Chimeric cytokine receptors

Abstract

To provide: variant cytokine receptors that will be able to specifically undergo activation, proliferation, and other immune functions in response to an administered cytokine; and methods of producing cells that express variant cytokine receptors.SOLUTION: Described herein are chimeric receptors comprising extracellular domains of G-CSFR and intracellular domains of various multi-subunit cytokine receptors for selective activation of cytokine signaling in cells of interest. In certain aspects, the selective activation of cytokine signaling in cells expressing the chimeric receptors described herein includes an ability to specifically stimulate adoptively transferred cells.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 912,223, filed October 8, 2019, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that was submitted via EFS-Web and is incorporated herein by reference in its entirety. The above ASCII copy, created on October 7, 2020, is named IKE001WO_SL.txt and is 60,383 bytes in size.

[0003] FIELD OF THE INVENTION Described herein are chimeric cytokine receptors comprising the extracellular domain of G-CSFR (granulocyte colony-stimulating factor receptor) and the intracellular domain of various multisubunit cytokine receptors for selectively activating cytokine signaling in cells of interest. Specifically, the present disclosure describes novel chimeric cytokine receptors comprising multisubunit intracellular signaling domains for delivering specific cytokine-like signals to cells of interest. The present disclosure also includes methods, cells, and kits for use in adoptive cell transfer (ACT), comprising cells expressing the chimeric cytokine receptor, and / or expression vectors encoding the chimeric cytokine receptor, and / or cytokines that bind to the chimeric cytokine receptor. [Background technology]

[0004] 2. Description of Related Art Patients undergoing cell-based immunotherapy treatments often receive cytokine therapy in the form of interleukin-2 (IL-2). IL-2 therapy is beneficial to these patients because it provides signals for proliferation, survival, and effector function to adoptively transferred immune cells (e.g., T lymphocytes or NK lymphocytes), improving their efficacy. However, patients receiving IL-2 therapy can experience significant and severe toxicity due to the effects of IL-2 on host immune cells. Therefore, there is a clinical need for variant cytokine receptors and methods for producing cells expressing variant cytokine receptors that specifically respond to administered cytokines to undergo activation, proliferation, and other immune functions, thereby reducing or eliminating dose-limiting toxicity and reducing or eliminating the need for lymphodepleting chemotherapy prior to immune cell infusion. Summary of the Invention

[0005] In certain embodiments, described herein are chimeric receptors, wherein the chimeric receptor comprises: (a) an extracellular domain (ECD) of G-CSFR (granulocyte colony-stimulating factor receptor) operably linked to a second domain; (b) comprising at least a portion of the intracellular domain (ICD) of a multisubunit cytokine receptor selected from the group consisting of IL-2R (interleukin-2 receptor), IL-7R (interleukin-7 receptor), IL-12R (interleukin-12 receptor), and IL-21R (interleukin-21 receptor), optionally wherein the IL-2R is selected from the group consisting of IL-2Rβ and IL-2Rγc, and optionally wherein the second domain comprises at least a portion of the C-terminal region of IL-2Rβ, IL-7Rα, IL-12Rβ2, or IL-21R; wherein at least a portion of the ICD of the cytokine receptor comprises at least one signaling molecule binding site of the intracellular domain of the cytokine receptor, optionally wherein the at least one signaling molecule binding site is selected from the group consisting of a STAT3 binding site of G-CSFR; a STAT3 binding site of gp130; a SHP-2 binding site of gp130; a SHC binding site of IL-2Rβ; STAT5-binding site in IL-2Rβ; STAT3-binding site in IL-2Rβ; STAT1-binding site in IL-2Rβ; STAT5-binding site in IL-7Rα; phosphatidylinositol 3-kinase (PI3K)-binding site in IL-7Rα; STAT4-binding site in IL-12Rβ2; STAT5-binding site in IL-12Rβ2; STAT3-binding site in IL-12Rβ2; STAT5-binding site in IL-21R; STAT3-binding site in IL-21R; and STAT1-binding site in IL-21R. and optionally, the ICD comprises the Box 1 and Box 2 regions of a protein selected from the group consisting of G-CSFR and gp130; and optionally, the chimeric receptor comprises a third domain comprising at least a portion of a transmembrane domain of a protein selected from the group consisting of G-CSFR, gp130 (glycoprotein 130), and IL-2Rβ, wherein optionally the transmembrane domain is a wild-type transmembrane domain.

[0006] In certain aspects, described herein are chimeric receptors comprising the ECD of G-CSFR operably linked to a second domain, wherein the second domain is (i) (a) The transmembrane domain of gp130; (b) the Box1 and Box2 regions of gp130, and (c) the C-terminal region of IL-2Rβ, or (ii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-2Rβ, or (iii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-12Rβ2, or (iv) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-21R, or (v) (a) The transmembrane domain of IL-2Rβ+γc; (b) the Box1 and Box2 regions of IL-2Rβ+γc, and (c) the C-terminal region of IL-2Rβ+γc, or (vi) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) A chimeric receptor containing the C-terminal region of IL-7Rα.

[0007] In certain embodiments, the activated chimeric receptor forms a homodimer, and optionally activation of the chimeric receptor causes a cellular response selected from the group consisting of proliferation, survival, and enhanced activity of a cell expressing the chimeric receptor, and optionally the chimeric receptor is activated by contact with G-CSF, and optionally the G-CSF is wild-type G-CSF, and optionally the extracellular domain of G-CSFR is a wild-type extracellular domain. In certain embodiments, the activated chimeric receptor forms a homodimer, and optionally activation of the chimeric receptor causes a cellular response selected from the group consisting of proliferation, survival, and enhanced activity of a cell expressing the chimeric receptor, and optionally the chimeric receptor is activated by contact with G-CSF, and optionally the G-CSF is wild-type G-CSF, and optionally the extracellular domain of G-CSFR is a wild-type extracellular domain.

[0008] In certain aspects, the chimeric receptor is expressed in a cell, optionally an immune cell, optionally a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, wherein the cell is a stem cell, optionally the cell is a primary cell, optionally the cell is a human cell.

[0009] In certain embodiments, the ICD is selected from the group consisting of (a) at least a portion of the ICD of IL-2Rβ having the amino acid sequence of SEQ ID NO: 16, 19, 21, 29, 31, 33, 35, 37, or 39; or (b) at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 41; or (c) at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO: 25 or 27; or (d) at least a portion of the ICD of IL-12Rβ2 having the amino acid sequence of SEQ ID NO: 23, 32, or 26. or (e) at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 34, 40, or 42; or (f) at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO: 18 or 38; or (g) at least a portion of the ICD of IL-7R having the amino acid sequence of SEQ ID NO: 43; or (h) at least a portion of the ICD of IL-2RG having the amino acid sequence of SEQ ID NO: 17.

[0010] In certain embodiments, the transmembrane domain is (a) SEQ ID NO: 8, or (b) SEQ ID NO: 9, or (c) SEQ ID NO: 10, or (d) SEQ ID NO: 11. In certain aspects, described herein is a cell comprising a nucleic acid encoding a chimeric receptor, the chimeric receptor comprising: (a) an extracellular domain (ECD) of G-CSFR (granulocyte colony-stimulating factor receptor) operably linked to a second domain; and (b) a multisubunit subunit selected from the group consisting of IL-2R (interleukin-2 receptor), IL-7R (interleukin-7 receptor), IL-12R (interleukin-12 receptor), and IL-21R (interleukin-21 receptor). and optionally, the second domain comprises at least a portion of the intracellular domain (ICD) of a cytokine receptor, wherein optionally the IL-2R is selected from the group consisting of IL-2Rβ and IL-2Rγc, and optionally the second domain comprises at least a portion of the C-terminal region of IL-2Rβ, IL-7Rα, IL-12Rβ2, or IL-21R; wherein at least a portion of the ICD of the cytokine receptor comprises at least one signaling molecule binding site of the intracellular domain of the cytokine receptor, and optionally the ICD comprises at least one signaling molecule binding site of G-CSFR; STAT3 binding site in IL-2Rβ; SHP-2 binding site in gp130; SHC binding site in IL-2Rβ; STAT5 binding site in IL-2Rβ; STAT3 binding site in IL-2Rβ; STAT1 binding site in IL-2Rβ; STAT5 binding site in IL-7Rα; phosphatidylinositol 3-kinase (PI3K) binding site in IL-7Rα; STAT4 binding site in IL-12Rβ2; STAT5 binding site in IL-12Rβ2; STAT3 binding site in IL-12Rβ2; STAT5 binding site in IL-21R; STAT3 binding site in IL-21R; and the STAT1 binding site of IL-21R; optionally, the ICD comprises the Box 1 and Box 2 regions of a protein selected from the group consisting of G-CSFR and gp130; optionally, the chimeric receptor comprises a third domain comprising at least a portion of a transmembrane domain of a protein selected from the group consisting of G-CSFR, gp130 (glycoprotein 130), and IL-2Rβ, wherein optionally the transmembrane domain is a wild-type transmembrane domain.In certain embodiments, the ECD of G-CSFR is encoded by the nucleic acid sequence set forth in SEQ ID NO: 5 or 6. In certain embodiments, the nucleic acid is: (a) a sequence encoding at least a portion of the ICD of IL-2Rβ having the amino acid sequence of SEQ ID NO: 16, 19, 21, 29, 31, 33, 35, 37, or 39; or (b) a sequence encoding at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 41; or (c) a sequence encoding at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO: 25 or 27; or (d) a sequence encoding at least a portion of the ICD of IL-12Rβ2 having the amino acid sequence of SEQ ID NO: 23, 32, or 26; or (e) a sequence encoding at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 34, 40, or 42; or (f) a sequence encoding at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO: 18 or 38; or (g) a sequence encoding at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 43; or (h) a sequence encoding at least a portion of the ICD of IL-2Rγc having the amino acid sequence of SEQ ID NO: 17.

[0011] In certain embodiments, the present disclosure describes an expression vector comprising a nucleic acid encoding a chimeric receptor described herein, hi certain embodiments, the vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and a plasmid. In certain aspects, described herein is a nucleic acid encoding a chimeric receptor, wherein the chimeric receptor comprises an ECD of G-CSFR operably linked to a second domain; and the second domain comprises: (i) (a) The transmembrane domain of gp130; (b) the Box1 and Box2 regions of gp130, and (c) the C-terminal region of IL-2Rβ, or (ii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-2Rβ, or (iii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-12Rβ2, or (iv) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-21R, or (v) (a) The transmembrane domain of IL-2Rβ+γc; (b) the Box1 and Box2 regions of IL-2Rβ+γc, and (c) the C-terminal region of IL-2Rβ+γc, or (vi) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) a nucleic acid encoding a chimeric receptor comprising the C-terminal region of IL-7Rα.

