rα; (II) TD of IL-9Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an ED 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 of SEQ ID NO: 13; (II) a TD 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 of SEQ ID NO: 18; and (III) a TD 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 of SEQ ID NO: 19. NO:20 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an ED comprising the amino acid sequence of SEQ ID NO:13; (II) a TD comprising the amino acid sequence of SEQ ID NO:18; and (III) an ID comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence shown in SEQ ID NO:3.

[0082] In some embodiments, the recombinant cytokine receptor comprises the ED of IL-9Rα. In some embodiments, the ED 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 with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the ED comprises the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-9Rα; (II) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-9Rα; (II) TD of IL-2Rβ; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an ED 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 of SEQ ID NO: 13; (II) a TD 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 of SEQ ID NO: 21; and (III) a TD 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 of SEQ ID NO: 22. NO:20 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an ED comprising the amino acid sequence of SEQ ID NO:13; (II) a TD comprising the amino acid sequence of SEQ ID NO:21; and (III) an ID comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence shown in SEQ ID NO:5.

[0083] In some embodiments, the IL-21Rα ED comprises the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the ED 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 with the amino acid sequence of SEQ ID NO: 14. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-21Rα; (II) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-21Rα; (II) TD of IL-21Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an ED 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 of SEQ ID NO: 14; (II) a TD 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 of SEQ ID NO: 19; and (III) a TD 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 of SEQ ID NO: 19. NO:20 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an ED comprising the amino acid sequence of SEQ ID NO:14; (II) a TD comprising the amino acid sequence of SEQ ID NO:19; and (III) an ID comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the amino acid sequence shown in SEQ ID NO:4.

[0084] In some embodiments, the recombinant cytokine receptor comprises the ED of IL-21Rα. In some embodiments, the ED comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the amino acid sequence of SEQ ID NO:14. In some embodiments, the ED comprises the amino acid sequence shown in SEQ ID NO:14. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-21Rα; (II) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-21Rα; (II) TD of IL-2Rβ; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an ED 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 of SEQ ID NO: 14; (II) a TD 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 of SEQ ID NO: 21; and (III) a TD 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 of SEQ ID NO: 22. NO:20 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the ID of the amino acid sequence. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an ED comprising the amino acid sequence of SEQ ID NO:14; (II) a TD comprising the amino acid sequence of SEQ ID NO:21; and (III) an ID comprising the amino acid sequence of SEQ ID NO:20.

[0085] In some embodiments, the recombinant cytokine receptor comprises the ED of IL-4Rα. In some embodiments, the ED comprises the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the ED 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 with the amino acid sequence of SEQ ID NO: 11. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) IL-4 cytokine; (II) ED of IL-4Rα; (III) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; and (IV) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) IL-4 cytokine; (II) ED of IL-4Rα; (III) TD of IL-4Rα or IL-2Rβ; and (IV) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) a cytokine 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 of SEQ ID NO: 22; (II) an ED 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 of SEQ ID NO: 11; (III) a cytokine 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 of SEQ ID NO: 16 or SEQ ID NO: 17. NO:21 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; and (IV) 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 of SEQ ID NO:20.In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) a cytokine comprising the amino acid sequence of SEQ ID NO: 22; (II) an ED comprising the amino acid sequence of SEQ ID NO: 11; (III) a TD comprising the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 21; and (IV) an ID comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the cytokine is tethered to the ED via a linker. 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 shown in SEQ ID NO: 6. In some embodiments, the recombinant cytokine receptor comprises a 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 shown in SEQ ID NO:22.

[0086] In some embodiments, the recombinant cytokine receptor comprises the ED of IL-7Rα. In some embodiments, the IL-7Rα ED comprises the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the ED 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 with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) IL-7 cytokine; (II) ED of IL-7Rα; (III) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; and (IV) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) IL-7 cytokine; (II) ED of IL-7Rα or IL-2Rβ; (III) TD of IL-7Rα; and (IV) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) a cytokine 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 of SEQ ID NO:23; (II) an ED 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 of SEQ ID NO:12; (III) a cytokine 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 of SEQ ID NO:17; NO:21 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; and (IV) 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 of SEQ ID NO:20.In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) a cytokine comprising the amino acid sequence of SEQ ID NO: 23; (II) an ED comprising the amino acid sequence of SEQ ID NO: 12; (III) a TD comprising the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 21; and (IV) an ID comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the cytokine is tethered to the ED via a linker. 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 shown in SEQ ID NO: 7. In some embodiments, the recombinant cytokine receptor comprises a 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 shown in SEQ ID NO:23.

[0087] In some embodiments, the IL-9Rα ED comprises the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the ED 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 with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) IL-9 cytokine; (II) ED of IL-9Rα; (III) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; and (IV) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) IL-9 cytokine; (II) ED of IL-9Rα; (III) TD of IL-9Rα; and (IV) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) a cytokine 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 of SEQ ID NO: 24; (II) an ED 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 of SEQ ID NO: 13; (III) a cytokine 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 of SEQ ID NO: 14. NO:18 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity; and (IV) comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:20. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) a cytokine comprising the amino acid sequence of SEQ ID NO:24; (II) an ED comprising the amino acid sequence of SEQ ID NO:13; (III) an amino acid sequence comprising SEQ ID NO:18; and (IV) an ID comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the cytokine is tethered to the ED via a linker.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 shown in SEQ ID NO: 8. In some embodiments, the recombinant cytokine receptor comprises a 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 shown in SEQ ID NO: 24.

[0088] In some embodiments, the recombinant cytokine receptor comprises the ED of IL-9Rα. In some embodiments, the ED comprises the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the ED 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 with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) IL-9 cytokine; (II) ED of IL-9Rα; (III) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; and (IV) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) IL-9 cytokine; (II) ED of IL-9Rα; (III) TD of IL-2Rβ; and (IV) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) a cytokine 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 of SEQ ID NO: 24; (II) an ED 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 of SEQ ID NO: 13; (III) a cytokine 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 of SEQ ID NO: 14. NO:21 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity; and (IV) comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:20. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) a cytokine comprising the amino acid sequence of SEQ ID NO:24; (II) an ED comprising the amino acid sequence of SEQ ID NO:13; (III) a TD comprising the amino acid sequence of SEQ ID NO:21; and (IV) an ID comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the cytokine is tethered to the ED via a linker.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 shown in SEQ ID NO: 15. In some embodiments, the recombinant cytokine receptor comprises a 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 shown in SEQ ID NO: 24.

[0089] In some embodiments, the IL-21Rα ED comprises the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the ED 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 with the amino acid sequence of SEQ ID NO: 14. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) IL-21 cytokine; (II) ED of IL-21Rα; (III) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; and (IV) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) IL-21 cytokine; (II) ED of IL-21Rα; (III) TD of IL-21Rα or IL-2Rβ; and (IV) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) a cytokine 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 of SEQ ID NO:25; (II) an ED 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 of SEQ ID NO:14; (III) a cytokine 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 of SEQ ID NO:19; NO:21 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity; and (IV) comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:20. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) a cytokine comprising the amino acid sequence of SEQ ID NO:25; (II) an ED comprising the amino acid sequence of SEQ ID NO:14; (III) a TD comprising the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:21; and (IV) an ID comprising the amino acid sequence of SEQ ID NO:20.In some embodiments, the cytokine is tethered to the ED via a linker. 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 shown in SEQ ID NO: 9. In some embodiments, the recombinant cytokine receptor comprises a 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 shown in SEQ ID NO: 25.

[0090] In some embodiments, a recombinant cytokine receptor comprising the aforementioned ED is expressed in a cell. In some embodiments, a recombinant cytokine receptor comprising the aforementioned ED 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.

[0091] In some embodiments, a recombinant cytokine receptor comprising the aforementioned ED is expressed in Treg. In some embodiments, Treg cells are CD4+, CD25+, and CD127lo. In some embodiments, Tregs expressing recombinant cytokine receptors comprising the aforementioned ED also express FOXP3 and HELIOS. In some embodiments, Tregs expressing recombinant cytokine receptors comprising the aforementioned ED also express high levels of FOXP3 and HELIOS. In some embodiments, Tregs expressing recombinant cytokine receptors comprising the aforementioned ED can survive and proliferate in the absence of IL-2. In some embodiments, at least about 60% (such as 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 the Tregs transduced with a recombinant cytokine receptor comprising the aforementioned ED remain viable in vitro 2-20 days after transduction. In some embodiments, at least about 60% (such as at least about 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 the recombinant cytokine receptor comprising the aforementioned ED can persist in vivo for at least about 3 days or longer. In some embodiments, the recombinant cytokine receptor comprising the aforementioned ED expressed in Tregs transduces intracellular IL-2 signaling within Tregs. In some embodiments, the signaling of the recombinant cytokine receptor comprising the aforementioned ED expressed in Tregs induces phosphorylation of STAT5. In some embodiments, Treg expressing a recombinant cytokine receptor comprising the aforementioned ED can suppress the activity of effector T cells in the absence of IL-2, to an extent at least the same or higher than wild-type Treg 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, Treg expressing a recombinant cytokine receptor comprising the aforementioned ED also expresses a chimeric antigen receptor (CAR).

[0092] 2. Intracellular domain

[0093] IL-2Rβ is mainly expressed in hematopoietic cells, and its involvement in immune cell-mediated immune responses is well described. The IL-2Rβ polypeptide interacts with JAK1 through its C-terminal cytoplasmic domain (which contains amino acids 240-525). The IL-2Rβ intracellular domain (ID) lacks intrinsic catalytic activity and therefore relies on its constitutive interaction with JAK1 at the box 1 and box 3 motifs in its juxtamembrane region to promote IL-2 signal transduction (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 through JAK1 kinase, (2) PI3K-AKT pathway activation through Shc phosphorylation, and (3) Ras / MAPK pathway activation through Shc phosphorylation (see, e.g., Ye, C et al. (2018), Signal Transduct Target Ther; 3:2, hereby incorporated by reference in its entirety). Of these three pathways, JAK1 / STAT5 signaling is dominant. Activation of the IL-2 pathway in all T cell lineages has been shown to induce T cell proliferation and survival. However, within CD4+CD25+Treg cells, IL-2Rβ signaling only participates in the JAK1 / STAT5 pathway (see, e.g., Bensinger, SJ et al. (2010), J Immunol; 172(9): 5287-5296, hereby incorporated 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). Although CD4+ and to a lesser extent CD8+ effector T cells can produce IL-2 to signal their own proliferation and survival after activation, Tregs cannot produce IL-2. Therefore, Treg survival depends on IL-2 produced by effector T cells.

[0094] In some embodiments, the intracellular domain of the recombinant cytokine receptor provided herein comprises an active IL-2Rβ intracellular domain. In some embodiments, the intracellular domain of the recombinant cytokine receptor is an active IL-2Rβ intracellular domain. In some embodiments, the intracellular domain of the recombinant cytokine receptor provided herein can participate in downstream signal transduction. In some embodiments, the IL-2Rβ intracellular domain phosphorylates STAT5 by activating JAK1 kinase after IL-2RβID activation. In some embodiments, the IL-2 intracellular domain phosphorylates Shc after IL-2Rβ receptor activation, thereby activating the downstream PI3K-AKT pathway. In some embodiments, the IL-2 intracellular domain phosphorylates Shc after IL-2RβID activation, thereby activating the downstream Ras / MAPK pathway. In some embodiments, the IL-2Rβ intracellular domain phosphorylates STAT5 by activating JAK1 kinase after recombinant cytokine receptor activation. In some embodiments, the IL-2 intracellular domain phosphorylates Shc after activation of the recombinant cytokine receptor, thereby activating the downstream PI3K-AKT pathway. In some embodiments, the IL-2 intracellular domain phosphorylates Shc after activation of the recombinant cytokine receptor, thereby activating the downstream Ras / MAPK pathway. In some embodiments, the recombinant cytokine receptor comprises an IL-4Rα / IL-2Rβ, IL-7Rα / IL-2Rβ, IL-9Rα / IL-2Rβ or IL-21Rα / IL-2Rβ extracellular / intracellular receptor domain. In some embodiments, the recombinant cytokine receptor is IL-4Rα / IL-2Rβ, IL-7Rα / IL-2Rβ, IL-9Rα / IL-2Rβ or IL-21Rα / IL-2Rβ.

[0095] In some embodiments, the IL-2Rβ intracellular domain phosphorylates STAT5 by activating JAK1 kinase after activation of the recombinant cytokine receptor. In some embodiments, the IL-2Rβ intracellular domain phosphorylates Shc after activation of the recombinant cytokine receptor, thereby activating the downstream PI3K-AKT pathway. In some embodiments, the IL-2Rβ intracellular domain phosphorylates Shc after activation of the recombinant cytokine receptor, thereby activating the downstream Ras / MAPK pathway. In some embodiments, the recombinant cytokine receptor comprises an IL-4Rα / IL-2Rβ extracellular / intracellular receptor domain. In some embodiments, the recombinant cytokine receptor is IL-4Rα / IL-2Rβ.

[0096] In some embodiments, the IL-2Rβ intracellular domain phosphorylates STAT5 by activating JAK1 kinase after activation of the recombinant cytokine receptor. In some embodiments, the IL-2Rβ intracellular domain phosphorylates Shc after activation of the recombinant cytokine receptor, thereby activating the downstream PI3K-AKT pathway. In some embodiments, the IL-2Rβ intracellular domain phosphorylates Shc after activation of the recombinant cytokine receptor, thereby activating the downstream Ras / MAPK pathway. In some embodiments, the recombinant cytokine receptor comprises an IL-7Rα / IL-2Rβ extracellular / intracellular receptor domain. In some embodiments, the recombinant cytokine receptor is IL-7Rα / IL-2Rβ.

[0097] In some embodiments, the IL-2Rβ intracellular domain phosphorylates STAT5 by activating JAK1 kinase after activation of the recombinant cytokine receptor. In some embodiments, the IL-2Rβ intracellular domain phosphorylates Shc after activation of the recombinant cytokine receptor, thereby activating the downstream PI3K-AKT pathway. In some embodiments, the IL-2Rβ intracellular domain phosphorylates Shc after activation of the recombinant cytokine receptor, thereby activating the downstream Ras / MAPK pathway. In some embodiments, the recombinant cytokine receptor comprises an IL-9Rα / IL-2Rβ extracellular / intracellular receptor domain. In some embodiments, the recombinant cytokine receptor is IL-9Rα / IL-2Rβ.

[0098] In some embodiments, the IL-2Rβ intracellular domain phosphorylates STAT5 by activating JAK1 kinase after recombinant cytokine receptor activation. In some embodiments, the IL-2Rβ intracellular domain phosphorylates Shc after recombinant cytokine receptor activation, thereby activating the downstream PI3K-AKT path. In some embodiments, the IL-2Rβ intracellular domain phosphorylates Shc after recombinant cytokine receptor activation, thereby activating the downstream Ras / MAPK path. In some embodiments, the recombinant cytokine receptor comprises an IL-21Rα / IL-2Rβ extracellular / intracellular receptor domain. In some embodiments, the recombinant cytokine receptor is IL-21Rα / IL-2Rβ.

[0099] 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 is capable of signaling in the presence of a cytokine other than IL-2. In some embodiments, the IL-2Rβ ID comprises the amino acid sequence set forth in SEQ ID NO: 20. 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% of SEQ ID NO: 20. In some embodiments, the recombinant cytokine receptor comprises: (I) ED of IL-4Rα, IL-7Rα, IL-9Rα, or IL-21Rα; (II) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα, or IL-21Rα; (III) ID of IL-2Rβ; and (IV) optionally, a homologous cytokine tethered to the ED of the recombinant cytokine receptor via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) ED of IL-4Rα; (II) TD of IL-4Rα; (III) ID of IL-2Rβ; and (IV) optionally, an IL-4 cytokine tethered to the ED of the recombinant cytokine receptor via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-7Rα; (II) TD of IL-7Rα; (III) ID of IL-2Rβ; and (IV) optionally, an IL-7 cytokine tethered to the ED of the recombinant cytokine receptor via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-9Rα; (II) TD of IL-9Rα; (III) ID of IL-2Rβ; and (IV) optionally, an IL-9 cytokine tethered to the ED of the recombinant cytokine receptor via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-21Rα; (II) TD of IL-21Rα; (III) ID of IL-2Rβ; and (IV) optionally, an IL-21 cytokine tethered to the ED of the recombinant cytokine receptor via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises: (I) a cognate cytokine tethered to the ED of the recombinant cytokine receptor, optionally via a polypeptide linker; (II) the ED of IL-4Rα, IL-7Rα, IL-9Rα, or IL-21Rα; (III) the TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα, or IL-21Rα; and (IV) the ID of IL-2Rβ.In some embodiments, the ED is from the same cytokine receptor as the TD (e.g., an ED from IL-4Rα and a TD from IL-4Rα). In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an IL-4 cytokine, optionally tethered to the ED of the recombinant cytokine receptor via a polypeptide linker; (II) the ED of IL-4Rα; (III) the TD of IL-4Rα; and (IV) the ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an IL-4 cytokine, optionally tethered to the ED of the recombinant cytokine receptor via a polypeptide linker; (II) the ED of IL-4Rα; (III) the TD of IL-2Rβ; and (IV) the ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an IL-7 cytokine, optionally tethered to the ED of the recombinant cytokine receptor via a polypeptide linker; (II) the ED of IL-7Rα; (III) the TD of IL-7Rα; and (IV) the ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an IL-7 cytokine, optionally tethered to the ED of the recombinant cytokine receptor via a polypeptide linker; (II) the ED of IL-7Rα; (III) the TD of IL-2Rβ; and (IV) the ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an IL-9 cytokine, optionally tethered to the ED of the recombinant cytokine receptor via a polypeptide linker; (II) the ED of IL-9Rα; (III) the TD of IL-9Rα; and (IV) the ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an IL-9 cytokine, optionally tethered to the ED of the recombinant cytokine receptor via a polypeptide linker; (II) the ED of IL-9Rα; (III) the TD of IL-2Rβ; and (IV) the ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an IL-21 cytokine, optionally tethered to the ED of the recombinant cytokine receptor via a polypeptide linker; (II) the ED of IL-21Rα; (III) the TD of IL-21Rα; and (IV) the ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) an IL-21 cytokine, optionally tethered to the ED of the recombinant cytokine receptor via a polypeptide linker; (II) the ED of IL-21Rα; (III) the TD of IL-2Rβ; and (IV) the ID of IL-2Rβ.In some embodiments, the recombinant cytokine receptor comprises: (I) ED of IL-4Rα, IL-7Rα, IL-9Rα, or IL-21Rα; (II) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα, or IL-21Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-4Rα; (II) TD of IL-4Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-4Rα; (II) TD of IL-2Rβ; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-7Rα; (II) TD of IL-7Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-7Rα; (II) TD of IL-2Rβ; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-9Rα; (II) TD of IL-9Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-9Rα; (II) TD of IL-2Rβ; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-21Rα; (II) TD of IL-21Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) the ED of IL-21Rα; (II) the TD of IL-2Rβ; and (III) the ID of IL-2Rβ.

[0100] 3. Transmembrane domain

[0101] The TDs of the IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα, and IL-21Rα receptors, which are members of the class I cytokine receptors, comprise modular single-pass TDs. The lengths of the TDs of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα, and IL-21Rα are 25 residues, 25 residues, 24 residues, 25 residues, 21 residues, and 21 residues.

[0102] In some embodiments, the recombinant cytokine receptor comprises a transmembrane domain. In some embodiments, the transmembrane domain is located between the extracellular domain and the intracellular domain of the recombinant cytokine. In some embodiments, the transmembrane domain is from a cytokine receptor. In some embodiments, the transmembrane domain is from a class I cytokine receptor. In some embodiments, the transmembrane domain is from a class II cytokine receptor. In some embodiments, the transmembrane domain is a transmembrane domain selected from the group consisting of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα and IL-21Rα. In some embodiments, the transmembrane domain is a transmembrane domain from IL-2Rβ. In some embodiments, the transmembrane domain is a transmembrane domain from IL-4Rα. In some embodiments, the transmembrane domain is a transmembrane domain from IL-7Rα. In some embodiments, the transmembrane domain is a transmembrane domain from IL-9Rα. In some embodiments, the transmembrane domain is a transmembrane domain from IL-21Rα. In some embodiments, TD is selected from the same cytokine receptor as ED. In some embodiments, TD is selected from a different cytokine receptor than ED. In some embodiments, TD comprises any one of the amino acid sequences shown in SEQ ID NOs: 16-19 and 21.

[0103] In some embodiments, the IL-2Rβ TD comprises the amino acid sequence shown in SEQ ID NO: 21. 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 with the amino acid sequence shown in SEQ ID NO: 21. In some embodiments, the recombinant cytokine receptor comprises a TD of about 20-30 amino acids in length. In some embodiments, the recombinant cytokine receptor comprises a TD of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. In some embodiments, the recombinant cytokine receptor comprises: (I) ED of IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; (II) TD of IL-2Rβ; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-4Rα; (II) TD of IL-2Rβ; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) IL-4 cytokine; (II) ED of IL-4Rα; (III) TD of IL-2Rβ; and (IV) ID of IL-2Rβ. In some embodiments, the IL-4 cytokine is tethered to the ED of IL-4Rα via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-7Rα; (II) TD of IL-2Rβ; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) IL-7 cytokine; (II) ED of IL-7Rα; (III) TD of IL-2Rβ; and (IV) ID of IL-2Rβ. In some embodiments, the IL-7 cytokine is tethered to the ED of IL-7Rα via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-9Rα; (II) TD of IL-2Rβ; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) IL-9 cytokine; (II) ED of IL-9Rα; (III) TD of IL-2Rβ; and (IV) ID of IL-2Rβ. In some embodiments, the IL-9 cytokine is tethered to the ED of IL-9Rα via a polypeptide linker. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-21Rα; (II) TD of IL-2Rβ; and (III) ID of IL-2Rβ.In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) IL-21 cytokine; (II) ED of IL-21Rα; (III) TD of IL-2Rβ; and (IV) ID of IL-2Rβ. In some embodiments, the IL-21 cytokine is tethered to the ED of IL-21Rα via a polypeptide linker.

[0104] In some embodiments, the IL-4Rα TD comprises the amino acid sequence shown in SEQ ID NO: 16. 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 with the amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the recombinant cytokine receptor comprises a TD of about 20-30 amino acids in length. In some embodiments, the recombinant cytokine receptor comprises a TD of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. In some embodiments, the recombinant cytokine receptor comprises: (I) ED of IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; (II) TD of IL-4Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) ED of IL-4Rα; (II) TD of IL-4Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) IL-4 cytokine; (II) ED of IL-4Rα; (III) TD of IL-4Rα; and (IV) ID of IL-2Rβ. In some embodiments, the IL-4 cytokine is tethered to the ED of IL-4Rα via a polypeptide linker.

[0105] In some embodiments, the IL-7RαTD comprises the amino acid sequence shown in SEQ ID NO: 17. 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 with the amino acid sequence shown in SEQ ID NO: 17. In some embodiments, the recombinant cytokine receptor comprises a TD of about 20-30 amino acids in length. In some embodiments, the recombinant cytokine receptor comprises a TD of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. In some embodiments, the recombinant cytokine receptor comprises: (I) ED of IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; (II) TD of IL-7Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) ED of IL-7Rα; (II) TD of IL-7Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) IL-7 cytokine; (II) ED of IL-7Rα; (III) TD of IL-7Rα; and (IV) ID of IL-2Rβ. In some embodiments, the IL-7 cytokine is tethered to the ED of IL-7Rα via a polypeptide linker.

[0106] In some embodiments, the IL-9RαTD comprises the amino acid sequence shown in SEQ ID NO: 18. 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 with the amino acid sequence shown in SEQ ID NO: 18. In some embodiments, the recombinant cytokine receptor comprises a TD of about 20-30 amino acids in length. In some embodiments, the recombinant cytokine receptor comprises a TD of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. In some embodiments, the recombinant cytokine receptor comprises: (I) ED of IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; (II) TD of IL-9Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-9Rα; (II) TD of IL-9Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) IL-9 cytokine; (II) ED of IL-9Rα; (III) TD of IL-9Rα; and (IV) ID of IL-2Rβ. In some embodiments, the IL-9 cytokine is tethered to the ED of IL-9Rα via a polypeptide linker.

[0107] In some embodiments, IL-21RαTD comprises the amino acid sequence shown in SEQ ID NO: 19. In some embodiments, 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 with the amino acid sequence shown in SEQ ID NO: 19. In some embodiments, the recombinant cytokine receptor comprises a TD of about 20-30 amino acids in length. In some embodiments, the recombinant cytokine receptor comprises a TD of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. In some embodiments, the recombinant cytokine receptor comprises: (I) ED of IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; (II) TD of IL-21Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) ED of IL-21Rα; (II) TD of IL-21Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) IL-21 cytokine; (II) ED of IL-21Rα; (III) TD of IL-21Rα; and (IV) ID of IL-2Rβ. In some embodiments, the IL-21 cytokine is tethered to the ED of IL-21Rα via a polypeptide linker.

[0108] In some embodiments, 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 any one of the amino acid sequences shown in SEQ ID NOs: 16-19 and 21. In some embodiments, TD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-19 and 21.

[0109] In some embodiments, a recombinant cytokine receptor comprising the aforementioned TD is expressed in an immune cell. In some embodiments, a recombinant cytokine receptor comprising the aforementioned TD is expressed in a T cell. In some embodiments, a recombinant cytokine receptor comprising the aforementioned TD is expressed in a regulatory T cell (Treg). In some embodiments, Treg cells are CD4+, CD25+, and CD127lo (i.e., CD127low). In some embodiments, Treg expressing a recombinant cytokine receptor comprising the aforementioned TD expresses FOXP3 and / or HELIOS. In some embodiments, Treg expressing a recombinant cytokine receptor comprising the aforementioned TD also expresses high levels of FOXP3 and / or HELIOS. In some embodiments, Treg expressing a recombinant cytokine receptor comprising the aforementioned TD can survive and proliferate in the absence of IL-2. In some embodiments, at least about 60% (such as 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 the Tregs transduced with a recombinant cytokine receptor comprising the aforementioned TD remain viable in vitro 12-20 days after transduction. In some embodiments, at least about 60% (such as at least about 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 the recombinant cytokine receptor comprising the aforementioned TD can persist in vivo for at least about 3 days or longer. In some embodiments, the recombinant cytokine receptor comprising the aforementioned TD expressed in Tregs transduces intracellular IL-2 signaling within Tregs. In some embodiments, the signaling of the recombinant cytokine receptor comprising the aforementioned TD expressed in Tregs induces phosphorylation of STAT5. In some embodiments, Treg expressing a recombinant cytokine receptor comprising the aforementioned TD can suppress the activity of effector T cells in the absence of IL-2, to an extent at least the same as or higher than wild-type Treg 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, Treg expressing a recombinant cytokine receptor comprising the aforementioned TD also expresses a chimeric antigen receptor (CAR).

[0110] 4. Tethered Cytokines and Linkers

[0111] The aforementioned recombinant cytokine receptors can be optionally tethered to their cognate cytokines via a polypeptide linker. In some embodiments, IL-4 can be tethered to the recombinant cytokine receptor IL-4RαED. In some embodiments, the recombinant cytokine receptor tethered to IL-4 comprises the amino acid sequence shown in SEQ ID NO:6. In some embodiments, the recombinant cytokine receptor tethered to IL-4 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the amino acid sequence shown in SEQ ID NO:6. In some embodiments, IL-7 can be tethered to the recombinant cytokine receptor IL-7RαED. In some embodiments, the recombinant cytokine receptor tethered to IL-7 comprises the amino acid sequence shown in SEQ ID NO:7. In some embodiments, the IL-7 tethered 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 shown in SEQ ID NO: 7. In some embodiments, IL-9 can be tethered to the recombinant cytokine receptor IL-9RαED. In some embodiments, the IL-9 tethered recombinant cytokine receptor comprises the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the IL-9 tethered 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 shown in SEQ ID NO: 8. In some embodiments, the IL-9 tethered recombinant cytokine receptor comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the recombinant cytokine receptor tethered to IL-9 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 shown in SEQ ID NO: 15. In some embodiments, IL-21 can be tethered to the recombinant cytokine receptor IL-21RαED. In some embodiments, the recombinant cytokine receptor tethered to IL-21 comprises the amino acid sequence shown in SEQ ID NO: 9.In some embodiments, the IL-21 tethered 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 shown in SEQ ID NO: 9. 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 is capable of signaling in the absence of exogenous interleukin cytokines.

[0112] In some embodiments, the homologous cytokine tethered to the recombinant cytokine receptor via a polypeptide linker comprises any one of SEQ ID NOs: 22-25. In some embodiments, the homologous 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 shown in SEQ ID NOs: 22-25.

[0113] In some embodiments, the recombinant cytokine receptor comprises an IL-4 cytokine tethered to the extracellular domain of IL-4Rα. In some embodiments, IL-4 comprises the amino acid sequence shown in SEQ ID NO: 22. In some embodiments, IL-4 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 shown in SEQ ID NO: 22. In some embodiments, the IL-4 tethered 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 shown in SEQ ID NO:6, wherein the recombinant cytokine receptor comprises an IL-4 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 shown in SEQ ID NO:22, and wherein the recombinant cytokine receptor comprises an IL-4 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 shown in SEQ ID NO:23. The amino acid sequence shown in NO:11 comprises an ED of an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity. In some embodiments, the IL-4 tethered recombinant cytokine receptor comprises an amino acid sequence comprising 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 shown in SEQ ID NO:6, wherein the recombinant cytokine receptor comprises an IL-4 cytokine having an amino acid sequence comprising 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 shown in SEQ ID NO:22, and wherein the recombinant cytokine receptor comprises an ED having an amino acid sequence comprising 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 shown in SEQ ID NO:11.