[0012] In certain embodiments, the ECD of G-CSFR is encoded by the nucleic acid sequence set forth in SEQ ID NO: 5 or 6. In certain embodiments, the nucleic acid is (a) a sequence encoding at least a portion of the ICD of IL-2Rβ having the amino acid sequence of SEQ ID NO: 16, 19, 21, 29, 31, 33, 35, 37, or 39; or (b) a sequence encoding at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 41; or (c) a sequence encoding at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO: 25 or 27; or (d) a sequence encoding at least a portion of the ICD of IL-12Rβ2 having the amino acid sequence of SEQ ID NO: 23, 32, or 26; or (e) a sequence encoding at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 34, 40, or 42; or (f) a sequence encoding at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO: 18 or 38; or (g) a sequence encoding at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 43; or (h) a sequence encoding at least a portion of the ICD of IL-2Rγc having the amino acid sequence of SEQ ID NO: 17.

[0013] In certain aspects, described herein is an expression vector comprising a nucleic acid described herein, hi certain embodiments, the vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and a plasmid.

[0014] In certain aspects, described herein is a cell comprising a nucleic acid encoding a chimeric receptor, wherein the chimeric receptor is (a) an extracellular domain (ECD) of G-CSFR (granulocyte colony-stimulating factor receptor) operably linked to a second domain; (b) comprising at least a portion of the intracellular domain (ICD) of a multisubunit cytokine receptor selected from the group consisting of IL-2R (interleukin-2 receptor), IL-7R (interleukin-7 receptor), IL-12R (interleukin-12 receptor), and IL-21R (interleukin-21 receptor), optionally wherein the IL-2R is selected from the group consisting of IL-2Rβ and IL-2Rγc, and optionally wherein the second domain comprises at least a portion of the C-terminal region of IL-2Rβ, IL-7Rα, IL-12Rβ2, or IL-21R; at least a portion of the ICD of the cytokine receptor comprises at least one signaling molecule binding site of the intracellular domain of the cytokine receptor, optionally wherein the ICD comprises at least one of: a STAT3 binding site of G-CSFR; a STAT3 binding site of gp130; a SHP-2 binding site of gp130; a SHC binding site of IL-2Rβ; a STAT5 binding site of IL-2Rβ; a STAT3-binding site of IL-2Rβ; a STAT1-binding site of IL-2Rβ; a STAT5-binding site of IL-7Rα; a phosphatidylinositol 3-kinase (PI3K)-binding site of IL-7Rα; a STAT4-binding site of IL-12Rβ2; a STAT5-binding site of IL-12Rβ2; a STAT3-binding site of IL-12Rβ2; a STAT5-binding site of IL-21R; a STAT3-binding site of IL-21R; and a STAT1-binding site of IL-21R. at least one signal transduction molecule binding site; optionally, the ICD comprises a Box 1 region and a Box 2 region of a protein selected from the group consisting of G-CSFR and gp130; optionally, the chimeric receptor comprises a third domain comprising at least a portion of a transmembrane domain of a protein selected from the group consisting of G-CSFR, gp130 (glycoprotein 130), and IL-2Rβ, wherein optionally the transmembrane domain is a wild-type transmembrane domain; Optionally, the cell is an immune cell, optionally a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, optionally wherein the cell is a stem cell, optionally wherein the cell is a primary cell, optionally wherein the cell is a human cell, a cell comprising a nucleic acid encoding a chimeric receptor.

[0015] In certain aspects, described herein is a cell comprising a nucleic acid encoding a chimeric receptor, wherein the chimeric receptor comprises an ECD of G-CSFR operably linked to a second domain; and the second domain comprises: (i) (a) The transmembrane domain of gp130; (b) the Box1 and Box2 regions of gp130, and (c) the C-terminal region of IL-2Rβ, or (ii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-2Rβ, or (iii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-12Rβ2, or (iv) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-21R, or (v) (a) The transmembrane domain of IL-2Rβ+γc; (b) the Box1 and Box2 regions of IL-2Rβ+γc, and (c) the C-terminal region of IL-2Rβ+γc, or (vi) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) C-terminal region of IL-7Rα Includes; Optionally, the cell is an immune cell, optionally a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, wherein the cell is a stem cell, optionally wherein the cell is a primary cell, optionally wherein the cell is a human cell, wherein the cell comprises a nucleic acid encoding a chimeric receptor.

[0016] In certain embodiments, the ECD of G-CSFR is encoded by a nucleic acid contained in a cell having the sequence set forth in SEQ ID NO: 5 or 6. In certain embodiments, the nucleic acid is (a) a sequence encoding at least a portion of the ICD of IL-2Rβ having the amino acid sequence of SEQ ID NO: 16, 19, 21, 29, 31, 33, 35, 37, or 39; or (b) a sequence encoding at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 41; or (c) a sequence encoding at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO: 25 or 27; or (d) a sequence encoding at least a portion of the ICD of IL-12Rβ2 having the amino acid sequence of SEQ ID NO: 23, 32, or 26; or (e) a sequence encoding at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 34, 40, or 42; or (f) a sequence encoding at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO: 18 or 38; or (g) a sequence encoding at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 43; or (h) a sequence encoding at least a portion of the ICD of IL-2Rγc having the amino acid sequence of SEQ ID NO: 17.

[0017] In certain aspects, described herein are cells comprising an expression vector described herein, optionally the cell is an immune cell, optionally a T cell or an NK cell. In certain aspects, described herein are cells comprising the chimeric receptor of claim 1, optionally the cell is an immune cell, optionally a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, optionally the cell is a stem cell, optionally the cell is a primary cell, optionally the cell is a human cell. In certain aspects, described herein is a cell comprising a chimeric receptor described herein, optionally the cell is an immune cell, optionally a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, optionally the cell is a stem cell, optionally the cell is a primary cell, optionally the cell is a human cell.

[0018] In certain aspects, described herein are methods for selectively activating a chimeric receptor expressed on the surface of a cell, comprising contacting the chimeric receptor with G-CSF, which selectively activates the chimeric receptor; the chimeric receptor comprising (a) an extracellular domain (ECD) of G-CSFR (granulocyte colony-stimulating factor receptor) operably linked to a second domain; the second domain comprising (b) an extracellular domain (ECD) of IL-2R (interleukin-2 receptor), IL-7R (interleukin-7 receptor), or the like. and optionally, the second domain comprises at least a portion of the intracellular domain (ICD) of a multisubunit cytokine receptor selected from the group consisting of IL-2R (interleukin-12 receptor), IL-12R (interleukin-12 receptor), and IL-21R (interleukin-21 receptor), wherein the IL-2R is selected from the group consisting of IL-2Rβ and IL-2Rγc, and optionally, the second domain comprises at least a portion of the C-terminal region of IL-2Rβ, IL-7Rα, IL-12Rβ2, or IL-21R; At least a portion of the receptor's ICD comprises at least one signaling molecule binding site of the intracellular domain of the cytokine receptor, and optionally the at least one signaling molecule binding site is selected from the group consisting of: a STAT3 binding site of G-CSFR; a STAT3 binding site of gp130; a SHP-2 binding site of gp130; a SHC binding site of IL-2Rβ; a STAT5 binding site of IL-2Rβ; a STAT3 binding site of IL-2Rβ; a STAT1 binding site of IL-2Rβ; and a STAT5 binding site of IL-7Rα. the phosphatidylinositol 3-kinase (PI3K) binding site of IL-7Rα; the STAT4 binding site of IL-12Rβ2; the STAT5 binding site of IL-12Rβ2; the STAT3 binding site of IL-12Rβ2; the STAT5 binding site of IL-21R; the STAT3 binding site of IL-21R; and the STAT1 binding site of IL-21R; optionally, the ICD comprises the Box 1 and Box 2 regions of a protein selected from the group consisting of G-CSFR and gp130;Optionally, the method of selectively activating a chimeric receptor, wherein the chimeric receptor comprises a third domain comprising at least a portion of a transmembrane domain of a protein selected from the group consisting of G-CSFR, gp130 (glycoprotein 130), and IL-2Rβ, and optionally the transmembrane domain is a wild-type transmembrane domain;

[0019] In certain aspects, described herein are methods of selectively activating a chimeric receptor expressed on the surface of a cell, comprising contacting the chimeric receptor with G-CSFR, which selectively activates the chimeric receptor; the chimeric receptor comprising an ECD of G-CSFR operably linked to a second domain; and the second domain comprising: (i) (a) The transmembrane domain of gp130; (b) the Box1 and Box2 regions of gp130, and (c) the C-terminal region of IL-2Rβ, or (ii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-2Rβ, or (iii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-12Rβ2, or (iv) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-21R, or (v) (a) The transmembrane domain of IL-2Rβ+γc; (b) the Box1 and Box2 regions of IL-2Rβ+γc, and (c) the C-terminal region of IL-2Rβ+γc, or (vi) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) C-terminal region of IL-7Rα A method for selectively activating a chimeric receptor, comprising:

[0020] In certain embodiments of the methods described herein, the activated chimeric receptor forms a homodimer, and optionally activation of the chimeric receptor results in a cellular response selected from the group consisting of proliferation, survival, and enhanced activity of a cell expressing the chimeric receptor; optionally, the chimeric receptor is activated by contact with G-CSF, optionally the G-CSF is wild-type G-CSF, and optionally the extracellular domain of G-CSFR is a wild-type extracellular domain; the chimeric receptor is expressed in a cell, optionally an immune cell, optionally a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, and optionally the cell is a stem cell, and optionally the cell is a primary cell, and optionally the cell is a human cell.

[0021] In certain embodiments of the methods described herein, the chimeric receptor comprises: (a) at least a portion of the ICD of IL-2Rβ having the amino acid sequence of SEQ ID NO: 16, 19, 21, 29, 31, 33, 35, 37, or 39; or (b) at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 41; or (c) at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO: 25 or 27; or (d) at least a portion of the ICD of IL-12Rβ2 having the amino acid sequence of SEQ ID NO: 23, 32, or 26; or (e) at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 34, 40, or 42; or (f) at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO: 18 or 38; or (g) at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 43; or (h) comprises at least a portion of the ICD of IL-2Rγc having the amino acid sequence of SEQ ID NO: 17; and the transmembrane domain comprises the sequence set forth in (a) SEQ ID NO: 8, or (b) SEQ ID NO: 9, or (c) SEQ ID NO: 10, or (d) SEQ ID NO: 11.

[0022] In certain aspects, described herein are methods of making a chimeric receptor in a cell, comprising introducing into the cell a nucleic acid of any one of claims 13-16, or 19-22, or an expression vector of any one of claims 17, 18, 23, or 24; optionally, the method comprises gene editing; optionally, the cell is an immune cell, optionally a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, optionally the cell is a stem cell, optionally the cell is a primary cell, and optionally the cell is a human cell. In certain embodiments, described herein are methods of treating a subject in need thereof, comprising injecting into the subject cells expressing a chimeric receptor and administering a cytokine that binds to the chimeric receptor; wherein the chimeric receptor (a) an extracellular domain (ECD) of G-CSFR (granulocyte colony-stimulating factor receptor) operably linked to a second domain; (b) comprising at least a portion of the intracellular domain (ICD) of a multisubunit cytokine receptor selected from the group consisting of IL-2R (interleukin-2 receptor), IL-7R (interleukin-7 receptor), IL-12R (interleukin-12 receptor), and IL-21R (interleukin-21 receptor), optionally wherein the IL-2R is selected from the group consisting of IL-2Rβ and IL-2Rγc, and optionally wherein the second domain is selected from the group consisting of IL-2Rβ, IL-2Rγc, ...γc, IL-2Rγc, IL-2 at least a portion of the C-terminal region of IL-7Rα, IL-12Rβ2, or IL-21R; wherein at least a portion of the ICD of the cytokine receptor comprises at least one signaling molecule binding site of the intracellular domain of the cytokine receptor, and optionally the ICD comprises at least one signaling molecule binding site of the intracellular domain of the cytokine receptor, and optionally the ICD comprises at least one signaling molecule binding site of the STAT3 binding site of G-CSFR; a STAT3 binding site of gp130; a SHP-2 binding site of gp130; a SHC binding site of IL-2Rβ; a STAT5 binding site of IL-2Rβ; a STAT3 binding site of IL-2Rβ a STAT1-binding site of IL-2Rβ; a STAT5-binding site of IL-7Rα; a phosphatidylinositol 3-kinase (PI3K)-binding site of IL-7Rα; a STAT4-binding site of IL-12Rβ2; a STAT5-binding site of IL-12Rβ2; a STAT3-binding site of IL-12Rβ2; a STAT5-binding site of IL-21R; a STAT3-binding site of IL-21R; and a STAT1-binding site of IL-21R. and optionally, the chimeric receptor comprises a binding site; optionally, the ICD comprises the Box 1 and Box 2 regions of a protein selected from the group consisting of G-CSFR and gp130; and optionally, the chimeric receptor comprises a third domain comprising at least a portion of a transmembrane domain of a protein selected from the group consisting of G-CSFR, gp130 (glycoprotein 130), and IL-2Rβ, wherein optionally the transmembrane domain is a wild-type transmembrane domain.

[0023] In certain aspects, described herein are methods of treating a subject in need thereof, comprising injecting into the subject cells expressing a chimeric receptor and administering a cytokine that binds to the chimeric receptor; the chimeric receptor comprising an ECD of G-CSFR operably linked to a second domain; and the second domain comprising: (i) (a) The transmembrane domain of gp130; (b) the Box1 and Box2 regions of gp130, and (c) the C-terminal region of IL-2Rβ, or (ii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-2Rβ, or (iii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-12Rβ2, or (iv) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-21R, or (v) (a) The transmembrane domain of IL-2Rβ+γc; (b) the Box1 and Box2 regions of IL-2Rβ+γc, and (c) the C-terminal region of IL-2Rβ+γc, or (vi) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-7Rα.

[0024] In certain embodiments of the method, the activated chimeric receptor forms a homodimer, and optionally activation of the chimeric receptor causes a cellular response selected from the group consisting of proliferation, survival, and enhanced activity of a cell expressing the chimeric receptor; optionally, the chimeric receptor is activated by contact with G-CSF; optionally, the G-CSF is wild-type G-CSF; optionally, the extracellular domain of the G-CSFR is a wild-type extracellular domain; the chimeric receptor is expressed in a cell; optionally, the cell is an immune cell, optionally a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, optionally, the cell is a stem cell, optionally, the cell is a primary cell, and optionally, the cell is a human cell. In certain embodiments, the chimeric receptor optionally comprises: (a) at least a portion of the ICD of IL-2Rβ having the amino acid sequence of SEQ ID NO: 16, 19, 21, 29, 31, 33, 35, 37, or 39; or (b) at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 41; or (c) at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO: 25 or 27; or (d) at least a portion of the ICD of IL-12Rβ2 having the amino acid sequence of SEQ ID NO: 23, 32, or 26; or (e) at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 34, 40, or 42; or (f) at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO: 18 or 38; or (g) at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 43; or (h) at least a portion of the ICD of IIL-γc having the amino acid sequence of SEQ ID NO: 17 the transmembrane domain comprises the sequence set forth in (a) SEQ ID NO:8, or (b) SEQ ID NO:9, or (c) SEQ ID NO:10, or (d) SEQ ID NO:11. In certain embodiments, the methods described herein are used to treat cancer. In certain embodiments, the methods are used to treat autoimmune diseases. In certain embodiments, the methods are used to treat inflammatory conditions. In certain embodiments, the methods are used to prevent or treat transplant rejection. In certain embodiments, the methods are used to treat infectious diseases. In certain embodiments, the methods further comprise administering at least one additional active agent, optionally, the additional active agent is an additional cytokine.

[0025] In certain embodiments, the methods described herein include: i) isolating a sample containing immune cells; (ii) transducing or transfecting the immune cells with a nucleic acid sequence encoding a chimeric cytokine receptor; (iii) administering or injecting the immune cells into a subject; and (iv) contacting the immune cells with a cytokine that binds to the chimeric receptor. In certain embodiments, before administering or injecting the cells into the subject, the subject has undergone immunoablative therapy. In certain embodiments, the sample containing immune cells is isolated from the subject to whom the cells will be administered or infused. In certain embodiments, the immune cells are contacted with the cytokine in vitro before the cells are administered or infused into the subject. In certain embodiments, the immune cells are contacted with the cytokine that binds to the chimeric receptor for a time sufficient to activate signaling from the chimeric receptor.

[0026] Described herein are kits for treating a subject in need thereof, comprising cells encoding a chimeric receptor described herein, optionally being immune cells, and instructions for use, optionally comprising a cytokine that binds to the chimeric receptor. Described herein are kits for producing a chimeric receptor expressed on a cell, comprising an expression vector encoding a chimeric receptor described herein, and instructions for use, optionally comprising a cytokine that binds to the chimeric receptor.