[0114] In some embodiments, the recombinant cytokine receptor comprises an IL-7 cytokine tethered to the extracellular domain of IL-7Rα. In some embodiments, IL-7 comprises the amino acid sequence shown in SEQ ID NO: 23. In some embodiments, IL-7 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 shown in SEQ ID NO: 23. In some embodiments, the IL-7 tethered 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 shown in SEQ ID NO:7, wherein the recombinant cytokine receptor comprises an IL-7 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 shown in SEQ ID NO:23, and wherein the recombinant cytokine receptor comprises an IL-7 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 shown in SEQ ID NO:23. The amino acid sequence shown in NO:12 comprises an ED of an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity. In some embodiments, the IL-7 tethered recombinant cytokine receptor comprises an amino acid sequence comprising 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 shown in SEQ ID NO:7, wherein the recombinant cytokine receptor comprises an IL-7 cytokine having an amino acid sequence comprising 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 shown in SEQ ID NO:23, and wherein the recombinant cytokine receptor comprises an ED having an amino acid sequence comprising 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 shown in SEQ ID NO:12.

[0115] In some embodiments, the recombinant cytokine receptor comprises an IL-9 cytokine tethered to the extracellular domain of IL-9Rα. In some embodiments, IL-9 comprises the amino acid sequence shown in SEQ ID NO: 24. In some embodiments, IL-9 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 shown in SEQ ID NO: 24. In some embodiments, the IL-9 tethered 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 shown in SEQ ID NO:8, wherein the recombinant cytokine receptor comprises an IL-9 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 shown in SEQ ID NO:24, and wherein the recombinant cytokine receptor comprises an IL-9 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 shown in SEQ ID NO:24. The amino acid sequence shown in NO:13 comprises an ED of an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity. In some embodiments, the IL-9 tethered recombinant cytokine receptor comprises an amino acid sequence comprising 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 shown in SEQ ID NO:8, wherein the recombinant cytokine receptor comprises an IL-9 cytokine having an amino acid sequence comprising 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 shown in SEQ ID NO:24, and wherein the recombinant cytokine receptor comprises an ED having an amino acid sequence comprising 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 shown in SEQ ID NO:13.In some embodiments, the IL-9 tethered 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 shown in SEQ ID NO: 15, wherein the recombinant cytokine receptor comprises an IL-9 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 shown in SEQ ID NO: 24, and wherein the recombinant cytokine receptor comprises an IL-9 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 shown in SEQ ID NO: The amino acid sequence shown in NO:13 comprises an ED of an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity. In some embodiments, the IL-9 tethered recombinant cytokine receptor comprises an amino acid sequence comprising 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 shown in SEQ ID NO: 15, wherein the recombinant cytokine receptor comprises an IL-9 cytokine having an amino acid sequence comprising 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 shown in SEQ ID NO: 24, and wherein the recombinant cytokine receptor comprises an ED having an amino acid sequence comprising 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 shown in SEQ ID NO: 13.

[0116] In some embodiments, the recombinant cytokine receptor comprises an IL-21 cytokine tethered to the extracellular domain of IL-21Rα. In some embodiments, IL-21 comprises the amino acid sequence shown in SEQ ID NO:25. In some embodiments, IL-21 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the amino acid sequence shown in SEQ ID NO:25. In some embodiments, the IL-21 tethered 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 shown in SEQ ID NO:9, wherein the recombinant cytokine receptor comprises an IL-21 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 shown in SEQ ID NO:25, and wherein the recombinant cytokine receptor comprises an IL-21 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 shown in SEQ ID NO:25. The amino acid sequence shown in NO:14 comprises an ED of an amino acid sequence having at least 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. In some embodiments, the IL-21 tethered recombinant cytokine receptor comprises an amino acid sequence comprising 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 shown in SEQ ID NO:9, wherein the recombinant cytokine receptor comprises an IL-21 cytokine having an amino acid sequence comprising 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 shown in SEQ ID NO:25, and wherein the recombinant cytokine receptor comprises an ED having an amino acid sequence comprising 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 shown in SEQ ID NO:14.

[0117] The linker can be a polypeptide linker of any length. In some embodiments, the length of the polypeptide linker is about 1 amino acid to about 10 amino acids, about 2 amino acids to about 15 amino acids, about 3 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 5 amino acids to about 9 amino acids, about 6 amino acids to about 8 amino acids, about 1 amino acid to about 20 amino acids, about 21 amino acids to about 30 amino acids, about 1 amino acid to about 30 amino acids, about 2 amino acids to about 20 amino acids, about 10 amino acids to about 30 amino acids, about 2 amino acids to about 19 amino acids, about 2 amino acids to about 18 amino acids. In some embodiments, the length of the polypeptide linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the length of the polypeptide linker is 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. For example, in some embodiments, the length of the polypeptide linker is about 5 amino acids.In some embodiments, the N-terminus of the polypeptide linker is covalently linked to the C-terminus of the interleukin cytokine, and the C-terminus of the polypeptide linker is covalently linked to the N-terminus of the ED.

[0118] The polypeptide linker can have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody can 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 , where n is an integer of at least one (SEQ ID NO: 29; SEQ ID NO: 27 when n = 2). Glycine and glycine-serine polymers are relatively unstructured and thus may be able to act as neutral tethers between components. Glycine also acquires significantly more sterically and are much less constrained than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). Accordingly, exemplary flexible linkers include, but are not limited to: Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 27), Gly-Gly-Gly-Gly-Ser (SEQ IDNO: 30), Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 31) and Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Leu-Gln (SEQ ID NO: 32). One of ordinary skill will recognize that the design of a recombinant cytokine receptor tethered to its cognate cytokine may include a fully or partially flexible linker such that the linker may include a flexible linker portion and one or more portions that impart a less flexible structure to provide the desired recombinant cytokine receptor structure.

[0119] In some embodiments, the linker between the cytokine and the recombinant cytokine receptor is a stable linker.In some embodiments, the linker between the cytokine and the recombinant cytokine receptor is not cleavable by a protease.

[0120] II. Cells

[0121] The present disclosure provides a kind of adoptive cell immunotherapy composition, it comprises the cell preparation of genetic modification as described herein, for example, the immune cell preparation of genetic modification, for example, lymphocyte.These cells are for example pluripotent cells, such as various progenitor cells or precursor cells of hematopoietic stem cell, hematopoietic pedigree and various immune cells (for example, people's autologous or allogeneic T cell, natural killer (NK) cell, dendritic cell or B cell).These cells can also be pluripotent stem cells (PSC), such as human embryonic stem cell and induction PSC, it can be used to produce treatment cell group.In some embodiments, pluripotent cells and pluripotent cells are differentiated into desired cell type in vitro before being implanted, injected or infused into patient.

[0122] In some alternatives, the genetically modified cell preparation is a T lymphocyte preparation. In some embodiments, the T lymphocyte preparation comprises CD4+T cells, which have (i) a chimeric receptor, which comprises an extracellular antibody domain, a spacer, a transmembrane domain and an intracellular signaling domain of a T cell receptor specific to a ligand associated with a disease or disorder, and (ii) a recombinant cytokine receptor as described herein. In other alternatives, the adoptive cellular immunotherapy composition comprises a CD8+ cytotoxic T lymphocyte preparation modified by a chimeric receptor providing a cellular immune response, wherein the cytotoxic T lymphocyte preparation comprises CD8+T cells, which have (i) a chimeric receptor, which comprises an extracellular antibody domain, a spacer, a transmembrane domain and an intracellular signaling domain of a T cell receptor specific to a ligand associated with a disease or disorder, and (ii) a recombinant cytokine receptor as described herein. In some alternatives, the T cell population modified by the chimeric receptor of the present disclosure can persist in vivo for at least about 3 days or longer. In some alternatives, each of these populations can be combined with each other or with one or more other cell types to provide a composition. In some alternatives, the genetically modified cells are Treg cells.

[0123] Regulatory T (Treg) cells participate in maintaining immune self-tolerance and reducing harmful immune responses to self and non-self (allogeneic) antigens. Treg includes both natural subtypes and induced subtypes. Natural Treg (nTreg) is a cell that originates as a separate cell lineage during development. Peripheral or induced Treg (iTreg) differentiates from conventional T cells. In some embodiments, CD4+T cells of non-nTreg and non-iTreg can be engineered into Treg using the methods and compositions of the present disclosure. In some embodiments, CD4+T cells are used to prepare Treg cells expressing recombinant cytokine receptors using the methods and compositions of the present disclosure.

[0124] In some embodiments, the cells expressing the recombinant cytokine receptors of the present disclosure are not 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 (Treg).

[0125] In some embodiments, the Treg expressing the recombinant cytokine receptor of the present disclosure is not naturally occurring (not nTreg and / or not iTreg). In some embodiments, the cell expresses a unique one or marker of Treg. Treg markers include high levels of CD25 (CD25+), low levels of CD127 (CD127lo), or high CD25 and low CD127. For example, the levels of CD25 and CD127 are compared with CD4+T cells of non-Treg. In some embodiments, Treg cells expressing recombinant cytokine receptors have a high CD25, high CD4 and low CD127 phenotype.

[0126] In some embodiments, the Treg expressing the tethered IL9Rα / IL-2Rβ recombinant cytokine receptor is not naturally occurring (not nTreg and / or not iTreg). In some embodiments, the cell expresses one or markers unique to Treg. Treg markers include high levels of CD25 (CD25+), low levels of CD127 (CD127lo or CD127low), or high CD25 and low CD127. In some embodiments, Treg cells expressing recombinant cytokine receptors have a high CD25, high CD4, and low CD127 phenotype.

[0127] In some embodiments, Tregs expressing tethered IL9Rα / IL-2Rβ recombinant cytokine receptors survive in the absence of exogenous cytokines (e.g., IL-2). In some embodiments, Tregs expressing tethered IL9Rα / IL-2Rβ recombinant cytokine receptors proliferate in the absence of exogenous cytokines (e.g., IL-2). In some embodiments, Tregs expressing tethered IL9Rα / IL-2Rβ recombinant cytokine receptors proliferate in the absence of exogenous cytokines. In some embodiments, Tregs expressing tethered IL9Rα / IL-2Rβ recombinant cytokine receptors are transplanted into individuals, such as rodents (such as mice) or humans. In some embodiments, individuals express IL-2. In some embodiments, Tregs expressing tethered IL9Rα / IL-2Rβ recombinant cytokine receptors survive and proliferate after adoptive cell transfer into individuals (e.g., rodents (such as mice) or humans, e.g., individuals expressing IL-2). In some embodiments, adoptively transferred Tregs expressing tethered IL9Rα / IL-2Rβ recombinant cytokine receptors prevent, treat, ameliorate, or otherwise reduce the severity of a disease, such as an autoimmune disease (eg, graft-versus-host disease).

[0128] Also provided herein are cells expressing recombinant cytokine receptors. In some embodiments, the recombinant cytokine receptor comprises an intracellular IL-2 receptor β chain domain, a transmembrane domain, and an extracellular domain that binds to a cytokine other than IL-2. In some embodiments, the recombinant cytokine receptor comprises an extracellular domain selected from the group consisting of: an IL-4 extracellular domain, an IL-7 extracellular domain, an IL-9 extracellular domain, and an IL-21 extracellular domain. In some embodiments, the cytokine is tethered to the recombinant cytokine receptor. In some embodiments, the cytokine is not IL-2.

[0129] In some embodiments, the cell expresses one or more proteins associated with the Treg phenotype. In some embodiments, the cell expresses FOXP3. FOXP3 plays a key role in the development and function of Treg cells (see Yagi et al. 2004, Int Immunol. 16 (11): 1643-56; Sadlon et al. 2018, Clin Transl Immunology 7 (2): e1011). FOXP3 was initially expressed during the first expansion of T cells, but then FOXP3 expression was lost after polyclonal stimulation. This is in contrast to Treg, in which FOXP3 expression rises and maintains over time. The expression level of FOXP3 can be assessed by conventional methods (such as Western blotting, flow cytometry or ELISA). The expression level can also be assessed by using technology (such as RT-qPCR) to analyze mRNA. In some embodiments, compared with untransduced cells, the expression of FOXP3 increases. In some embodiments, compared with cells without recombinant cytokine receptors, the expression of FOXP3 increases.

[0130] In some embodiments, compared with cells without recombinant cytokine receptors, FOXP3 expression increases by at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times or more. In some embodiments, compared with cells not transduced with recombinant receptors, FOXP3 expression increases by at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times or more. In some embodiments, compared with untransduced cells cultured in the absence of one or more cytokines, the expression of FOXP3 in Tregs transduced with recombinant cytokine receptors increases. In some embodiments, compared with untransduced cells cultured with cytokines that are not IL-2, the expression of FOXP3 in Tregs transduced with recombinant cytokine receptors increases. In some embodiments, FOXP3 expression is maintained at a level approximately the same as the highest expression day of FOXP3 after transduction with recombinant cytokine receptors.

[0131] In some embodiments, the Treg cells are CD4 positive (CD4+). In some embodiments, the cells are CD4+ / CD25+. In some embodiments, the cells are CD4+ / CD127 low (i.e., CD4+ / CD127lo). In some embodiments, the cells are CD4+ / CD25+ / CD127 low (i.e., CD4+ / CD25+ / CD127lo).

[0132] Treg can be characterized by CD25+ expression. In some embodiments, the cell is a Treg cell is CD25+. In some embodiments, the cell is CD4+ / CD25+. In some embodiments, the cell is CD25+ / CD127 low (i.e., CD25+ / CD127lo). In some embodiments, the cell is CD4+ / CD25+ / CD127 low (i.e., CD4+ / CD25+ / CD127lo).

[0133] In some embodiments, the cells express low levels of CD127 (CD127lo). In some embodiments, the cells are CD4+ / CD127 low (i.e., CD4+ / CD127lo). In some embodiments, the cells are CD25+ / CD127 low (i.e., CD25+ / CD127lo). In some embodiments, the cells are CD4+ / CD25+ / CD127 low (i.e., CD4+ / CD25+ / CD127lo). In some embodiments, cells transduced with the recombinant cytokine receptors provided herein maintain a CD127 low (CD127lo) state after transduction.

[0134] HELIOS is a transcription factor expressed in a Treg. In some embodiments, the Treg cells of the present disclosure express HELIOS. In some embodiments, compared with conventional T cells, the expression of HELIOS increases. In some embodiments, compared with conventional T cells, the expression of HELIOS is detectable. The expression level of HELIOS can be assessed by conventional methods (such as Western blot, flow cytometry or ELISA). The expression level can also be assessed by using technology (such as RT-qPCR) to analyze mRNA. In some embodiments, compared with conventional T cells, HELIOS expression increases by at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times or more. In some embodiments, compared with untransduced cells cultured in the absence of cytokines, the expression of HELIOS in Treg transduced with recombinant cytokine receptors increases. In some embodiments, Treg transduced with recombinant cytokine receptors maintains HELIOS expression after transduction.

[0135] In some embodiments, the cells express or secrete low levels of IL-2. In some embodiments, the expression or secretion of IL-2 in transduced Treg cells is reduced compared to conventional T cells. In some embodiments, the expression or secretion of IL-2 in Tregs transduced with recombinant cytokine receptors is reduced compared to untransduced cells cultured in the absence of cytokines. In some embodiments, the expression or secretion of IL-2 in Tregs transduced with recombinant cytokine receptors is reduced compared to untransduced cells.

[0136] In some embodiments, the cell has an immunosuppressive phenotype. In one embodiment, the cell produces an immunosuppressive effect in an individual suffering from an immune-related disorder. In some embodiments, the individual is a human. In some embodiments, the individual expresses IL-2. In some embodiments, the cell is autologous to the individual.