[0027] Described herein is a kit for producing a chimeric receptor expressed on a cell, the kit comprising a cell, optionally a bacterial cell, that contains an expression vector encoding a chimeric receptor described herein, and instructions for use, and optionally the kit comprises a cytokine that binds to the chimeric receptor. [The present invention 1001] A chimeric receptor comprising: (a) comprising the extracellular domain (ECD) of G-CSFR (granulocyte colony-stimulating factor receptor) operably linked to a second domain; The second domain is (b) comprising at least a portion of the intracellular domain (ICD) of a multisubunit cytokine receptor selected from the group consisting of IL-2R (interleukin-2 receptor), IL-7R (interleukin-7 receptor), IL-12R (interleukin-12 receptor), and IL-21R (interleukin-21 receptor); Optionally, the IL-2R is selected from the group consisting of IL-2Rβ and IL-2Rγc; Optionally, the second domain comprises at least a portion of the C-terminal region of IL-2Rβ, IL-7Rα, IL-12Rβ2, or IL-21R; at least a portion of the ICD of the cytokine receptor comprises at least one signaling molecule binding site of the intracellular domain of the cytokine receptor; Optionally, the at least one signal transduction molecule binding site is selected from the group consisting of a STAT3 binding site of G-CSFR; a STAT3 binding site of gp130; a SHP-2 binding site of gp130; a SHC binding site of IL-2Rβ; a STAT5 binding site of IL-2Rβ; a STAT3 binding site of IL-2Rβ; a STAT1 binding site of IL-2Rβ; a STAT5 binding site of IL-7Rα; a phosphatidylinositol 3-kinase (PI3K) binding site of IL-7Rα; a STAT4 binding site of IL-12Rβ2; a STAT5 binding site of IL-12Rβ2; a STAT3 binding site of IL-12Rβ2; a STAT5 binding site of IL-21R; a STAT3 binding site of IL-21R; and a STAT1 binding site of IL-21R; Optionally, the ICD comprises a Box1 and Box2 region of a protein selected from the group consisting of G-CSFR and gp130; Optionally, the chimeric receptor comprises a third domain comprising at least a portion of a transmembrane domain of a protein selected from the group consisting of G-CSFR, gp130 (glycoprotein 130), and IL-2Rβ, and optionally, the transmembrane domain is a wild-type transmembrane domain. The chimeric receptor. [The present invention 1002] A chimeric receptor comprising: comprising the ECD of G-CSFR operably linked to a second domain; The second domain is (i) (a) The transmembrane domain of gp130; (b) the Box1 and Box2 regions of gp130, and (c) the C-terminal region of IL-2Rβ, or (ii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-2Rβ, or (iii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-12Rβ2, or (iv) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-21R, or (v) (a) The transmembrane domain of IL-2Rβ+γc; (b) the Box1 and Box2 regions of IL-2Rβ+γc, and (c) the C-terminal region of IL-2Rβ+γc, or (vi) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) C-terminal region of IL-7Rα The chimeric receptor comprising: [The present invention 1003] the activated chimeric receptor forms a homodimer, Optionally, said activation of said chimeric receptor results in a cellular response selected from the group consisting of proliferation, survival, and enhanced activity of a cell expressing said chimeric receptor; Optionally, the chimeric receptor is activated by contact with G-CSF; Optionally, the G-CSF is wild-type G-CSF; Optionally, the extracellular domain of the G-CSFR is a wild-type extracellular domain. The chimeric receptor of the present invention. [The present invention 1004] the activated chimeric receptor forms a homodimer, Optionally, said activation of said chimeric receptor results in a cellular response selected from the group consisting of proliferation, survival, and enhanced activity of a cell expressing said chimeric receptor; Optionally, the chimeric receptor is activated by contact with G-CSF; Optionally, the G-CSF is wild-type G-CSF; Optionally, the extracellular domain of the G-CSFR is a wild-type extracellular domain. The chimeric receptor of the present invention 1002. [The present invention 1005] The chimeric receptor is capable of expressing, in a cell, Optionally in immune cells: Optionally in T cells: Optionally in NK cells: Optionally, in NKT cells: Optionally in B cells: Optionally in plasma cells: Optionally in macrophages, Optionally in dendritic cells Expressed, Optionally, the cells are stem cells; Optionally, the cells are primary cells; Optionally, the cell is a human cell. The chimeric receptor of the present invention. [The present invention 1006] The chimeric receptor is capable of expressing, in a cell, Optionally in T cells: Optionally in NK cells: Optionally, in NKT cells: Optionally in B cells: Optionally in plasma cells: Optionally in macrophages, Optionally in dendritic cells Expressed, Optionally, the cells are stem cells; Optionally, the cells are primary cells; Optionally, the cell is a human cell. The chimeric receptor of the present invention 1002. [The present invention 1007] The chimeric receptor is capable of expressing, in a cell, Optionally in T cells: Optionally in NK cells: Optionally, in NKT cells: Optionally in B cells: Optionally in plasma cells: Optionally in macrophages, Optionally in dendritic cells Expressed, Optionally, the cells are stem cells; Optionally, the cells are primary cells; Optionally, the cell is a human cell. The chimeric receptor of the present invention 1003. [The present invention 1008] The chimeric receptor is capable of expressing, in a cell, Optionally in T cells: Optionally in NK cells: Optionally, in NKT cells: Optionally in B cells: Optionally in plasma cells: Optionally in macrophages, Optionally in dendritic cells Expressed, Optionally, the cells are stem cells; Optionally, the cells are primary cells; Optionally, the cell is a human cell. The chimeric receptor of the present invention 1004. [The present invention 1009] The ICD is (a) at least a portion of the ICD of IL-2Rβ having the amino acid sequence of SEQ ID NO: 16, 19, 21, 29, 31, 33, 35, 37, or 39; or (b) at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 41; or (c) at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO: 25 or 27; or (d) at least a portion of the ICD of IL-12Rβ2 having the amino acid sequence of SEQ ID NO: 23, 32, or 26; or (e) at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 34, 40, or 42; or (f) at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO: 18 or 38; or (g) at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 43; or (h) at least a portion of the ICD of IL-2Rγc having the amino acid sequence of SEQ ID NO: 17 1001. A chimeric receptor of the present invention comprising: [The present invention 1010] The ICD is (a) at least a portion of the ICD of IL-2Rβ having the amino acid sequence of SEQ ID NO: 16, 19, 21, 29, 31, 33, 35, 37, or 39; or (b) at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 41; or (c) at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO: 25 or 27; or (d) at least a portion of the ICD of IL-12Rβ2 having the amino acid sequence of SEQ ID NO: 23, 32, or 26; or (e) at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 34, 40, or 42; or (f) at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO: 18 or 38; or (g) at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 43; or (h) at least a portion of the ICD of IL-2Rγc having the amino acid sequence of SEQ ID NO: 17 1002. A chimeric receptor of the present invention comprising: [The present invention 1011] the transmembrane domain is (a) SEQ ID NO: 8, or (b) SEQ ID NO: 9, or (c) SEQ ID NO: 10, or (d) SEQ ID NO: 11 1001. A chimeric receptor of the present invention, comprising the sequence set forth in [The present invention 1012] the transmembrane domain is (a) SEQ ID NO: 8, or (b) SEQ ID NO: 9, or (c) SEQ ID NO: 10, or (d) SEQ ID NO: 11 1002. A chimeric receptor of the present invention, comprising the sequence set forth in [The present invention 1013] A nucleic acid encoding a chimeric receptor, said chimeric receptor comprising: (a) comprising the extracellular domain (ECD) of G-CSFR (granulocyte colony-stimulating factor receptor) operably linked to a second domain; The second domain is (b) comprising at least a portion of the intracellular domain (ICD) of a multisubunit cytokine receptor selected from the group consisting of IL-2R (interleukin-2 receptor), IL-7R (interleukin-7 receptor), IL-12R (interleukin-12 receptor), and IL-21R (interleukin-21 receptor); Optionally, the IL-2R is selected from the group consisting of IL-2Rβ and IL-2Rγc; Optionally, the second domain comprises at least a portion of the C-terminal region of IL-2Rβ, IL-7Rα, IL-12Rβ2, or IL-21R; at least a portion of the ICD of the cytokine receptor comprises at least one signaling molecule binding site of the intracellular domain of the cytokine receptor; Optionally, the ICD comprises at least one signaling molecule binding site selected from the group consisting of a STAT3-binding site of G-CSFR; a STAT3-binding site of gp130; a SHP-2-binding site of gp130; a SHC-binding site of IL-2Rβ; a STAT5-binding site of IL-2Rβ; a STAT3-binding site of IL-2Rβ; a STAT1-binding site of IL-2Rβ; a STAT5-binding site of IL-7Rα; a phosphatidylinositol 3-kinase (PI3K)-binding site of IL-7Rα; a STAT4-binding site of IL-12Rβ2; a STAT5-binding site of IL-12Rβ2; a STAT3-binding site of IL-12Rβ2; a STAT5-binding site of IL-21R; a STAT3-binding site of IL-21R; and a STAT1-binding site of IL-21R; Optionally, the ICD comprises a Box1 and Box2 region of a protein selected from the group consisting of G-CSFR and gp130; Optionally, the chimeric receptor comprises a third domain comprising at least a portion of a transmembrane domain of a protein selected from the group consisting of G-CSFR, gp130 (glycoprotein 130), and IL-2Rβ; Optionally, the transmembrane domain is a wild-type transmembrane domain. A nucleic acid encoding the chimeric receptor. [The present invention 1014] 1013. The nucleic acid of the present invention, wherein the ECD of the G-CSFR is encoded by the nucleic acid sequence set forth in SEQ ID NO: 5 or 6. [The present invention 1015] (a) a sequence encoding at least a portion of the ICD of IL-2Rβ having the amino acid sequence of SEQ ID NO: 16, 19, 21, 29, 31, 33, 35, 37, or 39; or (b) a sequence encoding at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 41; or (c) a sequence encoding at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO: 25 or 27; or (d) a sequence encoding at least a portion of the ICD of IL-12Rβ2 having the amino acid sequence of SEQ ID NO: 23, 32, or 26; or (e) a sequence encoding at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 34, 40, or 42; or (f) a sequence encoding at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO: 18 or 38; or (g) a sequence encoding at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 43; or (h) a sequence encoding at least a portion of the ICD of IL-2Rγc having the amino acid sequence of SEQ ID NO: 17 The nucleic acid of the present invention, comprising: [The present invention 1016] (a) a sequence encoding at least a portion of the ICD of IL-2Rβ having the amino acid sequence of SEQ ID NO: 16, 19, 21, 29, 31, 33, 35, 37, or 39; or (b) a sequence encoding at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 41; or (c) a sequence encoding at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO: 25 or 27; or (d) a sequence encoding at least a portion of the ICD of IL-12Rβ2 having the amino acid sequence of SEQ ID NO: 23, 32, or 26; or (e) a sequence encoding at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 34, 40, or 42; or (f) a sequence encoding at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO: 18 or 38; or (g) a sequence encoding at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 43; or (h) a sequence encoding at least a portion of the ICD of IL-2Rγc having the amino acid sequence of SEQ ID NO: 17 1014. The nucleic acid of the present invention, comprising: [The present invention 1017] An expression vector comprising any one of the nucleic acids of the present inventions 1013 to 1016. [The present invention 1018] The expression vector of the present invention 1017, which is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and a plasmid. [The present invention 1019] A nucleic acid encoding a chimeric receptor, the chimeric receptor comprises an ECD of G-CSFR operably linked to a second domain; The second domain is (i) (a) The transmembrane domain of gp130; (b) the Box1 and Box2 regions of gp130, and (c) the C-terminal region of IL-2Rβ, or (ii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-2Rβ, or (iii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-12Rβ2, or (iv) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-21R, or (v) (a) The transmembrane domain of IL-2Rβ+γc; (b) the Box1 and Box2 regions of IL-2Rβ+γc, and (c) the C-terminal region of IL-2Rβ+γc, or (vi) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) C-terminal region of IL-7Rα A nucleic acid encoding the chimeric receptor comprising: [The present invention 1020] 1019. The nucleic acid of the present invention, wherein the ECD of the G-CSFR is encoded by the nucleic acid sequence set forth in SEQ ID NO: 5 or 6. [The present invention 1021] (a) a sequence encoding at least a portion of the ICD of IL-2Rβ having the amino acid sequence of SEQ ID NO: 16, 19, 21, 29, 31, 33, 35, 37, or 39; or (b) a sequence encoding at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 41; or (c) a sequence encoding at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO: 25 or 27; or (d) a sequence encoding at least a portion of the ICD of IL-12Rβ2 having the amino acid sequence of SEQ ID NO: 23, 32, or 26; or (e) a sequence encoding at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 34, 40, or 42; or (f) a sequence encoding at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO: 18 or 38; or (g) a sequence encoding at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 43; or (h) a sequence encoding at least a portion of the ICD of IL-2Rγc having the amino acid sequence of SEQ ID NO: 17 The nucleic acid of the present invention, comprising: [The present invention 1022] (a) a sequence encoding at least a portion of the ICD of IL-2Rβ having the amino acid sequence of SEQ ID NO: 16, 19, 21, 29, 31, 33, 35, 37, or 39; or (b) a sequence encoding at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 41; or (c) a sequence encoding at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO: 25 or 27; or (d) a sequence encoding at least a portion of the ICD of IL-12Rβ2 having the amino acid sequence of SEQ ID NO: 23, 32, or 26; or (e) a sequence encoding at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 34, 40, or 42; or (f) a sequence encoding at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO: 18 or 38; or (g) a sequence encoding at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 43; or (h) a sequence encoding at least a portion of the ICD of IL-2Rγc having the amino acid sequence of SEQ ID NO: 17 The nucleic acid of the present invention 1020, comprising: [The present invention 1023] An expression vector comprising any one of the nucleic acids of the present inventions 1019 to 1022. [The present invention 1024] An expression vector of the present invention 1023, selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and a plasmid. [The present invention 1025] 1. A cell comprising a nucleic acid encoding a chimeric receptor, said chimeric receptor comprising: (a) comprising the extracellular domain (ECD) of G-CSFR (granulocyte colony-stimulating factor receptor) operably linked to a second domain; The second domain is (b) comprising at least a portion of the intracellular domain (ICD) of a multisubunit cytokine receptor selected from the group consisting of IL-2R (interleukin-2 receptor), IL-7R (interleukin-7 receptor), IL-12R (interleukin-12 receptor), and IL-21R (interleukin-21 receptor); Optionally, the IL-2R is selected from the group consisting of IL-2Rβ and IL-2Rγc; Optionally, the second domain comprises at least a portion of the C-terminal region of IL-2Rβ, IL-7Rα, IL-12Rβ2, or IL-21R; at least a portion of the ICD of the cytokine receptor comprises at least one signaling molecule binding site of the intracellular domain of the cytokine receptor; Optionally, the ICD comprises at least one signaling molecule binding site selected from the group consisting of a STAT3-binding site of G-CSFR; a STAT3-binding site of gp130; a SHP-2-binding site of gp130; a SHC-binding site of IL-2Rβ; a STAT5-binding site of IL-2Rβ; a STAT3-binding site of IL-2Rβ; a STAT1-binding site of IL-2Rβ; a STAT5-binding site of IL-7Rα; a phosphatidylinositol 3-kinase (PI3K)-binding site of IL-7Rα; a STAT4-binding site of IL-12Rβ2; a STAT5-binding site of IL-12Rβ2; a STAT3-binding site of IL-12Rβ2; a STAT5-binding site of IL-21R; a STAT3-binding site of IL-21R; and a STAT1-binding site of IL-21R; Optionally, the ICD comprises a Box1 and Box2 region of a protein selected from the group consisting of G-CSFR and gp130; Optionally, the chimeric receptor comprises a third domain comprising at least a portion of a transmembrane domain of a protein selected from the group consisting of G-CSFR, gp130 (glycoprotein 130), and IL-2Rβ; Optionally, the transmembrane domain is a wild-type transmembrane domain; Optionally, the cell is an immune cell; optionally a T cell; optionally NK cells; optionally NKT cells; optionally a B cell; optionally plasma cells; optionally macrophages; optionally dendritic cells; Optionally, the cells are stem cells; Optionally, the cells are primary cells; Optionally, the