[0137] In some embodiments, the cells suppress, block or inhibit graft-versus-host disease in an individual. In one embodiment, the cells suppress, block or inhibit an immune-related disorder in an individual. In some embodiments, the cells are administered prior to the onset of an immune-related disorder. In some embodiments, the individual is a human. In some embodiments, the individual expresses IL-2. In some embodiments, the cells are autologous to the individual. In some embodiments, the cells are allogeneic to the individual.

[0138] The measuring method for the marker of characterizing Treg cells will be apparent to those of ordinary skill in the art.For example, the T cell populations of Treg or comprising Treg can be cultivated using the method described herein.After cultivation, Treg can be collected and stained using antibodies for Treg markers, such as FOXP3 (for example, labeled by PE), CD25 (for example, labeled by APC) and CD127 (for example, labeled by BV421), and analyzed using fluorescence activated flow cytometry (FACS) or fluorescence microscopy to express.Gene expression can be measured by methods such as RT-qPCR.

[0139] III. Preparation Method

[0140] The recombinant cytokine receptors described herein can be prepared and transduced into host cells, such as Treg cells, by any known protein expression method in the art. For example, see Examples 1-3. A DNA sequence encoding a 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 cells are recovered and cultured to obtain living host cells that stably express the recombinant cytokine receptor of the present invention.

[0141] In some embodiments, the application provides an isolated nucleic acid encoding one or more polypeptides of any one of the recombinant cytokine receptors. The isolated nucleic acid can 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 the expression of the nucleic acid, the vector can be introduced into a host cell to allow the expression of the nucleic acid in the host cell. The expression vector can contain a variety of elements for controlling expression, including but not limited to a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selectable marker and a signal sequence. These elements can be selected as appropriate by a person of ordinary skill in the art. For example, a promoter sequence can be selected to promote the transcription of a polynucleotide in the vector. Suitable promoter sequences include but are not limited to T7 promoters, T3 promoters, SP6 promoters, and β-actin promoters. EF1a promoter, CMV promoter and SV40 promoter. An enhancer sequence can be selected to enhance the transcription of the nucleic acid. The selectable marker may be chosen to allow selection of host cells into which the vector has been inserted from those into which the vector has not been inserted, for example, the selectable marker may be a transmembrane gene, such as EGFR, which can be identified by flow cytometry and FACS analysis.

[0142] 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. In some embodiments, the nucleic acid (e.g., a vector, such as an expression vector, a viral vector, or a cloning vector) encodes an antigen receptor and / or another additional polypeptide. The antigen receptor can be, for example, an antibody, an engineered antibody (such as a scFv, CAR, an engineered TCR, a TCR mimetic, or a chimeric antibody-T cell receptor), or a chimeric signaling receptor. The antigen receptor can target a target antigen (e.g.,Antigens may include, but are not limited to, AFP (alpha-fetoprotein), αvβ6 or other integrins, 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, CD74, CD79a, CD79b, CD98, CD123, CD138, CD171, CD352, CEA (carcinoembryonic antigen), Claudin, c-MET, DLL3 (delta-like protein 3), DLL4, ENPP3 (extracellular nucleotide pyrophosphatase / phosphodiesterase family member 3), EpCAM, EPG-2 (epithelial glycoprotein 2), EPG-40, ephrinB2, EPHa2 (ephrine receptor A2), ERBB dimer, estrogen receptor, ETBR (endothelial reductase angiopeptide B receptor), FAP-α (fibroblast activation protein α), fetal AchR (fetal acetylcholine receptor), FBP (folate binding protein), FCRL5, FR-α (folate receptor α), GCC (guanylate cyclase C), GD2, GD3, GPC2 (glypican-2), GPC3, gp100 (glycoprotein 100), GPNMB (glycoprotein NMB), GPRC5D (G protein-coupled receptor 5D), HER2, HER3, HER4, hepatitis B surface antigen, HL A-A1 (human leukocyte antigen Al), 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 alpha), IL-13Ra2 (IL-13 receptor alpha 2), KDR (kinase insert domain receptor), LI cell adhesion molecule (LI-CAM), Liv-1, LRRC8A (leucine-rich repeat protein 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 complex (e.g.,HLA-A complexed with peptides derived from AFP, KRAS, NY-ESO, MAGE-A, and WT1), NCAM (neural cell adhesion molecule), connexin-4, NKG2D (natural killer cell family 2 member D) ligand, NY-ESO, carcinoembryonic antigen, PD-1, PD-L1, PRAME (melanoma preferentially expressed antigen), 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 of HIV, HBV, HCV, HPV, and other pathogens.

[0143] In some embodiments, the construct comprising a recombinant cytokine receptor described herein comprises a cleavable linker. In some embodiments, the cleavable linker is a 2A polypeptide. In some embodiments, the construct comprising a recombinant cytokine receptor comprises a nucleotide sequence encoding a P2A linker between the nucleotide sequences encoding the recombinant cytokine receptor and a marker (such as EGFR). In some embodiments, 2A-like sequences or "peptide bond skipping" 2A sequences are derived from, for example, many different viruses, including, for example, from the bright vein flat moth. These sequences are sometimes also referred to as "peptide skipping sequences". When this type of sequence is located within a cistron between two polypeptides to be separated, the ribosome appears to skip the peptide bond; in the case of the bright vein flat moth sequence, the bond between the Gly and Pro amino acids at the carboxyl terminal "PGP" is omitted. This can leave two to three polypeptides, such as 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 encoding the 5' of the 2A sequence can end with additional amino acids at the carboxyl terminus, including a Gly residue and any upstream residue in the 2A sequence. The peptide encoding the 3' of the 2A sequence can end with additional amino acids at the amino terminus, including a Pro residue and any downstream residues after the 2A sequence. In some embodiments, the cleavable linker is a 2A polypeptide derived from porcine teschovirus-1 (P2A). In some embodiments, the 2A co-translational sequence is a 2A-like sequence. In some embodiments, the 2A co-translational sequence is T2A (bright vein flat moth virus 2A), F2A (foot-and-mouth disease virus 2A), P2A (porcine teschovirus-1 2A), BmCPV 2A (cytoplasmic polyhedrosis virus 2A), BmIFV 2A (softening disease virus of silkworm 2A) or E2A (equine rhinitis A virus 2A). In some embodiments, the 2A co-translational sequence is T2A-GSG, F2A-GSG, P2A-GSG or E2A-GSG. In some embodiments, the 2A co-translational sequence is selected from the group consisting of T2A, P2A and F2A. By "cleavable linker" is meant a linker that is cleaved by any means, including, for example, non-enzymatic means (such as peptide hopping) or enzymatic means (see, e.g., Donnelly, ML 2001, J. Gen. Virol. 82: 1013-25, hereby incorporated by reference in its entirety). In a specific embodiment, P2A comprises (or consists of) a sequence disclosed herein (e.g., a sequence disclosed in the Examples below).

[0144] In some embodiments, the 2A linker comprises the amino acid sequence of SEQ ID NO: 10 (SGATNFSLLKQAGDVEENPGP). In certain embodiments, the 2A linker further comprises the GSG amino acid sequence at the amino terminus of the polypeptide; in other embodiments, the 2A linker comprises the GSGPR (SEQ ID NO: 28) amino acid sequence at the amino terminus of the polypeptide. Therefore, the term "2A" sequence can refer to the 2A sequence in the examples described herein, or can also refer to the 2A sequence as listed herein, which further comprises the GSG or GSGPR (SEQ ID NO: 28) sequence at the amino terminus of the linker.

[0145] In some embodiments, host cells (e.g., Treg cells) contain the above-mentioned vectors. 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 method, cationic lipid-mediated delivery, liposome-mediated transfection, electroporation, gene gun method, receptor-mediated gene delivery, delivery mediated by polylysine, histone, chitosan and peptide. Standard methods for transducing cells to express target vectors are well known in the art. In some embodiments, the host cell comprises a vector comprising an isolated nucleic acid encoding a recombinant cytokine receptor.

[0146] In some embodiments, the application provides a method for expressing any recombinant cytokine receptor described herein, the method comprising cultivating a host cell containing a separation of a vector, and recovering the recombinant cytokine receptor from the cell culture. Cultivate the separated host cell under conditions allowing the expression of the separated nucleic acid inserted into the vector. Conditions suitable for the expression of polynucleotides may include, but are not limited to, suitable culture medium, 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. One of ordinary skill in the art may appropriately select suitable conditions for the purpose of expression.

[0147] 1. Carrier

[0148] In certain aspects, the disclosure provides nucleic acid molecules encoding any one or more recombinant cytokine receptors described herein. Such nucleic acid molecules can be inserted into appropriate vectors (e.g., viral vectors or non-viral plasmid vectors) for introduction into target host regulatory T cells (Treg).

[0149] As used herein, the term "recombinant" or "non-natural" refers to an organism, microorganism, cell, nucleic acid molecule or vector including at least one genetic change or modified by introducing an exogenous nucleic acid molecule, wherein such change or modification is introduced by genetic engineering. Genetic changes include, for example, the introduction of modifications of expressible nucleic acid molecules encoding proteins, fusion proteins, enzymes, other nucleic acid molecules adding, missing, replacing, or other functional destruction of cellular genetic material. Additional modifications include, for example, non-coding regulatory regions, wherein modifications change the expression of genes or operons. In some embodiments, cells obtained from a subject (such as regulatory T cells (Treg)) can be converted into non-natural or recombinant regulatory T cells (Treg) (e.g., non-natural or recombinant Treg) by introducing nucleic acids encoding recombinant cytokine receptors as described herein, whereby cells express recombinant cytokine receptors located on the cell surface.

[0150] The vector encoding the core virus is referred to herein as a "viral vector". There are a large number of 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, Ann. Rev. Genomics Hum. Genet. 2: 177, 2001). Suitable viral vectors include RNA virus-based vectors, such as retrovirus-derived vectors, e.g., Maloney murine leukemia virus (MLV)-derived vectors, and include more complex retrovirus-derived vectors, e.g., lentivirus-derived vectors. HIV-1-derived vectors belong to this category. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Vesna virus (sheep lentivirus). Methods for using retroviral and lentiviral vectors and packaging cells to transduce mammalian host 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., pLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther. 18:1748, 2010; and Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available.

[0151] In some embodiments, a non-endogenous nucleic acid sequence encoding a recombinant cytokine receptor is introduced using a viral vector. The viral vector can be a retroviral vector or a lentiviral vector. The viral vector can also include a nucleic acid sequence encoding a marker for transduction. The transduction marker of the viral vector is known in the art, including a selection marker that can confer drug resistance, or a detectable marker, such as a fluorescent marker or a cell surface protein that can be detected by a method (such as flow cytometry). In a specific embodiment, the viral vector also includes a gene marker for transduction, which includes a green fluorescent protein, an extracellular domain of human CD2 or a truncated human EGFR (huEGFRt; see Wang et al., Blood 118: 1255, 2011). When the viral vector genome includes multiple nucleic acid sequences to be expressed as a single transcript in a host cell (e.g., T cells, such as Treg), the viral vector can also include another sequence between two (or more) transcripts, allowing bicistronic or polycistronic expression. Examples of such sequences for viral vectors include an internal ribosome entry site (IRES), a furin cleavage site, a viral 2A peptide, or any combination thereof.

[0152] Other vectors can also be used for polynucleotide delivery, including DNA viral vectors, including, for example, adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex virus (HSV), including amplicon vectors, replication-defective HSV, and attenuated HSV (Krisky et al., Gene Ther. 5:1517, 1998).

[0153] Other vectors recently developed for gene therapy use can also be used with the compositions and methods of the present disclosure. Such vectors include those derived from baculovirus and alpha-virus (Jolly, DJ. 1999. EmergingViralVectors. pp. 209-40 Friedmann T. ed. The Development of HumanGeneTherapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as Sleeping Beauty or other transposon vectors).

[0154] In certain embodiments, hematopoietic progenitor cells or embryonic stem cells are modified to include non-endogenous nucleic acid molecules encoding recombinant cytokine receptors disclosed herein. Hematopoietic progenitor cells may include induced pluripotent stem cells, which may be derived from or derived from fetal liver tissue, bone marrow, umbilical cord blood or peripheral blood. Hematopoietic progenitor cells may be from humans, mice, rats or other mammals.

[0155] In certain embodiments, host cells transfected to express a recombinant cytokine receptor of the present disclosure are functional Tregs.

[0156] One or more growth factor cytokines that promote the proliferation of Tregs expressing the recombinant cytokine receptors 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 Tregs include IL-2, TGFβ or the like.

[0157] IV. Methods of Expanding and Cultivating Cells

[0158] In some embodiments, the cell transfected to express the recombinant cytokine receptor of the present disclosure is a eukaryotic cell. In some embodiments, the cell transfected to express the recombinant cytokine receptor of the present disclosure is a human cell. In some embodiments, the cell transfected to express the recombinant cytokine receptor of the present disclosure is a human embryonic kidney (HEK) cell. In certain embodiments, the cell transfected to express the recombinant cytokine receptor of the present disclosure is an immune cell. In certain embodiments, the cell transfected to express the recombinant cytokine receptor of the present disclosure is a T cell. In certain embodiments, the cell transfected to express the recombinant cytokine receptor of the present disclosure is a functional Treg.

[0159] In some embodiments, Treg cells are separated from peripheral blood mononuclear (PBMC) cells. In some embodiments, Treg cells are separated from PBMC 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 CD25+. In some embodiments, Treg cells are enriched by positive selection for CD4+CD25+CD127lo cells. In some embodiments, enrichment occurs by FACS. In some embodiments, Treg is stimulated by anti-CD3 antibodies and / or anti-CD28 antibodies on the 0th day after positive selection. In some embodiments, Treg is stimulated again by anti-CD3 antibodies and / or anti-CD28 antibodies on the 9th day of cultivation after positive selection.

[0160] One or more growth factor cytokines that promote the proliferation of Tregs expressing the recombinant cytokine receptor of the present disclosure can be added to the culture. The cytokine can be a human or non-human cytokine. Exemplary growth factor cytokines that can be used to promote Treg proliferation include IL-4, IL-7, IL-9, IL-21, etc. In some embodiments, the cytokine is added to the culture medium before selection. In some embodiments, the cytokine is added to the culture medium after cell separation. In some embodiments, the cytokine is added to the culture medium for about 15-60 minutes. In some embodiments, the cytokine is added to the culture medium for about 40 minutes. In some embodiments, the cytokine is added to the cell culture about every 12 to 60 hours, such as about 24 to about 48 hours. In some embodiments, the cytokine is added to the culture medium during the culture period. In some embodiments, cells are treated with cytokines, wherein the cells are cultured at a cell density of about 0.25-0.3 million cells / mL. In some embodiments, the concentration of the cytokine during cytokine treatment is about 0.15ng / mL–100ng / mL. In some embodiments, the concentration of IL-2 during cytokine treatment is about 300 IU / mL. In some embodiments, the concentration of IL-2 during cytokine treatment is 300 IU / mL.

[0161] In some embodiments, the cell culture of the recombinant cytokine receptor of the present disclosure is expressed enough time to induce proliferation or differentiation. Generally, the cell is maintained in culture for about 3 days 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 should be understood that the cell can be maintained to reach the desired result (that is, the desired cell composition or proliferation level) required for the appropriate amount of time. For example, in order to produce a cell composition mainly comprising Treg, the cell can be maintained in culture for about 30 days.