cell is a human cell. A cell containing a nucleic acid encoding said chimeric receptor. [The present invention 1026] A cell comprising a nucleic acid encoding a chimeric receptor, said chimeric receptor comprising an ECD of G-CSFR operably linked to a second domain; said second domain comprising: (i) (a) The transmembrane domain of gp130; (b) the Box1 and Box2 regions of gp130, and (c) the C-terminal region of IL-2Rβ, or (ii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-2Rβ, or (iii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-12Rβ2, or (iv) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-21R, or (v) (a) The transmembrane domain of IL-2Rβ+γc; (b) the Box1 and Box2 regions of IL-2Rβ+γc, and (c) the C-terminal region of IL-2Rβ+γc, or (vi) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) C-terminal region of IL-7Rα Includes; Optionally, the cell is an immune cell; optionally a T cell; optionally NK cells; optionally NKT cells; optionally a B cell; optionally plasma cells; optionally macrophages; optionally dendritic cells; Optionally, the cells are stem cells; Optionally, the cells are primary cells; Optionally, the cell is a human cell. A cell containing a nucleic acid encoding said chimeric receptor. [The present invention 1027] The cell of claim 1025 or 1026, wherein the ECD of the G-CSFR is encoded by the nucleic acid sequence set forth in SEQ ID NO: 5 or 6. [The present invention 1028] The nucleic acid (a) a sequence encoding at least a portion of the ICD of IL-2Rβ having the amino acid sequence of SEQ ID NO: 16, 19, 21, 29, 31, 33, 35, 37, or 39; or (b) a sequence encoding at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 41; or (c) a sequence encoding at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO: 25 or 27; or (d) a sequence encoding at least a portion of the ICD of IL-12Rβ2 having the amino acid sequence of SEQ ID NO: 23, 32, or 26; or (e) a sequence encoding at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 34, 40, or 42; or (f) a sequence encoding at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO: 18 or 38; or (g) a sequence encoding at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 43; or (h) a sequence encoding at least a portion of the ICD of IL-2RG having the amino acid sequence of SEQ ID NO: 17; 1027. The cell of the present invention, comprising: [The present invention 1029] A cell comprising an expression vector of the present invention 1017, 1018, 1023, or 1024, Optionally, the cell is an immune cell; optionally a T cell; optionally NK cells; optionally NKT cells; optionally a B cell; optionally plasma cells; optionally macrophages; optionally dendritic cells; Optionally, the cells are stem cells; Optionally, the cells are primary cells; Optionally, the cell is a human cell. The cells. [The present invention 1030] A cell comprising the chimeric receptor of the present invention, Optionally, the cell is an immune cell; optionally a T cell; optionally NK cells; optionally NKT cells; optionally a B cell; optionally plasma cells; optionally macrophages; optionally dendritic cells; Optionally, the cells are stem cells; Optionally, the cells are primary cells; Optionally, the cell is a human cell. The cells. [The present invention 1031] A cell comprising the chimeric receptor of the present invention 1002, Optionally, the cell is an immune cell; optionally a T cell; optionally NK cells; optionally NKT cells; optionally a B cell; optionally plasma cells; optionally macrophages; optionally dendritic cells; Optionally, the cells are stem cells; Optionally, the cells are primary cells; Optionally, the cell is a human cell. The cells. [The present invention 1032] 1. A method for selectively activating a chimeric receptor expressed on the surface of a cell, comprising: contacting the chimeric receptor with G-CSF, which selectively activates said chimeric receptor; the chimeric receptor (a) comprising the extracellular domain (ECD) of G-CSFR (granulocyte colony-stimulating factor receptor) operably linked to a second domain; The second domain is (b) comprising at least a portion of the intracellular domain (ICD) of a multisubunit cytokine receptor selected from the group consisting of IL-2R (interleukin-2 receptor), IL-7R (interleukin-7 receptor), IL-12R (interleukin-12 receptor), and IL-21R (interleukin-21 receptor); Optionally, the IL-2R is selected from the group consisting of IL-2Rβ and IL-2Rγc; Optionally, the second domain comprises at least a portion of the C-terminal region of IL-2Rβ, IL-7Rα, IL-12Rβ2, or IL-21R; at least a portion of the ICD of the cytokine receptor comprises at least one signaling molecule binding site of the intracellular domain of the cytokine receptor; Optionally, the at least one signal transduction molecule binding site is selected from the group consisting of a STAT3 binding site of G-CSFR; a STAT3 binding site of gp130; a SHP-2 binding site of gp130; a SHC binding site of IL-2Rβ; a STAT5 binding site of IL-2Rβ; a STAT3 binding site of IL-2Rβ; a STAT1 binding site of IL-2Rβ; a STAT5 binding site of IL-7Rα; a phosphatidylinositol 3-kinase (PI3K) binding site of IL-7Rα; a STAT4 binding site of IL-12Rβ2; a STAT5 binding site of IL-12Rβ2; a STAT3 binding site of IL-12Rβ2; a STAT5 binding site of IL-21R; a STAT3 binding site of IL-21R; and a STAT1 binding site of IL-21R; Optionally, the ICD comprises a Box1 and Box2 region of a protein selected from the group consisting of G-CSFR and gp130; Optionally, the chimeric receptor comprises a third domain comprising at least a portion of a transmembrane domain of a protein selected from the group consisting of G-CSFR, gp130 (glycoprotein 130), and IL-2Rβ; Optionally, the transmembrane domain is a wild-type transmembrane domain. The method. [The present invention 1033] 1. A method for selectively activating a chimeric receptor expressed on the surface of a cell, comprising: contacting the chimeric receptor with G-CSF, which selectively activates said chimeric receptor; the chimeric receptor comprises an ECD of G-CSFR operably linked to a second domain; The second domain is (i) (a) The transmembrane domain of gp130; (b) the Box1 and Box2 regions of gp130, and (c) the C-terminal region of IL-2Rβ, or (ii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-2Rβ, or (iii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-12Rβ2, or (iv) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-21R, or (v) (a) The transmembrane domain of IL-2Rβ+γc; (b) the Box1 and Box2 regions of IL-2Rβ+γc, and (c) the C-terminal region of IL-2Rβ+γc, or (vi) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) C-terminal region of IL-7Rα The method comprising: [The present invention 1034] the activated chimeric receptor forms a homodimer, Optionally, said activation of said chimeric receptor results in a cellular response selected from the group consisting of proliferation, survival, and enhanced activity of a cell expressing said chimeric receptor; Optionally, the chimeric receptor is activated by contact with G-CSF; Optionally, the G-CSF is wild-type G-CSF; Optionally, the extracellular domain of the G-CSFR is a wild-type extracellular domain; The chimeric receptor is capable of expressing, in a cell, Optionally in immune cells: Optionally in T cells: Optionally in NK cells: Optionally, in NKT cells: Optionally in B cells: Optionally in plasma cells: Optionally in macrophages, Optionally in dendritic cells Expressed, Optionally, the cells are stem cells; Optionally, the cells are primary cells; Optionally, the cell is a human cell. The method of the present invention 1032 or 1033. [This invention 1035] the chimeric receptor (a) at least a portion of the ICD of IL-2Rβ having the amino acid sequence of SEQ ID NO: 16, 19, 21, 29, 31, 33, 35, 37, or 39; or (b) at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 41; or (c) at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO: 25 or 27; or (d) at least a portion of the ICD of IL-12Rβ2 having the amino acid sequence of SEQ ID NO: 23, 32, or 26; or (e) at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 34, 40, or 42; or (f) at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO: 18 or 38; or (g) at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 43; or (h) at least a portion of the ICD of IL-2Rγc having the amino acid sequence of SEQ ID NO: 17 Includes; the transmembrane domain is (a) SEQ ID NO: 8, or (b) SEQ ID NO: 9, or (c) SEQ ID NO: 10, or (d) SEQ ID NO: 11 1034. The method of claim 1034, comprising the sequence set forth in [The present invention 1036] 1. A method for producing a chimeric receptor in a cell, comprising: introducing into said cells any one of the nucleic acids of the present inventions 1013 to 1016 or 1019 to 1022, or any one of the expression vectors of the present inventions 1017, 1018, 1023, or 1024; Optionally, the method comprises gene editing; Optionally, the cell is an immune cell; optionally a T cell; optionally NK cells; optionally NKT cells; optionally a B cell; optionally plasma cells; optionally macrophages; optionally dendritic cells; Optionally, the cells are stem cells; Optionally, the cells are primary cells; Optionally, the cell is a human cell. The method. [This invention 1037] 1. A method of treating a subject in need thereof, comprising: injecting cells expressing a chimeric receptor into the subject and administering a cytokine that binds to the chimeric receptor; the chimeric receptor (a) comprising the extracellular domain (ECD) of G-CSFR (granulocyte colony-stimulating factor receptor) operably linked to a second domain; The second domain is (b) comprising at least a portion of the intracellular domain (ICD) of a multisubunit cytokine receptor selected from the group consisting of IL-2R (interleukin-2 receptor), IL-7R (interleukin-7 receptor), IL-12R (interleukin-12 receptor), and IL-21R (interleukin-21 receptor); Optionally, the IL-2R is selected from the group consisting of IL-2Rβ and IL-2Rγc; Optionally, the second domain comprises at least a portion of the C-terminal region of IL-2Rβ, IL-7Rα, IL-12Rβ2, or IL-21R; at least a portion of the ICD of the cytokine receptor comprises at least one signaling molecule binding site of the intracellular domain of the cytokine receptor; Optionally, the ICD comprises at least one signaling molecule binding site selected from the group consisting of a STAT3-binding site of G-CSFR; a STAT3-binding site of gp130; a SHP-2-binding site of gp130; a SHC-binding site of IL-2Rβ; a STAT5-binding site of IL-2Rβ; a STAT3-binding site of IL-2Rβ; a STAT1-binding site of IL-2Rβ; a STAT5-binding site of IL-7Rα; a phosphatidylinositol 3-kinase (PI3K)-binding site of IL-7Rα; a STAT4-binding site of IL-12Rβ2; a STAT5-binding site of IL-12Rβ2; a STAT3-binding site of IL-12Rβ2; a STAT5-binding site of IL-21R; a STAT3-binding site of IL-21R; and a STAT1-binding site of IL-21R; Optionally, the ICD comprises a Box1 and Box2 region of a protein selected from the group consisting of G-CSFR and gp130; Optionally, the chimeric receptor comprises a third domain comprising at least a portion of a transmembrane domain of a protein selected from the group consisting of G-CSFR, gp130 (glycoprotein 130), and IL-2Rβ; Optionally, the transmembrane domain is a wild-type transmembrane domain. The method. [The present invention 1038] 1. A method of treating a subject in need thereof, comprising: injecting cells expressing a chimeric receptor into the subject and administering a cytokine that binds to the chimeric receptor; the chimeric receptor comprises an ECD of G-CSFR operably linked to a second domain; The second domain is (i) (a) The transmembrane domain of gp130; (b) the Box1 and Box2 regions of gp130, and (c) the C-terminal region of IL-2Rβ, or (ii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-2Rβ, or (iii) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-12Rβ2, or (iv) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-21R, or (v) (a) The transmembrane domain of IL-2Rβ+γc; (b) the Box1 and Box2 regions of IL-2Rβ+γc, and (c) the C-terminal region of IL-2Rβ+γc, or (vi) (a) The transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) C-terminal region of IL-7Rα The method comprising: [This invention 1039] the activated chimeric receptor forms a homodimer, Optionally, said activation of said chimeric receptor results in a cellular response selected from the group consisting of proliferation, survival, and enhanced activity of a cell expressing said chimeric receptor; Optionally, the chimeric receptor is activated by contact with G-CSF; Optionally, the G-CSF is wild-type G-CSF; Optionally, the extracellular domain of the G-CSFR is a wild-type extracellular domain; expressing the chimeric receptor in a cell; Optionally, the cell is an immune cell; optionally a T cell; optionally NK cells; optionally NKT cells; optionally a B cell; optionally plasma cells; optionally macrophages; optionally dendritic cells; Optionally, the cells are stem cells; Optionally, the cells are primary cells; Optionally, the cell is a human cell. The method of the present invention 1037 or 1038. [The present invention 1040] The chimeric receptor optionally comprises: (a) at least a portion of the ICD of IL-2Rβ having the amino acid sequence of SEQ ID NO: 16, 19, 21, 29, 31, 33, 35, 37, or 39; or (b) at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 41; or (c) at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO: 25 or 27; or (d) at least a portion of the ICD of IL-12Rβ2 having the amino acid sequence of SEQ ID NO: 23, 32, or 26; or (e) at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 34, 40, or 42; or (f) at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO: 18 or 38; or (g) at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 43; or (h) at least a portion of the ICD of IL-2Rγc having the amino acid sequence of SEQ ID NO: 17 Includes; the transmembrane domain is (a) SEQ ID NO: 8, or (b) SEQ ID NO: 9, or (c) SEQ ID NO: 10, or (d) SEQ ID NO: 11 1039. The method of claim 1039, comprising the sequence set forth in [The present invention 1041] The method of any one of claims 1037 to 1038, wherein the method is used to treat cancer. [The present invention 1042] The method of any one of claims 1037 to 1038, wherein the method is used to treat an autoimmune disease. [This invention 1043] The method of any one of claims 1037 to 1038, wherein the method is used to treat an inflammatory condition. [This invention 1044] The method of any one of claims 1037 to 1038, wherein the method is used to prevent or treat transplant rejection. [This invention 1045] The method of any one of claims 1037 to 1038, wherein the method is used to treat an infectious disease. [The present invention 1046] 1039. The method of any one of claims 1037 to 1038, wherein said method further comprises administering at least one additional active agent, optionally wherein said additional active agent is an additional cytokine. [This invention 1047] (i) isolating a sample containing immune cells; (ii) transducing or transfecting the immune cells with a nucleic acid sequence encoding the chimeric cytokine receptor; (iii) administering or injecting the immune cells of (ii) into the subject; and (iv) contacting the immune cells with the cytokine that binds to the chimeric receptor. The method of the present invention 1037, comprising: [This invention 1048] (i) isolating a sample containing immune cells; (ii) transducing or transfecting the immune cells with a nucleic acid sequence encoding the chimeric cytokine receptor; (iii) administering or injecting the immune cells of (ii) into the subject; and (iv) contacting the immune cells with the cytokine that binds to the chimeric receptor. The method of the present invention 1038, comprising: [This invention 1049] The method of any one of claims 1047 to 1048, wherein the subject has undergone immunoablative therapy prior to administering or injecting the cells into the subject. [The present invention 1050] 1049. The method of any one of claims 1047 to 1048, wherein the sample containing said immune cells is isolated from said subject to be administered or infused with said cells. [This invention 1051] The method of any one of claims 1047 to 1048, wherein said immune cells are contacted with said cytokine in vitro prior to administering or injecting said cells into said subject. [This invention 1052] 1048. The method of any one of claims 1046 to 1047, wherein said immune cell is contacted with said cytokine that binds to said chimeric receptor for a time sufficient to activate signaling from said chimeric receptor. [This invention 1053] 1. A kit for treating a subject in need thereof, comprising: A cell encoding any one of the chimeric receptors of the present inventions 1001 to 1012, which is optionally an immune cell; Instructions for use and Including, Optionally, the kit comprises a cytokine that binds to the chimeric receptor. The kit. [This invention 1054] 1. A kit for producing a chimeric receptor expressed on a cell, comprising: An expression vector encoding any one of the chimeric receptors of the present inventions 1001 to 1012; Instructions for use and Including, Optionally, the kit comprises a cytokine that binds to the chimeric receptor. The kit. [This invention 1055] 1. A kit for producing a chimeric receptor expressed on a cell, comprising: A cell, optionally a bacterial cell, comprising an expression vector encoding any one of the chimeric receptors of the present inventions 1001 to 1012; Instructions for use and Including, Optionally, the kit comprises a cytokine that binds to the chimeric receptor. The kit. [Brief explanation of the drawings]