[0162] 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.

[0163] In some embodiments, the inhibitory activity of the transduced Treg increases after transduction with the recombinant cytokine receptor provided herein. In some embodiments, the transduced Treg cells provided herein reduce the division rate of conventional (e.g., cytotoxic) T cells. In some embodiments, compared with CD4+T cells cultured in the absence of Treg, the transduced Treg cells provided herein reduce the division rate of CD4+T cells. In some embodiments, compared with CD8+T cells cultured in the absence of Treg, the transduced Treg cells provided herein reduce the division rate of CD8+T cells. In some embodiments, the activity of cytotoxic T cells is reduced.

[0164] In some embodiments, provided herein is a method for expanding transduced Treg cells in the absence of IL-2, the method comprising culturing cells expressing recombinant receptors provided herein. In some embodiments, the cells are cultured in a culture medium comprising a cytokine other than IL-2. In some embodiments, the cells are cultured in a cell culture medium comprising a cytokine that binds to the ED of a recombinant cytokine receptor. In some embodiments, the cells are cultured in a culture medium comprising two, three, four, five or more cytokines.

[0165] V. Pharmaceutical Compositions, Preparations and Kits

[0166] The present application also provides a pharmaceutical composition comprising cells containing the recombinant cytokine receptor described herein, for example, T cells such as Treg cells.

[0167] The pharmaceutical compositions can be suitable for a variety of modes of administration as described herein, including, for example, systemic or local administration. In some embodiments, the pharmaceutical compositions are formulated for intravenous administration.

[0168] The pharmaceutical composition to be used for in vivo administration is usually formulated into a sterile substantially isotonic pharmaceutical composition, and fully complies with all good manufacturing practices (GMP) regulations of the U.S. Food and Drug Administration. Sterility can be easily achieved by filtering with a sterile filtration membrane, such as filtering any liquid solution of Treg cells or pharmaceutical compositions described herein for reconstruction, storage, etc. as described herein. In some embodiments, the composition does not contain pathogens. For injection, the pharmaceutical composition can be in the form of a liquid solution, for example, in a physiologically compatible buffer such as Hanks solution (Hank's solution) or Ringer's solution (Ringer's solution).

[0169] In some embodiments, the pharmaceutical composition is suitable for administration to humans. In some embodiments, the pharmaceutical composition is suitable for administration to rodents (e.g., mice, rats) or non-human primates (e.g., cynomolgus monkeys). In some embodiments, the pharmaceutical composition is cryogenically preserved.

[0170] The application also provides a kit comprising a composition described herein (such as a pharmaceutical composition), and may further include one or more instructions for methods of using the composition, such as the uses described herein. The kit described herein may also include other materials that may be desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any method described herein.

[0171] VI. Treatment Methods

[0172] In some embodiments, there is provided herein a method for treating an immune-related disorder, the method comprising administering a cell (e.g., Treg cell) comprising a recombinant cytokine receptor provided herein. In some embodiments, the recombinant cytokine receptor comprises ED and TD, and the ID of interleukin-2 receptor β (IL-2Rβ), wherein ED is not combined with exogenous IL-2. In some embodiments, the recombinant cytokine receptor used in the method for treating an immune-related disorder can signal in the absence of exogenous IL-2. In some embodiments, the recombinant cytokine receptor used in the method for treating an immune-related disorder comprises interleukin ED, TD and ID. In some embodiments, ED is combined with a cytokine other than IL-2. Therefore, in some embodiments, a method for treating an immune-related disorder is provided, the method comprising administering a cell (e.g., Treg cell) comprising a recombinant cytokine receptor comprising: (I) ED of IL-4Rα, IL-7Rα, IL-9Rα, or IL-21Rα; (II) IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα, or IL-21Rα; and (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises the amino acid sequence shown in SEQ ID NO:1-5. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1-5. 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 cells are Tregs.

[0173] In some embodiments, the method for treating an immune-related disorder comprises administering a cell (e.g., a Treg cell, such as any Treg cell described herein) comprising a recombinant cytokine receptor comprising an ED of IL-4Rα. In some embodiments, the method comprises transducing a cell (e.g., a Treg cell) with a vector encoding a recombinant cytokine receptor. In some embodiments, the cell is autologous to the individual to be treated. In some embodiments, the individual to be treated expresses IL-2. In some embodiments, the ED comprises the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the ED 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% with SEQ ID NO: 11. In some embodiments, the method of treating an immune disorder comprises transducing a cell (e.g., a Treg cell) with a recombinant cytokine receptor comprising, in the N-terminal to C-terminal direction: (I) ED of IL-4Rα; (II) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα, or IL-21Rα; (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) ED of IL-4Rα; (II) TD of IL-4Rα or IL-2Rβ; (III) ID of IL-2Rβ. 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 shown in SEQ ID NO:1, and wherein the recombinant cytokine receptor comprises an ED 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 shown in SEQ ID NO:11. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence comprising 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 shown in SEQ ID NO: 1, and wherein the recombinant cytokine receptor comprises an ED having an amino acid sequence comprising 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 shown 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.

[0174] In some embodiments, the method for immune-related disorders comprises administering a cell (e.g., a Treg cell, such as any Treg cell described herein) comprising a recombinant cytokine receptor comprising the ED of IL-7Rα. In some embodiments, the method comprises transducing a cell (e.g., a Treg cell) with a vector encoding a recombinant cytokine receptor. In some embodiments, the cell is autologous to the individual to be treated. In some embodiments, the individual to be treated expresses IL-2. In some embodiments, the IL-7RαED comprises the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the ED 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% with SEQ ID NO: 12. In some embodiments, the method of treating an immune-related disorder comprises administering a cell (e.g., a Treg cell) comprising a recombinant cytokine receptor comprising, in the N-terminal to C-terminal direction: (I) ED of IL-7Rα; (II) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα, or IL-21Rα; (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) ED of IL-7Rα; (II) TD of IL-7Rα or IL-2Rβ; (III) ID of IL-2Rβ. 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 shown in SEQ ID NO:2, and wherein the recombinant cytokine receptor comprises an ED 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 shown in SEQ ID NO:12. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence comprising 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 shown in SEQ ID NO: 2, and wherein the recombinant cytokine receptor comprises an ED having an amino acid sequence comprising 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 shown in SEQ ID NO: 12. 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.

[0175] In some embodiments, the method for treating an immune-related disorder comprises administering a cell (e.g., a Treg cell, such as any Treg cell described herein) comprising a recombinant cytokine receptor comprising the ED of IL-9Rα. In some embodiments, the method comprises transducing a cell (e.g., a Treg cell) with a vector encoding a recombinant cytokine receptor. In some embodiments, the cell is autologous to the individual to be treated. In some embodiments, the individual to be treated expresses IL-2. In some embodiments, the IL-9RαED comprises the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the ED 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% with SEQ ID NO: 13. In some embodiments, the method of treating an immune-related disorder comprises administering a cell (e.g., a Treg cell) comprising a recombinant cytokine receptor comprising, in the N-terminal to C-terminal direction: (I) ED of IL-9Rα; (II) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα, or IL-21Rα; (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) ED of IL-9Rα; (II) TD of IL-9Rα or IL-2Rβ; (III) ID of IL-2Rβ. 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 shown in SEQ ID NO:3 or 5, and wherein the recombinant cytokine receptor comprises an ED 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 shown in SEQ ID NO:13. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence comprising 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 shown in SEQ ID NO: 3 or 5, and wherein the recombinant cytokine receptor comprises an ED having an amino acid sequence comprising 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 shown in SEQ ID NO: 13. 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.

[0176] In some embodiments, the method for treating immune-related disorders includes the use of a recombinant cytokine receptor comprising the ED of IL-21R α. In some embodiments, the method includes transducing cells (e.g., Treg cells, such as any Treg cells described herein) with a vector encoding a recombinant cytokine receptor. In some embodiments, cells are autologous for the individual to be treated. In some embodiments, the individual to be treated expresses IL-2. In some embodiments, IL-21R α ED includes the amino acid sequence shown in SEQ ID NO:14. In some embodiments, ED includes and SEQ ID NO:14 includes 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% amino acid sequence. In some embodiments, the method of treating an immune-related disorder comprises the use of a recombinant cytokine receptor comprising, in the N-terminal to C-terminal direction: (I) ED of IL-21Rα; (II) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα, or IL-21Rα; (III) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises, in the N-terminal to C-terminal direction: (I) ED of IL-21Rα; (II) TD of IL-21Rα or IL-2Rβ; (III) ID of IL-2Rβ. 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 shown in SEQ ID NO:4, and wherein the recombinant cytokine receptor comprises an ED 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 shown in SEQ ID NO:14. In some embodiments, the recombinant cytokine receptor comprises an amino acid sequence comprising 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 shown in SEQ ID NO: 4, and wherein the recombinant cytokine receptor comprises an ED having an amino acid sequence comprising 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 shown in SEQ ID NO: 14. 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 cells are Tregs.

[0177] In some embodiments, the method for treating an immune-related disorder comprises administering a cell (e.g., a Treg cell, such as any Treg cell described herein) comprising a recombinant cytokine receptor comprising an ED of IL-4Rα. In some embodiments, the method comprises transducing a cell (e.g., a Treg cell) with a vector encoding a recombinant cytokine receptor. In some embodiments, the cell is autologous to the individual to be treated. In some embodiments, the individual to be treated expresses IL-2. In some embodiments, the ED comprises the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the ED 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% with SEQ ID NO: 11. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) IL-4 cytokine; (II) ED of IL-4Rα; (III) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα, or IL-21Rα; and (IV) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) IL-4 cytokine; (II) ED of IL-4Rα; (III) TD of IL-4Rα or IL-2Rβ; and (IV) ID of IL-2Rβ. In some embodiments, the IL-4 cytokine is tethered to the ED of IL-4Rα via a polypeptide linker. In some embodiments, the IL-4 tethered 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 shown in SEQ ID NO:6, wherein the recombinant cytokine receptor comprises an IL-4 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 shown in SEQ ID NO:22, and wherein the recombinant cytokine receptor comprises an IL-4 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 shown in SEQ ID NO:23. The amino acid sequence shown in NO:11 comprises an ED of an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity.In some embodiments, the IL-4 tethered recombinant cytokine receptor comprises an amino acid sequence comprising 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 shown in SEQ ID NO: 6, wherein the recombinant cytokine receptor comprises an IL-4 cytokine having an amino acid sequence comprising 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 shown in SEQ ID NO: 22, and wherein the recombinant cytokine receptor comprises an ED having an amino acid sequence comprising 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 shown 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 cells are Tregs.

[0178] In some embodiments, the method for treating an immune-related disorder comprises administering a cell (e.g., a Treg cell, such as any Treg cell described herein) comprising a recombinant cytokine receptor comprising the ED of IL-7Rα. In some embodiments, the method comprises transducing a cell (e.g., a Treg cell) with a vector encoding a recombinant cytokine receptor. In some embodiments, the cell is autologous to the individual to be treated. In some embodiments, the individual to be treated expresses IL-2. In some embodiments, the IL-7RαED comprises the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the ED 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% with SEQ ID NO: 12. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) IL-7 cytokine; (II) ED of IL-7Rα; (III) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα, or IL-21Rα; and (IV) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) IL-7 cytokine; (II) ED of IL-7Rα; (III) TD of IL-7Rα or IL-2Rβ; and (IV) ID of IL-2Rβ. In some embodiments, the IL-7 cytokine is tethered to the ED of IL-7Rα via a polypeptide linker. In some embodiments, the IL-7 tethered 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 shown in SEQ ID NO:7, wherein the recombinant cytokine receptor comprises an IL-7 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 shown in SEQ ID NO:23, and wherein the recombinant cytokine receptor comprises an IL-7 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 shown in SEQ ID NO:23. The amino acid sequence shown in NO:12 comprises an ED of an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity.In some embodiments, the IL-7 tethered recombinant cytokine receptor comprises an amino acid sequence comprising 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 shown in SEQ ID NO:7, wherein the recombinant cytokine receptor comprises an IL-7 cytokine having an amino acid sequence comprising 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 shown in SEQ ID NO:23, and wherein the recombinant cytokine receptor comprises an ED having an amino acid sequence comprising 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 shown in SEQ ID NO:12. 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 cells are Tregs.

[0179] In some embodiments, the method for treating an immune-related disorder comprises administering a cell (e.g., a Treg cell, such as any Treg cell described herein) comprising a recombinant cytokine receptor comprising the ED of IL-9Rα. In some embodiments, the method comprises transducing a cell (e.g., a Treg cell) with a vector encoding a recombinant cytokine receptor. In some embodiments, the cell is autologous to the individual to be treated. In some embodiments, the individual to be treated expresses IL-2. In some embodiments, the IL-9RαED comprises the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the ED 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% with SEQ ID NO: 13. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) IL-9 cytokine; (II) ED of IL-9Rα; (III) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα, or IL-21Rα; and (IV) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) IL-9 cytokine; (II) ED of IL-9Rα; (III) TD of IL-9Rα or IL-2Rβ; and (IV) ID of IL-2Rβ. In some embodiments, the IL-9 cytokine is tethered to the ED of IL-9Rα via a polypeptide linker. In some embodiments, the IL-9 tethered 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 shown in SEQ ID NO:8 or 15, wherein the recombinant cytokine receptor comprises an IL-9 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 shown in SEQ ID NO:24, and wherein the recombinant cytokine receptor comprises an IL-9 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 shown in SEQ ID NO:25. The amino acid sequence shown in NO:13 comprises an ED of an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity.In some embodiments, the IL-9 tethered recombinant cytokine receptor comprises an amino acid sequence comprising 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 shown in SEQ ID NO: 8 or 15, wherein the recombinant cytokine receptor comprises an IL-9 cytokine having an amino acid sequence comprising 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 shown in SEQ ID NO: 24, and wherein the recombinant cytokine receptor comprises an ED having an amino acid sequence comprising 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 shown in SEQ ID NO: 13. 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.

[0180] In some embodiments, the method for treating immune-related disorders includes administering cells (e.g., Treg cells, such as any Treg cells described herein) comprising recombinant cytokine receptors, which include ED of IL-21R α. In some embodiments, the method includes transducing cells (e.g., Treg cells) with a vector encoding a recombinant cytokine receptor. In some embodiments, cells are autologous for the individual to be treated. In some embodiments, the individual to be treated expresses IL-2. In some embodiments, IL-21R α ED includes the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, ED includes and SEQ ID NO: 14 includes 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% amino acid sequence. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) IL-21 cytokine; (II) ED of IL-21Rα; (III) TD of IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα or IL-21Rα; and (IV) ID of IL-2Rβ. In some embodiments, the recombinant cytokine receptor comprises in the N-terminal to C-terminal direction: (I) IL-21 cytokine; (II) ED of IL-21Rα; (III) TD of IL-21Rα or IL-2Rβ; and (IV) ID of IL-2Rβ. In some embodiments, the IL-21 tethered 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 shown in SEQ ID NO:9, wherein the recombinant cytokine receptor comprises an IL-21 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 shown in SEQ ID NO:25, and wherein the recombinant cytokine receptor comprises an IL-21 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 shown in SEQ ID NO:25. The amino acid sequence shown in NO:14 comprises an ED of an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity.In some embodiments, the IL-21 tethered recombinant cytokine receptor comprises an amino acid sequence comprising 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 shown in SEQ ID NO:9, wherein the recombinant cytokine receptor comprises an IL-21 cytokine having an amino acid sequence comprising 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 shown in SEQ ID NO:25, and wherein the recombinant cytokine receptor comprises an ED having an amino acid sequence comprising 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 shown in SEQ ID NO:14. In some embodiments, the IL-21 cytokine is tethered to the ED of IL-21Rα via a polypeptide linker. 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.

[0181] In some embodiments, the recombinant cytokine receptors of the present disclosure are expressed in T cells. In some embodiments, one or more recombinant cytokine receptors are expressed in T cells. In some embodiments, the recombinant cytokine receptors can be co-expressed with a chimeric antigen receptor (CAR). In some embodiments, T cells are regulatory T cells (Treg). In some embodiments, Treg is CD4+, CD25+, CD127lo. In some embodiments, Treg expresses FOXP3 and / or HELIOS. In some embodiments, the recombinant cytokine receptors are expressed in Treg cells.

[0182] The immune disorders, diseases, disorders and reactions or responses treated according to the methods and compositions of the present invention are meant diseases in which the immune system contributes to the pathogenesis or can be part of the treatment. These reactions include, but are not limited to, cancer, inflammation, autoimmune disorders, disorders or diseases, and persistent and progressive immune responses to infectious non-self antigens from bacteria, viruses (e.g., HCV), fungi or parasitic organisms that invade and persist in mammals and humans. Such disorders and disorders include allergies and / or asthma. Allergies and asthma may be due to sensitization to foreign or non-self antigens such as pollen, animal dander and food proteins. The source of the stimulating foreign antigens may be plants, fungi, molds or other environmental pollutants.

[0183] Autoimmunity is defined as a persistent and progressive immune response to non-infectious self-antigens, as distinct from infectious non-self-antigens from bacteria, viruses, fungi, or parasitic organisms that invade and persist in mammals and humans. Autoimmune conditions include: graft-versus-host disease, autoimmune polyendocrine syndrome, type I diabetes mellitus (TIDM), autoimmune gastritis, autoimmune uveitis, autoimmune vasculitis, colitis, thyroiditis, Addison's disease, agammaglobulinemia, alopecia areata, amyloid storage disease, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune hepatitis, autoimmune inner ear disease, acute motor sensory axonal neuropathy, Behqet's syndrome, and leukemia. disease), bullous herpes, Castleman disease, celiac disease, Chagas disease, acute inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis, Churg-Strauss, cicatricial pemphigoid / benign mucositis pemphigus, Cogan syndrome, cold agglutinin disease, congenital heart block, coxsackievirus myocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, Devic's disease, discoid lupus, Dressler syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis, Graves disease, Guillian-Barre syndrome, Hashim otothyroiditis, hemolytic anemia, Sjögren's purpura, herpes gestationis or pemphigoid gestationis, 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 sclerosus, woody conjunctivitis, linear IgA disease, lupus, Lyme disease, chronic Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, Moren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatic disease, PANDAS (pediatric autoimmune neuropsychiatric disorder with streptococcal infection), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry'sRomber syndrome, pars planitis (intermediate uveitis), Parsonnage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, postcardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia, pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter syndrome, complex The present invention relates to autoantigens, such as chondroitinib, chondroitinib, leukopenia ...

[0184] Cells that can be "allogeneic cells" are those cells that are separated from one individual (donor) and infused into another; and "autologous cells" refer to those cells that are separated and infused back into the same individual. In some embodiments, the cells are autologous to the individual. In some embodiments, the individual is a human. In some embodiments, the individual expresses IL-2. In some embodiments, the cells are T cells. In some embodiments, the cells are Treg. In some embodiments, Treg cells are separated from human peripheral blood mononuclear cells (PBMC). In some embodiments, Treg cells are modified in vitro with vectors encoding one or more recombinant cytokine receptors. In some embodiments, Treg cells are expanded in vitro in the absence of IL-2. In some embodiments, Treg cells are modified with nucleic acids or vectors encoding recombinant cytokine receptors. In some embodiments, Treg cells are modified to proliferate in the absence of IL-2. In some embodiments, Treg cells have one or more IL-2 signaling markers detected. In some embodiments, the IL-2 signaling marker detected is phosphorylated STAT-5. In some embodiments, Tregs are CD4+, CD25+, CD127lo. In some embodiments, Tregs express FOXP3 and / or HELIOS. In some embodiments, Treg cells are administered to the same individual to treat immune-related disorders.

[0185] In some embodiments, the method for treating an immune-related disorder comprises administering Treg cells to an individual in need. In some embodiments, the individual expresses IL-2. In some embodiments, the recombinant cytokine receptor can be co-expressed with a chimeric antigen receptor (CAR). In some embodiments, Treg is CD4+, CD25+, CD127lo. In some embodiments, Treg expresses FOXP3 and / or HELIOS.

[0186] In some embodiments, the method for treating immune-related disorders includes administering autologous Treg cells of the individual. In some embodiments, the individual is a human. In some embodiments, the individual expresses IL-2. In some embodiments, the method for treating immune-related disorders includes administering Treg cells separated from human peripheral blood mononuclear cells (PBMC). In some embodiments, Treg cells are modified in vitro with a vector encoding one or more recombinant cytokine receptors. In some embodiments, Treg cells are expanded in vitro in the absence of IL-2. In some embodiments, the method for treating immune-related disorders includes administering Treg cells modified in vitro with a vector encoding one or more recombinant cytokine receptors. In some embodiments, the method for treating immune-related disorders includes administering Treg cells expanded in vitro in the absence of IL-2. In some embodiments, the method for treating immune-related disorders includes administering Treg cells modified with nucleic acids or vectors encoding recombinant cytokine receptors. In some embodiments, the method for treating immune-related disorders includes administering Treg cells modified with nucleic acids or vectors encoding recombinant cytokine receptors. In some embodiments, the method for treating immune-related disorders includes administering Treg cells modified to proliferate in the absence of IL-2. In some embodiments, the method for treating immune-related disorders includes administering Treg cells, wherein one or more IL-2 signaling markers are detected. In some embodiments, the method for treating an immune-related disorder comprises administering Treg cells, wherein the IL-2 signaling marker detected is phosphorylated STAT-5. In some embodiments, the method for treating an immune-related disorder comprises administering CD4+, CD25+, CD127lo Treg cells. In some embodiments, the method for treating an immune-related disorder comprises administering Treg cells expressing FOXP3 and / or HELIOS. In some embodiments, the method for treating an immune-related disorder comprises administering Treg cells described herein.

[0187] In some embodiments, the method for treating immune-related disorders includes administering Treg cells comprising a recombinant cytokine receptor of the present disclosure. In some embodiments, compared with wild-type Treg, the Treg has an increased inhibitory activity against CD8+ and / or CD4+T cells. In some embodiments, compared with wild-type Treg when cultured in the absence of IL-2, the Treg has an increased inhibitory activity against CD8+ and / or CD4+T cells. In some embodiments, compared with unmodified cells cultured in the absence of IL-2, the Treg has an increased inhibitory activity against CD8+ and / or CD4+T cells. In some embodiments, the inhibitory activity of the Treg against CD8+ and / or CD4+T cells cultured in the absence of IL-2 is roughly equivalent or approximately the same as that of unmodified Treg cells cultured with IL-2. In some embodiments, the inhibitory activity of Treg reduces the division rate of CD4+ and / or CD8+T cells. In some embodiments, the method further includes culturing Treg cells in a composition comprising cytokines. In some embodiments, the method further includes culturing Treg cells in a composition comprising homologous cytokines. In some embodiments, the method further comprises culturing the Treg cells in a composition comprising one or more additional cytokines in addition to IL-2.

[0188] In some embodiments, the method for treating immune-related disorders includes transducing a cell population with a recombinant cytokine receptor. In some embodiments, the method for treating immune-related disorders includes transducing a T cell population with a recombinant cytokine receptor. In some embodiments, the method for treating immune-related disorders includes transducing a Treg cell population with a recombinant cytokine receptor. In some embodiments, the viability of the cell is increased compared to untransduced Treg. In some embodiments, at least about 60%-99% of the cells in the Treg cell population transduced with a recombinant cytokine remain viable in vitro for about 7-20 days after transduction. In some embodiments, at least about 60%-99% of the cells in the Treg cell population transduced with a recombinant cytokine remain viable in vitro for about 7-14 days after transduction. In some embodiments, at least about 60% of the cells in the Treg cell population transduced with a recombinant cytokine remain viable in vitro for about 7 days after transduction. In some embodiments, the method further includes culturing Treg cells in a composition comprising cytokines. In some embodiments, the method further includes culturing Treg cells in a composition comprising homologous cytokines. In some embodiments, the method further comprises culturing the Treg cells in a composition comprising one or more cytokines in addition to IL-2.

[0189] In some embodiments, the method for treating immune-related disorders includes transducing a cell population with a recombinant cytokine receptor. In some embodiments, the method for treating immune-related disorders includes transducing a T cell population with a recombinant cytokine receptor. In some embodiments, the method for treating immune-related disorders includes transducing a Treg cell population with a recombinant cytokine receptor. In some embodiments, the viability of the cell is increased compared to untransduced Treg. In some embodiments, at least about 60%-99% of the cells in the Treg cell population transduced with a recombinant cytokine can persist in vivo for at least about 3 days or longer. In some embodiments, at least about 60%-99% of the cells in the Treg cell population transduced with a recombinant cytokine can persist in vivo for at least about 3 days or longer. In some embodiments, at least about 60% of the cells in the Treg cell population transduced with a recombinant cytokine can persist in vivo for at least about 3 days or longer. In some embodiments, at least about 60% of the cells in the Treg cell population transduced with a recombinant cytokine can persist in vivo for at least about 3 days or longer. In some embodiments, the Treg cell population transduced with a recombinant cytokine can persist in vivo for at least about 1, 2, 3, 5, 6, 8, 10 or 11 months, or at least one year or longer.

[0190] In some embodiments, the method for treating immune-related disorders includes transducing a cell population with a recombinant cytokine receptor. In some embodiments, the method for treating immune-related disorders includes transducing a T cell population with a recombinant cytokine receptor. In some embodiments, the method for treating immune-related disorders includes transducing a Treg cell population with a recombinant cytokine receptor, which produces a cell population containing more viable cells than a composition comprising the same Treg cell population not transduced with a recombinant cytokine receptor. In some embodiments, at least about 60%-99% of the cells in the Treg cell population transduced with a recombinant cytokine remain viable in vitro for about 7-20 days after transduction. In some embodiments, at least about 60%-99% of the cells in the Treg cell population transduced with a recombinant cytokine remain viable in vitro for about 7-14 days after transduction. In some embodiments, at least about 60% of the cells in the Treg cell population transduced with a recombinant cytokine remain viable in vitro for about 7 days after transduction. In some embodiments, the method further includes culturing Treg cells in a composition comprising cytokines. In some embodiments, the method further comprises culturing the Treg cells in a composition comprising a cognate cytokine.

[0191] In some embodiments, the method for treating immune-related disorders includes transducing a cell population with a recombinant cytokine receptor. In some embodiments, the method for treating immune-related disorders includes transducing a T cell population with a recombinant cytokine receptor. In some embodiments, the method for treating immune-related disorders includes transducing a Treg cell population with a recombinant cytokine receptor, which produces a cell population containing more viable cells than a composition comprising the same Treg cell population not transduced with a recombinant cytokine receptor. In some embodiments, at least about 60%-99% of the cells in the Treg cell population transduced with a recombinant cytokine can persist in vivo for at least about 3 days or longer. In some embodiments, at least about 60%-99% of the cells in the Treg cell population transduced with a recombinant cytokine can persist in vivo for at least about 3 days or longer. In some embodiments, at least about 60% of the cells in the Treg cell population transduced with a recombinant cytokine can persist in vivo for at least about 3 days or longer. In some embodiments, at least about 60% of the cells in the Treg cell population transduced with a recombinant cytokine can persist in vivo for at least about 3 days or longer. In some embodiments, a population of Treg cells transduced with a recombinant cytokine can persist in vivo for at least about 1, 2, 3, 5, 6, 8, 10, or 11 months, or at least a year or longer.

[0192] In some embodiments, the method for treating an immune-related disorder comprises transducing a transduced Treg cell population with a recombinant cytokine receptor, wherein the transduced Treg cell population expands at least twice as much as the same Treg cell population not transduced with the recombinant cytokine receptor. In some embodiments, the method for treating an immune-related disorder comprises a composition, wherein the Treg cell population transduced with a recombinant cytokine receptor expands at least twice, and wherein the Treg cell population transduced with a recombinant cytokine receptor maintains expression of at least one Treg marker selected from the group consisting of CD4+, CD25+, and CD127lo.

[0193] In some embodiments, the recombinant cytokine receptors of the present disclosure can also be used in combination with chimeric antigen receptors (CARs). In some embodiments, the modified Tregs comprise recombinant cytokine receptors and chimeric antigens.

[0194] VII. Definitions

[0195] It should be understood that certain features of the disclosure described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, various features of the disclosure described in the context of a single embodiment for the sake of brevity may also be provided separately or in any suitable sub-combination. All combinations of embodiments with respect to specific method steps, reagents, or conditions are specifically encompassed by the disclosure and are disclosed herein as if each combination were individually and explicitly disclosed.

[0196] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0197] "About" values ​​or parameters mentioned herein refer to the usual error ranges for the respective values ​​that are readily known to those skilled in the art. References herein to "about" a value or parameter include (and describe) aspects directed to the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0198] When used herein, the term "and / or" is considered to be a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include: "A and B"; "A or B"; "A" (alone) and "B" (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0199] It is to be understood that aspects and embodiments of the present invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0200] As used herein, the terms "including," "containing," and "comprising" are used in an open, non-limiting sense.

[0201] "Percentage (%) of amino acid sequence identity" or "homology" for polypeptides and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the compared polypeptides after the sequences are aligned and any conservative substitutions are considered as part of the sequence identity. Alignment for the purpose of determining percentage of amino acid sequence identity can be achieved in various ways that are within the skill of the art, such as using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequences. However, for the purposes of this article, the sequence comparison computer program ALIGN-2 is used to generate amino acid sequence identity % values. The ALIGN-2 sequence comparison computer program is written by Genentech, Inc., and the source code has been submitted to the U.S. Copyright Office, Washington DC, 20559, together with user documentation, and it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California. The ALIGN-2 program should be compiled for use with a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and cannot be changed.

[0202] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, livestock (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as rhesus monkeys and cynomolgus monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0203] "Treatment" or "therapy" of a subject refers to any type of intervention or procedure performed on a subject, or the administration of an active agent to a subject, with the intent to cure, reverse, alleviate, ameliorate, inhibit, slow, or prevent the onset, progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical markers associated with a disease.

[0204] An "effective amount" or "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of disease or promotes regression of disease (as manifested by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of impairment or disability caused by disease affliction). The ability of a therapeutic agent to promote regression of disease can be evaluated using a variety of methods known to the skilled artisan, such as in human subjects during clinical trials, in animal model systems predictive of human efficacy, or by measuring the activity of the agent in in vitro assays.