[0028] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description and accompanying drawings.

[0029] [Figure 1] FIG. 1 is a schematic representation of the native IL-2Rβ, IL-2Rγc, and G-CSFR subunits, and the G2R-1 receptor subunit design.

[0030] [Figure 2]Figure 2 shows the expansion (fold change in cell number) of 32D-IL-2Rβ cells (a 32D cell line stably expressing the human IL-2Rβ subunit) expressing the indicated G-CSFR chimeric receptor subunits and stimulated with WT G-CSF, IL-2, or no cytokine. G / γc is tagged with a Myc epitope at its N-terminus (Myc / G / γc), and G / IL-2Rβ is tagged with a Flag epitope at its N-terminus (Flag / G / IL-2Rβ); these epitope tags aid in detection by flow cytometry and do not affect receptor function. Additionally, the lower panels in B–D show the percentage of cells expressing the G-CSFR ECD (%G-CSFR+) under each culture condition. Squares represent cells stimulated with IL-2. Triangles represent cells stimulated with G-CSF. Circles represent cells not stimulated with cytokine.

[0031] [Figure 3] Figure 3 shows graphs showing the expansion (fold change in cell number) of human T cells expressing Flag-tagged G / IL-2R β subunit only, Myc-tagged G / γ subunit only, or full-length G-CSFR. A-D) PBMC-derived T cells; E-H) tumor-associated lymphocytes (TALs). Squares represent cells stimulated with IL-2. Triangles represent cells stimulated with G-CSF. Circles represent cells not stimulated with cytokines.

[0032] [Figure 4] Figure 4 is a schematic diagram of native and chimeric receptors showing JAK, STAT, Shc, SHP-2, and PI3K binding sites. The shaded scheme includes the receptors from Figure 1.

[0033] [Figure 5] Figure 5 is a schematic diagram of the chimeric receptors showing the Jak, STAT, Shc, SHP-2, and PI3K binding sites. The shaded scheme includes the receptors from Figures 1 and 4.

[0034] [Figure 6]FIG. 6 is a diagram of the lentiviral plasmid containing the G2R-2 cDNA insert.

[0035] [Figure 7] Figure 7 depicts graphs showing G-CSFR ECD expression assessed by flow cytometry in G2R-2 transduced cells: A) 32D-IL-2Rβ cell line; B) PBMC-derived human T cells and human tumor-associated lymphocytes (TAL).

[0036] [Figure 8] Figure 8 shows graphs showing the expansion (fold change in cell number) of cells expressing G2R-2 compared to non-transduced cells. A) Human PBMC-derived T cells; B, C) Human tumor-associated lymphocytes (TAL) from two independent experiments. Squares represent cells stimulated with IL-2. Triangles represent cells stimulated with G-CSF. Circles represent cells not stimulated with cytokines.

[0037] [Figure 9] Figure 9 depicts graphs showing the expansion (fold change in cell number) of CD4- or CD8-selected human tumor-associated lymphocytes expressing G2R-2 compared to non-transduced cells. A) Non-transduced CD4-selected cells; B) Non-transduced CD8-selected cells; C) G2R-2-transduced CD4-selected cells; D) G2R-2-transduced CD8-selected cells. The dotted gray line represents cells stimulated with IL-2. The solid black line represents cells stimulated with G-CSF. The dashed gray line represents cells not stimulated with cytokines.

[0038] [Figure 10]Figure 10 depicts a graph showing the expansion (fold change in cell number) of CD4+ or CD8+ tumor-associated lymphocytes expressing G2R-2. Cells were first expanded in G-CSF or IL-2 as indicated. Cells were then plated with IL-2, G-CSF, or medium alone. The solid gray line represents cells stimulated with IL-2. The solid black line represents cells stimulated with G-CSF. The dashed light gray line represents cells expanded with IL-2 and stimulated with medium alone. The dashed dark gray line represents cells expanded with G-CSF and stimulated with medium alone.

[0039] [Figure 11] Figure 11 depicts graphs showing the immunophenotype (by flow cytometry) of CD4- or CD8-selected tumor-associated lymphocytes (TALs) expressing G2R-2 chimeric receptor constructs versus untransduced cells after expansion with G-CSF or IL-2. A) Percentage of live cells displaying CD4+, CD8+, or CD3-CD56+ cell surface phenotypes. B) Percentage of live CD8+ cells displaying the indicated cell surface phenotypes based on CD45RA and CCR7 expression.

[0040] [Figure 12] Figure 12 depicts a graph showing the results of a BrdU incorporation assay to assess proliferation of primary human T cells expressing G2R-2 versus non-transduced cells. T cells were selected by culture in IL-2 or G-CSF, as indicated, prior to the assay. A) Tumor-associated lymphocytes; B) PBMC-derived T cells.

[0041] [Figure 13]Figure 13 depicts graphs showing the results of a BrdU incorporation assay to assess proliferation of primary murine T cells expressing G2R-2 or single-chain G / IL-2Rβ (a component of G2R-1) versus mock-transduced cells. A) Transduction efficiency, as reflected by the percentage of cells expressing the G-CSFR ECD (by flow cytometry) after culture with the indicated cytokines; B) Percentage of BrdU incorporation among all live cells in response to the indicated cytokines; C) Percentage of BrdU incorporation by cells expressing the G-CSFR ECD (G-CSFR+ cells). All cells were expanded with IL-2 for 3 days before assay. Squares represent cells stimulated with IL-2. Triangles represent cells stimulated with G-CSF. Circles represent cells not stimulated with cytokines.

[0042] [Figure 14] Figure 14 depicts Western blots to detect the indicated cytokine signaling events in human primary T cells expressing G2R-2 versus non-transduced cells. β-actin, total Akt, and histone H3 serve as protein loading controls. A, B) Tumor-associated lymphocytes (TAL); C) PBMC-derived T cells.

[0043] [Figure 15] Figure 15 shows Western blots for detecting the indicated cytokine signaling events in primary mouse T cells expressing G2R-2 or single-chain G / IL-2Rβ (derived from G2R-1) versus mock-transduced cells. The arrow indicates the specific phospho-Jak2 band; other larger bands are presumably the result of cross-reactivity of the primary anti-phospho-Jak2 antibody with phospho-Jak1. β-Actin and histone H3 served as protein loading controls.

[0044] [Figure 16]Figure 16 is a graph showing the results of a BrdU incorporation assay assessing cell cycle progression of 32D-IL-2Rβ cells (or non-transduced cells) expressing the indicated chimeric receptors in response to stimulation with no cytokine, IL-2 (300 IU / mL), or WT G-CSF (30 ng / mL), or 130 G-CSF (30 ng / mL).

[0045] [Figure 17] Figure 17 is a graph showing the results of a BrdU incorporation assay assessing cell cycle progression of primary murine T cells (or untransduced cells) expressing the indicated chimeric receptors in response to stimulation with no cytokine, IL-2, or WT, 130, 304, or 307 cytokines. A and B represent repeat experiments.

[0046] [Figure 18] Figure 18 depicts Western blots to detect the indicated cytokine signaling events in 32D-IL-2Rβ cells (or untransduced cells) expressing the indicated chimeric receptor subunits in response to stimulation with no cytokine, IL-2, WT G-CSF, or 130 G-CSF. β-Actin and histone H3 serve as protein loading controls.

[0047] [Figure 19] Figure 19 shows A) Western blots to detect the indicated cytokine signaling events in primary murine T cells expressing the indicated chimeric receptor subunits in response to stimulation with no cytokine, IL-2, WT G-CSF, 130 G-CSF, or 304 G-CSF. β-actin and histone H3 serve as protein loading controls. B) Transduction efficiency of the cells used in panel A, assessed by flow cytometry using an antibody specific for the extracellular domain of the human G-CSF receptor.

[0048] [Figure 20]Figure 20 presents plots showing G-CSFR ECD expression by flow cytometry in primary human tumor-associated lymphocytes (TALs) transduced with the indicated chimeric receptor constructs. Live CD3+, CD56- cells were gated on CD8 or CD4, and G-CSFR ECD expression is shown for each population.

[0049] [Figure 21] Figure 21 presents graphs and images showing the expansion, proliferation, and signaling of primary human tumor-associated lymphocytes (TALs) expressing G2R-3 versus untransduced cells. A) Graph showing the results of a T cell expansion assay in which cells were transduced with a lentivirus encoding G2R-3, washed, and then replated with IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. Viable cells were counted every 3–4 days. Squares represent cells stimulated with IL-2. Triangles represent cells stimulated with G-CSF. Circles represent cells not stimulated with cytokine. B) Western blot to assess intracellular signaling events. Cells were harvested from the expansion assay and stimulated with IL-2 (300 IU / ml) or wild-type G-CSF (100 ng / ml). The arrow indicates the specific phospho-Jak2 band at 125 kDa; the larger band is presumably the result of cross-reactivity of the primary anti-phospho-Jak2 antibody with phospho-Jak1. β-Actin and histone H3 served as protein loading controls. C) Graph showing the results of a BrdU incorporation assay to assess T cell proliferation. Cells were harvested from the expansion assay, washed, and then replated in IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine.

[0050] [Figure 22]Figure 22 presents graphs showing fold expansion and G-CSFR ECD expression of primary human PBMC-derived T cells expressing G2R-3 with WT ECD versus untransduced cells. A) Graph showing the results of a T cell expansion assay in which cells were transduced with a lentivirus encoding G2R-3. On day 1, WT G-CSF (100 ng / ml) or no cytokine (media only) was added to the culture medium. Cells were then supplemented with media containing WT G-CSF or no cytokines until day 21. On day 21 of expansion, cells were washed and replated with WT G-CSF (100 ng / mL), IL-7 (20 ng / mL), and IL-15 (20 ng / mL), or no cytokines. Viable cells were counted every 2–4 days. Squares represent cells stimulated with G-CSF. Triangles represent cells stimulated with G-CSF and replated with IL-7 and IL-15 on day 21. Circles represent cells not stimulated with cytokines. Diamonds represent cells stimulated with G-CSF and replated with medium alone on day 21. B) Graph showing expression of G-CSFR ECD measured by flow cytometry on days 21 or 42 of expansion.

[0051] [Figure 23] Figure 23 depicts graphs showing the intracellular signaling and immunophenotype of primary human PBMC-derived T cells expressing G2R-3 versus non-transduced cells. A) Western blot to assess intracellular signaling events. Cells were harvested from the expansion assay and stimulated with IL-2 (300 IU / ml) or wild-type G-CSF (100 ng / ml). β-actin serves as a protein loading control. B, C) Representative flow cytometry plots and graphs showing the immunophenotype, as assessed by flow cytometry, of cells expressing G2R-3 versus non-transduced cells at day 42 of expansion.

[0052] [Figure 24]Figure 24 depicts graphs showing the fold expansion of primary human PBMC-derived T cells expressing G2R-3 with either the 304 or 307 ECD versus untransduced cells. A) Graph showing the results of a T cell expansion assay in which cells were transduced with a lentivirus encoding the G2R-3 304 ECD. B) Graph showing the results of a T cell expansion assay in which cells were transduced with a lentivirus encoding the G2R-3 307 ECD. C) Graph showing the results of a T cell expansion assay using untransduced cells. On day 2, IL-2 (300 IU / mL), 304 G-CSF (100 ng / mL), 307 G-CSF (100 ng / mL), or no cytokine (media only) was added to the cultures as indicated and replenished every 2 days thereafter. Viable cells were counted every 3–4 days. Diamonds represent cells stimulated with 304 G-CSF. Squares represent cells stimulated with 307 G-CSF. Triangles represent cells stimulated with IL-2. Inverted triangles represent cells not stimulated with cytokines.

[0053] [Figure 25] Figure 25 depicts a graph showing the results of a BrdU incorporation assay to assess proliferation of primary human PBMC-derived T cells expressing G2R-3 with 304 or 307 ECD versus untransduced cells. Cells were transduced with lentivirus encoding the G2R-3 304 or 307 ECD and expanded in 304 or 307 G-CSF (100 ng / mL). Untransduced cells were expanded in IL-2 (300 IU / mL). On day 12 of expansion, cells were washed and replated with IL-2 (300 IU / mL), 130 G-CSF (100 ng / mL), 304 G-CSF (100 ng / mL), 307 G-CSF (100 ng / mL), or no cytokine.

[0054] [Figure 26] FIG. 26 depicts a graph showing G-CSFR ECD expression by flow cytometry in primary mouse T cells transduced with the indicated chimeric receptor constructs.

[0055] [Figure 27] Figure 27 shows G-CSF-induced phosphorylation of STAT3 (detected by flow cytometry) in primary PBMC-derived human T cells expressing G21R-1 or G21R-2. Cells were subdivided (i.e., gated) into G-CSFR-positive (upper panel) or G-CSFR-negative (lower panel) populations.

[0056] [Figure 28] Figure 28 presents graphs and images showing G-CSF-induced biochemical signaling events in primary mouse T cells expressing G21R-1 or G12R-1. A) Graph showing STAT3 phosphorylation (detected by flow cytometry) in CD4+ or CD8+ cells transduced with G21R-1 and stimulated with no cytokine, IL-21, or G-CSF. B) Graph showing the percentage of cells staining positive for phospho-STAT3 after stimulation with no cytokine (filled circles), IL-21 (squares), or WT G-CSF (gray circles). Live cells were gated by CD8 or CD4, and the percentage of phospho-STAT3-positive cells is shown for each population. C) Western blot to assess the indicated cytokine signaling events in cells expressing G21R-1 or G12R-1 and stimulated with IL-21, IL-12, or WT G-CSF. β-Actin and histone H3 serve as protein loading controls.