[0205] The phrase "pharmaceutically acceptable" means that the substance or composition must be compatible chemically and / or toxicologically with the other ingredients making up the formulation and / or the mammal to be treated therewith.

[0206] As used herein, the term "tethered" includes fusion, connection, linkage, attachment, etc., and can include any methods known to those skilled in the art for fusion, connection, linkage, attachment, etc. For example, two polypeptide sequences can be "tethered" or otherwise fused, linked, linked, attached, etc. using a polypeptide or peptide linker.

[0207] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range is understood to include any integer and fraction thereof (where appropriate) (such as one tenth and one hundredth of an integer) within the range. Descriptions of endpoints include ranges between all endpoints disclosed. For example, descriptions of 1, 2, or 3 include 1-2, 2-3, and 1-3 ranges.

[0208] Implementation

[0209] 1. A recombinant cytokine receptor comprising an extracellular cytokine receptor domain, a transmembrane domain and an intracellular IL-2 receptor β chain domain, wherein the extracellular cytokine receptor domain binds to a cytokine other than IL-2.

[0210] 2. A recombinant cytokine receptor comprising an extracellular cytokine receptor domain, a transmembrane domain and an intracellular IL-2 receptor β domain, wherein the extracellular cytokine receptor domain binds and is tethered to a cytokine other than IL-2.

[0211] 3. A recombinant cytokine receptor as described in embodiment 1 or 2, wherein the extracellular cytokine receptor domain is selected from the group consisting of: an IL-4 extracellular domain, an IL-7 extracellular domain, an IL-9 extracellular domain, and an IL-21 extracellular domain.

[0212] 4. A recombinant cytokine receptor as described in any of embodiments 2-3, wherein the extracellular cytokine receptor domain is tethered to the cytokine via a polypeptide linker.

[0213] 5. A recombinant cytokine receptor as described in embodiment 4, wherein the peptide linker comprises a glycine residue and a serine residue.

[0214] 6. A recombinant cytokine receptor as described in embodiment 1, wherein the extracellular cytokine receptor domain is not tethered to the cytokine.

[0215] 7. A recombinant cytokine receptor as described in any of embodiments 1-6, wherein the cytokine receptor participates in IL-2 signaling in the absence of IL-2.

[0216] 8. A cytokine receptor as described in any of embodiments 1-7, wherein the transmembrane domain is a transmembrane domain of a cytokine receptor.

[0217] 9. A recombinant cytokine receptor as described in embodiment 8, wherein the transmembrane domain is the transmembrane domain of IL-9 receptor, IL-2 receptor, IL-4 receptor, IL-7 receptor or IL-21 receptor.

[0218] 10. A recombinant cytokine receptor as described in any of embodiments 1-9, wherein the intracellular domain comprises 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 the amino acid sequence shown in SEQ ID NO:20.

[0219] 11. A recombinant cytokine receptor as described in any of embodiments 1-10, wherein the extracellular domain 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 the amino acid sequence shown in SEQ ID NO:11-14.

[0220] 12. A recombinant cytokine receptor as described in any of embodiments 1-11, wherein the transmembrane domain 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 the amino acid sequence shown in SEQ ID NOs:16-19.

[0221] 13. A recombinant cytokine receptor as described in any of embodiments 1-12, wherein the cytokine receptor comprises 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 the amino acid sequence shown in SEQ ID NO:6-9.

[0222] 14. A nucleic acid encoding a recombinant cytokine receptor as described in any one of embodiments 1-13.

[0223] 15. A vector comprising the nucleic acid of embodiment 14.

[0224] 16. The vector of embodiment 15, wherein the vector is a lentiviral vector.

[0225] 17. The vector of embodiment 15 or 16, further comprising a marker gene.

[0226] 18. The vector of embodiment 17, wherein the marker gene is a transmembrane protein.

[0227] 19. The vector of embodiment 18, wherein the transmembrane protein is EGFR.

[0228] 20. A T cell comprising a recombinant cytokine receptor as described in any one of embodiments 1-13, a nucleic acid as described in embodiment 14, or a vector as described in any one of embodiments 15-19.

[0229] 21. The T cell of embodiment 20, wherein the T cell is a regulatory T cell (Treg), wherein the Treg is CD4+, CD25+ and CD127lo.

[0230] 22. The T cell of embodiment 21, wherein the Treg expresses FOX3P and / or HELIOS.

[0231] 23. The T cell of any one of embodiments 20-22, further comprising a chimeric antigen receptor (CAR).

[0232] 24. A composition comprising the nucleic acid of embodiment 14, the vector of any one of embodiments 15-20, or the T cell of any one of embodiments 21-23.

[0233] 25. A method for treating an immune-related disorder, the method comprising administering the T cell of any one of embodiments 20-23 or the composition of embodiment 24 to an individual in need thereof.

[0234] 26. The method of embodiment 25, wherein the cells are autologous to the individual.

[0235] 27. The method of any one of embodiments 25 to 26, wherein the individual is a human.

[0236] 28. A method for expanding transduced Treg cells in the absence of IL-2, the method comprising introducing the nucleic acid of embodiment 14 or the vector of any one of embodiments 15-19 into the Treg cells, and culturing the cells in the absence of IL-2.

[0237] 29. The method of embodiment 28, further comprising detecting at least one Treg marker selected from the group consisting of CD4+, CD25+ and CD127.

[0238] 30. The method of embodiment 29, further comprising detecting FOXP3 and / or HELIOS.

[0239] 31. The method of any one of embodiments 28-30, wherein the Treg cells are capable of proliferating in the absence of IL-2.

[0240] 32. The method of any one of embodiments 28-31, wherein one or more IL-2 signaling markers are detected.

[0241] 33. The method of any one of embodiments 28-32, wherein the one or more IL-2 signaling markers include phosphorylated STAT5.

[0242] 34. A method as described in any one of embodiments 28-33, wherein the Treg has increased suppressive activity on CD8+ and / or CD4+ T cells.

[0243] 35. The method of embodiment 34, wherein the division rate of CD4+ and / or CD8+ T cells is reduced.

[0244] 36. The method of any one of embodiments 28-35, wherein the relative amount of Treg cells in the composition comprising a population of Treg cells transduced with a recombinant cytokine receptor increases over time.

[0245] 37. A method as described in any of embodiments 28-36, wherein the composition comprising a population of Treg cells transduced with the recombinant cytokine receptor contains more viable cells compared to a composition comprising the same population of Treg cells not transduced with the recombinant cytokine receptor.

[0246] 38. A method as described in embodiment 36 or embodiment 37, wherein at least 60% of the cells in the Treg cell population transduced with the recombinant cytokine receptor remain viable 7 days after transduction.

[0247] 39. The method of any one of embodiments 28-38, further comprising culturing the Treg cells in a composition comprising the cytokine.

[0248] 40. The method of any one of embodiments 28-39, further comprising culturing the Treg cells in a composition comprising one or more cytokines in addition to IL-2.

[0249] 41. A method as described in any of embodiments 28-40, wherein the expansion of the Treg cell population transduced with the recombinant cytokine receptor is at least twice that of the same Treg cell population not transduced with the recombinant cytokine receptor.

[0250] 42. A method as described in any of embodiments 28-41, wherein the Treg cell population transduced with the recombinant cytokine receptor is expanded at least two-fold, and wherein the Treg cell population transduced with the recombinant cytokine receptor maintains expression of at least one Treg marker selected from the group consisting of CD4+, CD25+ and CD1271o.

[0251] 43. A recombinant cytokine receptor comprising an extracellular cytokine receptor domain, a transmembrane domain and an intracellular IL-2 receptor β chain domain, wherein the extracellular cytokine receptor domain is capable of binding to a cytokine other than IL-2.

[0252] 44. A recombinant cytokine receptor comprising an extracellular cytokine receptor domain, a transmembrane domain and an intracellular IL-2 receptor β domain, wherein the extracellular cytokine receptor domain is capable of binding and tethering to a cytokine other than IL-2.

[0253] 45. A recombinant cytokine receptor as described in embodiment 43 or embodiment 44, wherein the extracellular cytokine receptor domain is selected from the group consisting of: an IL-4 extracellular domain, an IL-7 extracellular domain, an IL-9 extracellular domain, and an IL-21 extracellular domain.

[0254] 46. ​​A recombinant cytokine receptor as described in embodiment 44 or embodiment 45, wherein the extracellular cytokine receptor domain is tethered to the cytokine via a polypeptide linker.

[0255] 47. A recombinant cytokine receptor as described in embodiment 46, wherein the polypeptide linker comprises a glycine residue and a serine residue.

[0256] 48. The recombinant cytokine receptor of claim 43 or claim 45, wherein the extracellular cytokine receptor domain is not tethered to the cytokine.

[0257] 49. A recombinant cytokine receptor as described in any of embodiments 43-48, wherein the recombinant cytokine receptor participates in IL-2 signaling in the absence of IL-2.

[0258] 50. The cytokine receptor of any one of embodiments 43-49, wherein the transmembrane domain is a transmembrane domain of a cytokine receptor.

[0259] 51. A recombinant cytokine receptor as described in embodiment 50, wherein the transmembrane domain is the transmembrane domain of the IL-9 receptor, the IL-2 receptor, the IL-4 receptor, the IL-7 receptor or the IL-21 receptor.

[0260] 52. A recombinant cytokine receptor as described in any of embodiments 43-51, wherein the TM and the ED are from the same cytokine receptor.

[0261] 53. A recombinant cytokine receptor as described in any of embodiments 43-5, wherein the intracellular domain comprises 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 the amino acid sequence shown in SEQ ID NO:20.

[0262] 54. A recombinant cytokine receptor as described in any of embodiments 43-53, wherein the extracellular domain 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 the amino acid sequence shown in SEQ ID NO: 11-14 or 21.

[0263] 55. A recombinant cytokine receptor as described in any of embodiments 43-54, wherein the transmembrane domain 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 the amino acid sequence shown in SEQ ID NOs: 16-19.

[0264] 56. The recombinant cytokine receptor of any one of embodiments 43-55, wherein:

[0265] the intracellular domain 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 the amino acid sequence shown in SEQ ID NO:20;

[0266] The extracellular domain comprises 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 the amino acid sequence shown in SEQ ID NOs: 11-14; and / or

[0267] The transmembrane domain 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 shown in SEQ ID NOs: 16-19.

[0268] 57. The recombinant cytokine receptor of any one of embodiments 43-56, wherein the recombinant cytokine receptor comprises:

[0269] a) IL-4Rα extracellular domain, IL-4Rα transmembrane domain and IL-2Rβ intracellular domain;

[0270] b) IL-4Rα extracellular domain, IL-2Rβ transmembrane domain and IL-2Rβ intracellular domain;

[0271] c) IL-7Rα extracellular domain, IL-7Rα transmembrane domain and IL-2Rβ intracellular domain;

[0272] d) IL-7Rα extracellular domain, IL-2Rβ transmembrane domain and IL-2Rβ intracellular domain;

[0273] e) IL-9Rα extracellular domain, IL-9Rα transmembrane domain and IL-2Rβ intracellular domain;

[0274] f) IL-9Rα extracellular domain, IL-2Rβ transmembrane domain and IL-2Rβ intracellular domain;

[0275] g) IL-21Rα extracellular domain, IL-21Rα transmembrane domain, and IL-2Rβ intracellular domain; or

[0276] h) IL-21Rα extracellular domain, IL-2Rβ transmembrane domain and IL-2Rβ intracellular domain.

[0277] 58. The recombinant cytokine receptor of any one of embodiments 43-57, wherein:

[0278] a) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:16, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:11;

[0279] b) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:17, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:12;

[0280] c) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:18, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:13;

[0281] d) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:21, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:13;

[0282] e) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:19, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:14;

[0283] f) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:16, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:11, and the cytokine comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:22;

[0284] g) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:17, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:12, and the cytokine comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:23;

[0285] h) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:18, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:13, and the cytokine comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:24;

[0286] i) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:21, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:13, and the cytokine comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:24; or

[0287] j) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:19, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:14, and the cytokine comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:25.

[0288] 59. A recombinant cytokine receptor as described in any of embodiments 43, 45 and 48-58, 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 the amino acid sequence shown in SEQ ID NO:1-5.

[0289] 60. A recombinant cytokine receptor as described in any of embodiments 43-47 and 49-59, 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 the amino acid sequence shown in SEQ ID NO:6-9 or 15.

[0290] 61. A nucleic acid encoding a recombinant cytokine receptor as described in any one of embodiments 43-60.

[0291] 62. A vector comprising the nucleic acid of embodiment 61.

[0292] 63. The vector of embodiment 62, wherein the vector is a lentiviral vector.

[0293] 64. The vector of embodiment 62 or 63, further comprising a marker gene.

[0294] 65. The vector of embodiment 64, wherein the marker gene is a transmembrane protein.

[0295] 66. The vector of embodiment 65, wherein the transmembrane protein is EGFR.

[0296] 67. A T cell comprising a recombinant cytokine receptor as described in any of embodiments 43-60, a nucleic acid as described in embodiment 61, or a vector as described in any of embodiments 62-66.

[0297] 68. The T cell of embodiment 67, wherein the T cell is a regulatory T cell (Treg), wherein the Treg is CD4+, CD25+ and CD127lo.

[0298] 69. The T cell of embodiment 68, wherein the Treg expresses FOX3P and / or HELIOS.

[0299] 70. The T cell of any one of embodiments 67-69, further comprising a chimeric antigen receptor (CAR).

[0300] 71. A composition comprising the nucleic acid of embodiment 61, the vector of any one of embodiments 62-66, or the T cell of any one of embodiments 67-70.

[0301] 72. A method for expanding Treg cells in the absence of IL-2, the method comprising introducing the nucleic acid as described in embodiment 61 or the vector as described in any one of embodiments 62-66 into the Treg cells, and culturing the cells in vitro in the absence of IL-2.

[0302] 73. The method of embodiment 72, further comprising detecting at least one Treg marker selected from the group consisting of CD4+, CD25+ and CD127.

[0303] 74. The method of embodiment 73, further comprising detecting FOXP3 and / or HELIOS.

[0304] 75. The method of any one of embodiments 72-74, wherein the Treg cells are capable of proliferating in the absence of IL-2.

[0305] 76. The method of any one of embodiments 72-75, wherein one or more IL-2 signaling markers are detected.

[0306] 77. The method of any one of embodiments 72-76, wherein the one or more IL-2 signaling markers include phosphorylated STAT5.

[0307] 78. A method as described in any of embodiments 72-77, wherein the Treg has increased suppressive activity on CD8+ and / or CD4+ T cells.

[0308] 79. The method of embodiment 78, wherein the division rate of CD4+ and / or CD8+ T cells is reduced.

[0309] 80. The method of any one of embodiments 72-79, wherein the relative amount of Treg cells in the composition comprising a population of Treg cells transduced with a recombinant cytokine receptor increases over time.

[0310] 81. The method of any one of embodiments 72-80, wherein a composition comprising a population of Treg cells transduced with the recombinant cytokine receptor contains more viable cells than a composition comprising the same population of Treg cells not transduced with the recombinant cytokine receptor.

[0311] 82. A method as described in embodiment 80 or embodiment 81, wherein at least 60% of the cells in the Treg cell population transduced with the recombinant cytokine receptor remain viable 7 days after transduction.

[0312] 83. The method of any one of embodiments 72-82, further comprising culturing the Treg cells in a composition comprising the cytokine.

[0313] 84. The method of any one of embodiments 72-83, further comprising culturing the Treg cells in a composition comprising one or more cytokines in addition to IL-2.

[0314] 85. A method as described in any of embodiments 72-84, wherein the expansion of the Treg cell population transduced with the recombinant cytokine receptor is at least twice that of the same Treg cell population not transduced with the recombinant cytokine receptor.

[0315] 86. A method as described in any of embodiments 72-85, wherein the Treg cell population transduced with the recombinant cytokine receptor is expanded at least two-fold, and wherein the Treg cell population transduced with the recombinant cytokine receptor maintains expression of at least one Treg marker selected from the group consisting of CD4+, CD25+ and CD1271o.

[0316] 87. A regulatory T cell (Treg) comprising a recombinant cytokine receptor, wherein the recombinant cytokine receptor comprises:

[0317] An extracellular cytokine receptor domain, a transmembrane domain, and an intracellular IL-2 receptor β chain domain, wherein the extracellular cytokine receptor domain binds to a cytokine other than IL-2.

[0318] 88. The Treg of embodiment 87, wherein the extracellular cytokine receptor domain is tethered to the cytokine.

[0319] 89. The Treg of embodiment 87 or embodiment 88, wherein the extracellular cytokine receptor domain is selected from the group consisting of an IL-4 extracellular domain, an IL-7 extracellular domain, an IL-9 extracellular domain, and an IL-21 extracellular domain.

[0320] 90. The Treg of embodiment 88 or embodiment 89, wherein the extracellular cytokine receptor domain is tethered to the cytokine via a polypeptide linker, optionally wherein the polypeptide linker comprises a glycine residue and a serine residue.

[0321] 91. The Treg of embodiment 87 or embodiment 89, wherein the extracellular cytokine receptor domain is not tethered to the cytokine.

[0322] 92. The Treg of any one of embodiments 87-91, wherein the recombinant cytokine receptor participates in IL-2 signaling in the absence of IL-2.

[0323] 93. The Treg of any one of embodiments 87-92, wherein the transmembrane domain is a transmembrane domain of a cytokine receptor.

[0324] 94. The Treg of embodiment 93, wherein the transmembrane domain is the transmembrane domain of an IL-9 receptor, an IL-2 receptor, an IL-4 receptor, an IL-7 receptor, or an IL-21 receptor.

[0325] 95. The Treg of any one of embodiments 87-94, wherein the TM and the ED are from the same cytokine receptor.

[0326] 96. The Treg of any one of embodiments 87-95, wherein:

[0327] The intracellular domain 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 shown in SEQ ID NO:20; and / or

[0328] The extracellular domain comprises 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 the amino acid sequence shown in SEQ ID NOs: 11-14; and / or

[0329] The transmembrane domain comprises 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 the amino acid sequence shown in SEQ ID NO: 16-19 or 21.

[0330] 97. The Treg of any one of embodiments 87-96, wherein the recombinant cytokine receptor comprises:

[0331] a) IL-4Rα extracellular domain, IL-4Rα transmembrane domain and IL-2Rβ intracellular domain;

[0332] b) IL-4Rα extracellular domain, IL-2Rβ transmembrane domain and IL-2Rβ intracellular domain;

[0333] c) IL-7Rα extracellular domain, IL-7Rα transmembrane domain and IL-2Rβ intracellular domain;

[0334] d) IL-7Rα extracellular domain, IL-2Rβ transmembrane domain and IL-2Rβ intracellular domain;

[0335] e) IL-9Rα extracellular domain, IL-9Rα transmembrane domain and IL-2Rβ intracellular domain;

[0336] f) IL-9Rα extracellular domain, IL-2Rβ transmembrane domain and IL-2Rβ intracellular domain;

[0337] g) IL-21Rα extracellular domain, IL-21Rα transmembrane domain, and IL-2Rβ intracellular domain; or

[0338] h) IL-21Rα extracellular domain, IL-2Rβ transmembrane domain and IL-2Rβ intracellular domain.

[0339] 98. The Treg of any one of embodiments 87-97, wherein:

[0340] a) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:16, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:11;

[0341] b) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:17, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:12;

[0342] c) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:18, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:13;

[0343] d) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:21, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:13;

[0344] e) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:19, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:14;

[0345] f) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:16, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:11, and the cytokine comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:22;

[0346] g) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:17, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:12, and the cytokine comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:23;

[0347] h) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:18, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:13, and the cytokine comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:24;

[0348] i) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:21, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at leas...

Claims

1. A regulatory T cell (Treg) comprising a recombinant cytokine receptor, wherein the recombinant cytokine receptor comprises: An extracellular cytokine receptor domain, a transmembrane domain, and an intracellular IL-2 receptor β chain domain, wherein the extracellular cytokine receptor domain binds to a cytokine other than IL-2.

2. The Treg of claim 1, wherein the extracellular cytokine receptor domain is tethered to the cytokine.

3. The Treg according to claim 1 or 2, wherein the extracellular cytokine receptor domain is selected from the group consisting of an IL-4 extracellular domain, an IL-7 extracellular domain, an IL-9 extracellular domain, and an IL-21 extracellular domain.

4. The Treg according to any one of claims 2 to 3, wherein the extracellular cytokine receptor domain is tethered to the cytokine via a polypeptide linker, optionally wherein the polypeptide linker comprises a glycine residue and a serine residue. The Treg of claim 1 , wherein the extracellular cytokine receptor domain is not tethered to the cytokine.

6. The Treg of any one of claims 1-5, wherein the recombinant cytokine receptor participates in IL-2 signaling in the absence of IL-2.

7. The Treg of any one of claims 1-6, wherein the transmembrane domain is a transmembrane domain of a cytokine receptor.

8. The Treg of claim 7, wherein the transmembrane domain is a transmembrane domain of an IL-9 receptor, an IL-2 receptor, an IL-4 receptor, an IL-7 receptor, or an IL-21 receptor.

9. The Treg according to any one of claims 1 to 8, wherein the TM and the ED are from the same cytokine receptor.

10. The Treg according to any one of claims 1 to 9, wherein: The intracellular domain 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 shown in SEQ ID NO:20; and / or The extracellular domain comprises 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 the amino acid sequence shown in SEQ ID NOs: 11-14; and / or The transmembrane domain comprises 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 the amino acid sequence shown in SEQ ID NO: 16-19 or 21.

11. The Treg of any one of claims 1-10, wherein the recombinant cytokine receptor comprises: a) IL-4Rα extracellular domain, IL-4Rα transmembrane domain and IL-2Rβ intracellular domain; b) IL-4Rα extracellular domain, IL-2Rβ transmembrane domain and IL-2Rβ intracellular domain; c) IL-7Rα extracellular domain, IL-7Rα transmembrane domain and IL-2Rβ intracellular domain; d) IL-7Rα extracellular domain, IL-2Rβ transmembrane domain and IL-2Rβ intracellular domain; e) IL-9Rα extracellular domain, IL-9Rα transmembrane domain and IL-2Rβ intracellular domain; f) IL-9Rα extracellular domain, IL-2Rβ transmembrane domain and IL-2Rβ intracellular domain; g) IL-21Rα extracellular domain, IL-21Rα transmembrane domain, and IL-2Rβ intracellular domain; or h) IL-21Rα extracellular domain, IL-2Rβ transmembrane domain and IL-2Rβ intracellular domain.

12. The Treg according to any one of claims 1 to 11, wherein: a) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:16, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:11; b) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:17, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:12; c) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:18, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:13; d) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:21, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:13; e) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:19, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:14; f) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:16, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:11, and the cytokine comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:22; g) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:17, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:12, and the cytokine comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:23; h) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:18, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:13, and the cytokine comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:24; i) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:21, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:13, and the cytokine comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:24; or j) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:19, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:14, and the cytokine comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:

25.

13. The Treg of any one of claims 1, 3, and 5-12, 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: 1-5.

14. The Treg of any one of claims 1-4 and 6-13, wherein the recombinant cytokine receptor comprises an amino acid sequence 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-9 and 15.

15. The Treg of any one of claims 1-14, wherein the Treg is CD4+, CD25+ and CD127lo, optionally wherein the Treg expresses FOX3P and / or HELIOS.

16. The Treg of any one of claims 1-15, further comprising a chimeric antigen receptor (CAR).

17. The Treg according to any one of claims 1 to 16, for use in treating an immune-related disorder in an individual.

18. Tregs for use as claimed in claim 17, wherein the Treg cells are autologous to the individual, optionally wherein the individual is a human.

19. The Treg for use according to claim 17 or claim 18, wherein the Treg cell prevents, improves or cures immune-related disorders.

20. A recombinant cytokine receptor comprising an extracellular cytokine receptor domain, a transmembrane domain and an intracellular IL-2 receptor β chain domain, wherein the extracellular cytokine receptor domain is capable of binding to a cytokine other than IL-2.

21. A recombinant cytokine receptor comprising an extracellular cytokine receptor domain, a transmembrane domain and an intracellular IL-2 receptor β domain, wherein the extracellular cytokine receptor domain is capable of binding and tethering to a cytokine other than IL-2.

22. The recombinant cytokine receptor of claim 20 or 21, wherein the extracellular cytokine receptor domain is selected from the group consisting of an IL-4 extracellular domain, an IL-7 extracellular domain, an IL-9 extracellular domain, and an IL-21 extracellular domain.

23. A recombinant cytokine receptor as claimed in claim 21 or claim 22, wherein the extracellular cytokine receptor domain is tethered to the cytokine via a polypeptide linker, optionally wherein the polypeptide linker comprises a glycine residue and a serine residue.

24. The recombinant cytokine receptor of claim 20 or claim 22, wherein the extracellular cytokine receptor domain is not tethered to the cytokine.

25. The recombinant cytokine receptor of any one of claims 20-24, wherein the recombinant cytokine receptor participates in IL-2 signaling in the absence of IL-2.

26. A recombinant cytokine receptor as described in any one of claims 20-25, wherein the transmembrane domain is a transmembrane domain of a cytokine receptor, optionally wherein the transmembrane domain is a transmembrane domain of an IL-9 receptor, an IL-2 receptor, an IL-4 receptor, an IL-7 receptor or an IL-21 receptor.

27. The recombinant cytokine receptor of any one of claims 20-26, wherein the TM and the ED are from the same cytokine receptor.

28. The recombinant cytokine receptor according to any one of claims 20 to 27, wherein: the intracellular domain 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 the amino acid sequence shown in SEQ ID NO:20; The extracellular domain comprises 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 the amino acid sequence shown in SEQ ID NOs: 11-14; and / or The transmembrane domain comprises 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 the amino acid sequence shown in SEQ ID NO: 16-19 or 21.

29. The recombinant cytokine receptor of any one of claims 20-28, wherein the recombinant cytokine receptor comprises: a) IL-4Rα extracellular domain, IL-4Rα transmembrane domain and IL-2Rβ intracellular domain; b) IL-4Rα extracellular domain, IL-2Rβ transmembrane domain and IL-2Rβ intracellular domain; c) IL-7Rα extracellular domain, IL-7Rα transmembrane domain and IL-2Rβ intracellular domain; d) IL-7Rα extracellular domain, IL-2Rβ transmembrane domain and IL-2Rβ intracellular domain; e) IL-9Rα extracellular domain, IL-9Rα transmembrane domain and IL-2Rβ intracellular domain; f) IL-9Rα extracellular domain, IL-2Rβ transmembrane domain and IL-2Rβ intracellular domain; g) IL-21Rα extracellular domain, IL-21Rα transmembrane domain, and IL-2Rβ intracellular domain; or h) IL-21Rα extracellular domain, IL-2Rβ transmembrane domain and IL-2Rβ intracellular domain.

30. The recombinant cytokine receptor of any one of claims 20-29, wherein: a) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:16, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:11; b) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:17, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:12; c) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:18, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:13; d) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:21, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:13; e) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:19, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:14; f) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:16, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:11, and the cytokine comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:22; g) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:17, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:12, and the cytokine comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:23; h) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:18, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:13, and the cytokine comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:24; i) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:21, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:13, and the cytokine comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:24; or j) the intracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20, the transmembrane domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:19, and the extracellular domain comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:14, and the cytokine comprises an amino acid sequence comprising at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:

25.

31. A recombinant cytokine receptor as described in any of claims 20, 22, and 24-30, 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: 1-5.

32. A recombinant cytokine receptor as described in any of claims 20-23 and 25-31, 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: 6-9 and 15.

33. A nucleic acid encoding the recombinant cytokine receptor of any one of claims 20-32.

34. A vector comprising the nucleic acid of claim 33.

35. A method for expanding Treg cells in the absence of IL-2, the method comprising introducing the nucleic acid of claim 33 or the vector of claim 34 into the Treg cells, and culturing the cells in the absence of IL-2.

36. The method of claim 35, further comprising detecting at least one Treg marker selected from the group consisting of CD4+, CD25+ and CD127, and optionally further comprising detecting FOXP3 and / or HELIOS.

37. The method of claim 35 or claim 36, wherein the Treg cells are capable of proliferating in the absence of IL-2.

38. The method of any one of claims 35-37, wherein one or more endogenous IL-2 signaling markers are detected, optionally wherein the one or more endogenous IL-2 signaling markers comprise phosphorylated STAT-5.

39. The method of any one of claims 35-38, wherein the method produces Treg cells comprising a recombinant cytokine receptor, wherein: i. Compared with control Treg, the Treg has increased in vitro and / or in vivo inhibitory activity on CD8+ and / or CD4+ T cells; ii. the division rate of CD4+ and / or CD8+ T cells is reduced when cultured in the presence of said Tregs compared to the division rate of CD4+ and / or CD8+ T cells when cultured in the absence of said Tregs; iii. the relative amount of Treg cells in the 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 the recombinant cytokine receptor contains more viable cells than a composition comprising the same population of Treg cells not transduced with the recombinant cytokine receptor; v. At least 60% of the cells in the Treg cell population transduced with the recombinant cytokine receptor remain viable in vitro 7 days after transduction; vi. Treg proliferation levels are increased by one or more restimulations in vitro and / or in vivo; vii. The Tregs produce an increased level of IL-10 cytokine in vitro and / or in vivo compared to control Tregs; viii. the Tregs produce increased levels of IFN-γ cytokines in vitro and / or in vivo compared to control Tregs; and / or ix. The Tregs produce increased levels of Gr-B cytokines in vitro and / or in vivo compared to control Tregs.

40. The method of any one of claims 35-39, further comprising culturing the Treg cells in a composition comprising the cytokine, optionally wherein the method further comprises culturing the Treg cells in a composition comprising one or more additional cytokines in addition to IL-2.

41. The method of any one of claims 35-40, wherein the Treg cell population transduced with the recombinant cytokine receptor expands at least twice as much as the same Treg cell population not transduced with the recombinant cytokine receptor, optionally wherein the Treg cell population transduced with the recombinant cytokine receptor expands at least twice, and wherein the Treg cell population transduced with the recombinant cytokine receptor maintains expression of at least one Treg marker selected from the group consisting of CD4+, CD25+, and CD1271o.

42. The Treg for use as claimed in any one of claims 17 to 19, wherein the individual expresses IL-2.

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