[0057] [Figure 29A]Figure 29 presents graphs and images showing proliferation, G-CSFR ECD expression, and WT G-CSF-induced intracellular signaling events in primary murine T cells expressing G2R-2, G2R-3, G7R-1, G21 / 7R-1, and G27 / 2R-1, or mock-transduced T cells. A, B) Graphs showing the results of a BrdU incorporation assay to assess T cell proliferation. Cells were harvested, washed, and then replated in IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. Panels A and B represent independent replicate experiments. [Figure 29B] Figure 29 presents graphs and images showing proliferation, G-CSFR ECD expression, and WT G-CSF-induced intracellular signaling events in primary murine T cells expressing G2R-2, G2R-3, G7R-1, G21 / 7R-1, and G27 / 2R-1, or mock-transduced T cells. A, B) Graphs showing the results of a BrdU incorporation assay to assess T cell proliferation. Cells were harvested, washed, and then replated in IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. Panels A and B represent independent replicate experiments. [Figure 29C] Figure 29 presents graphs and images showing proliferation, G-CSFR ECD expression, and WT G-CSF-induced intracellular signaling events in primary murine T cells expressing G2R-2, G2R-3, G7R-1, G21 / 7R-1, and G27 / 2R-1, or mock-transduced T cells. C) Graph showing G-CSFR ECD expression by flow cytometry in primary murine T cells transduced with the indicated chimeric receptor constructs. [Figure 29D]Figure 29 presents graphs and images showing proliferation, G-CSFR ECD expression, and WT G-CSF-induced intracellular signaling events in primary murine T cells expressing G2R-2, G2R-3, G7R-1, G21 / 7R-1, and G27 / 2R-1, or mock-transduced T cells. D) Western blot to assess the indicated cytokine signaling events in cells expressing G2R-2, G2R-3, G7R-1, G21 / 7R-1, and G27 / 2R-1, or mock-transduced T cells. Cells were stimulated with IL-2 (300 IU / mL), IL-7 (10 ng / mL), IL-21 (10 ng / mL), IL-27 (50 ng / mL), or G-CSF (100 ng / mL). β-Actin and histone H3 serve as protein loading controls. Figure 30 presents graphs and images showing proliferation, G-CSFR ECD expression, and G-CSF-induced biochemical signaling events in primary murine T cells expressing G21 / 2R-1, G12 / 2R-1, and 21 / 12 / 2R-1, or mock-transduced T cells. A, B) Graphs showing the results of a BrdU incorporation assay to assess T cell proliferation. Cells were harvested, washed, and then replated in IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. Panels A and B represent independent replicate experiments. C) Graph showing G-CSFR ECD expression by flow cytometry in primary murine T cells transduced with the indicated chimeric receptor constructs. D) Western blot to assess the indicated cytokine signaling events in cells expressing G21 / 2R-1, G12 / 2R-1, and 21 / 12 / 2R-1, or mock-transduced T cells. Cells were stimulated with IL-2 (300 IU / mL), IL-21 (10 ng / mL), IL-12 (10 ng / mL), or G-CSF (100 ng / mL). β-actin and histone H3 served as protein loading controls.

[0058] [Figure 30]Figure 30 presents graphs and images showing proliferation, G-CSFR ECD expression, and G-CSF-induced biochemical signaling events in primary murine T cells expressing G21 / 2R-1, G12 / 2R-1, and 21 / 12 / 2R-1, or mock-transduced T cells. A, B) Graphs showing the results of a BrdU incorporation assay to assess T cell proliferation. Cells were harvested, washed, and then replated in IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. Panels A and B represent replicate experiments. C) Graph showing G-CSFR ECD expression by flow cytometry in primary murine T cells transduced with the indicated chimeric receptor constructs. D) Western blot to assess the indicated cytokine signaling events in cells expressing G21 / 2R-1, G12 / 2R-1, and 21 / 12 / 2R-1, or mock-transduced T cells. Cells were stimulated with IL-2 (300 IU / mL), IL-21 (10 ng / mL), IL-12 (10 ng / mL), or G-CSF (100 ng / mL). β-actin and histone H3 served as protein loading controls.

[0059] [Figure 31]Figure 31 shows graphs depicting fold expansion and G-CSFR ECD expression of primary human PBMC-derived T cells expressing G12 / 2R-1 with the 134 ECD versus untransduced cells. A) Graph showing the results of a T cell expansion assay in which cells were transduced with lentivirus encoding G12 / 2R-1_134-ECD and expanded with IL-2 (300 IU / ml), G-CSF (100 ng / ml), or medium. Viable cells were counted every 4–5 days. Squares represent cells unstimulated with cytokines. Triangles represent cells stimulated with G-CSF. Diamonds represent cells stimulated with IL-2. B) Graph showing the results of a T cell expansion assay in which cells were transduced as in panel A. On day 19 of expansion, cells were washed and replated with IL-2, G-CSF, or medium alone. Viable cells were counted every 4–5 days. Squares represent cells not stimulated with cytokines. Light gray diamonds represent cells stimulated with 130 G-CSF. Dark gray diamonds represent cells stimulated with IL-2. Light gray inverted triangles represent cells initially stimulated with IL-2 and then re-plated on day 19 with medium alone. Dark gray triangles represent cells initially stimulated with 130 G-CSF and then re-plated on day 19 with medium alone. C) Graph showing G-CSFR ECD expression measured by flow cytometry on days 4 or 16 of expansion.

[0060] [Figure 32]Figure 32 presents graphs showing the proliferation and immunophenotype of primary human PBMC-derived T cells expressing G12 / 2R-1 with a 134 ECD versus untransduced cells. A) Graph showing the results of a BrdU incorporation assay to assess T cell proliferation. Cells were harvested, washed, and then replated with IL-2 (300 IU / ml), IL-2 + IL-12 (300 IU / ml and 10 ng / ml, respectively), 130G-CSF (300 ng / ml), or medium alone. B, C) Representative flow cytometry plots and graphs showing the immunophenotype, as assessed by flow cytometry, of cells expressing G12 / 2R-1 with a 134 ECD versus untransduced cells on day 16 of expansion.

[0061] [Figure 33] Figure 33 presents graphs showing the fold expansion and proliferation of primary human PBMC-derived T cells expressing G12 / 2R-1 with the 304 ECD versus untransduced cells. A) Graph showing the results of a T cell expansion assay in which cells were transduced with lentivirus encoding G12 / 2R-1_134-ECD and expanded in IL-2 (300 IU / mL), 130 G-CSF (100 ng / ml), 304 G-CSF (100 ng / ml), or media alone. Untransduced cells were cultured in IL-2, 130 G-CSF, 304 G-CSF, or media alone. Viable cells were counted every 3-4 days. Inverted triangles represent cells not stimulated with cytokines. Triangles represent cells stimulated with IL-2. Circles represent cells stimulated with 130 G-CSF. Diamonds represent cells stimulated with 304 G-CSF. B) On day 12, cells were harvested from the expansion proliferation assay, washed, and then replated with IL-2 (300 IU / ml), 130 G-CSF (300 ng / ml), 304 G-CSF (100 ng / ml), 307 G-CSF (100 ng / ml), or medium alone.

[0062] [Figure 34]Figure 34 depicts Western blots to detect the indicated cytokine signaling events in primary PBMC-derived T cells expressing G2R-3 with 304 ECD, G12 / 2R-1 with 304 ECD, or untransduced T cells. Cells were harvested from expansion proliferation assays and stimulated with 304 G-CSF (100 ng / mL), IL-2 (300 IU / mL), IL-2 and IL-12 (10 ng / mL), or medium alone, as indicated. β-actin and histone H3 serve as protein loading controls. DETAILED DESCRIPTION OF THE INVENTION

[0063] Detailed Description of the Invention Briefly, as described in more detail below, described herein are chimeric receptors comprising the extracellular domain of G-CSFR and the intracellular domain of various multisubunit cytokine receptors for selectively activating cytokine signaling in cells of interest. In certain aspects, the selective activation of cytokine signaling in cells expressing the chimeric receptors described herein includes the ability to specifically stimulate adoptively transferred cells. Thus, described herein are novel processes and compositions of matter that have the potential to improve cell-based therapies for various disease indications.

[0064] definition Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0065] The term "treatment" means any therapeutically beneficial result in the treatment of a disease state, for example, a cancer disease state, including prevention, reduction in severity or progression, remission, or cure thereof.

[0066] The term "in vivo" refers to a process that occurs within a living organism.

[0067] As used herein, the term "mammal" includes both humans and non-humans, including, but not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0068] The term "sufficient amount" means an amount sufficient to produce a desired effect, for example, an amount sufficient to selectively activate a receptor expressed on a cell.

[0069] The term "therapeutically effective amount" is an amount effective to ameliorate symptoms of the disease.

[0070] The term "wild-type" refers to the naturally occurring amino acid sequence of a polypeptide or the native nucleic acid sequence of a gene encoding a polypeptide described herein. The wild-type sequence of a protein or gene is the most common sequence of the polypeptide or gene in that species of protein or gene.

[0071] As used herein, the term "chimeric receptor" refers to a transmembrane receptor that has been engineered to have at least a portion of at least one domain (e.g., the ECD, ICD, TMD, or C-terminal region) derived from the sequence of one or more different transmembrane proteins or receptors.

[0072] The term "operably linked" refers to a nucleic acid or amino acid sequence that is placed into a functional relationship with another nucleic acid or amino acid sequence, respectively. Generally, "operably linked" means that the nucleic acid or amino acid sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase.

[0073] As used herein, the term "extracellular domain" (ECD) refers to the domain of a receptor (e.g., G-CSFR) that is external to the plasma membrane when expressed on the surface of a cell. In certain embodiments, the ECD of G-CSFR comprises at least a portion of SEQ ID NO: 2 or 7.

[0074] As used herein, the term "intracellular domain" (ICD) refers to the domain of a receptor that is located intracellularly when the receptor is expressed on the cell surface.

[0075] As used herein, the term "transmembrane domain" (TMD or TM) refers to the domain or region of a cell surface receptor that is located within the plasma membrane when the receptor is expressed on the cell surface.

[0076] The term "cytokine" refers to a small protein (approximately 5-20 kDa) that can bind to a cytokine receptor expressed on a cell and induce cell signaling upon binding to and activating the cytokine receptor. Examples of cytokines include, but are not limited to, interleukins, lymphokines, colony-stimulating factors, and chemokines.

[0077] The term "cytokine receptor" refers to a receptor that binds to a cytokine, including cytokine receptors type 1 and type 2. Cytokine receptors include, but are not limited to, IL-2R (interleukin-2 receptor), IL-7R (interleukin-7 receptor), IL-12R (interleukin-12 receptor), and IL-21R (interleukin-21 receptor).

[0078] The term "G-CSFR" refers to granulocyte colony-stimulating factor receptor. G-CSFR may also be referred to as GCSFR, G-CSF receptor, colony-stimulating factor 3 receptor, CSF3R, CD114 antigen, or SCN7. Human G-CSFR is encoded by a gene having Ensembl identification number: ENSG00000119535. Human G-CSFR is encoded by a cDNA sequence corresponding to GeneBank accession number NM_156039.3.

[0079] The term "G-CSF" refers to granulocyte colony-stimulating factor. G-CSF may also be called colony-stimulating factor 3 and CSF3. Human G-CSF is encoded by the gene having Ensembl identification number: ENSG00000108342. Human G-CSF is encoded by the cDNA sequence corresponding to GeneBank accession number KP271008.1.

[0080] The term "at least a portion" refers to more than 50%, 75%, 80%, 90%, 95%, or 99% of the length of consecutive nucleotides or amino acids of a SEQ ID NO: described herein. At least a portion of a domain or binding site described herein (e.g., an ECD, ICD, transmembrane, C-terminal region, or signaling molecule binding site) can be more than 50%, 75%, 80%, 90%, 95%, or 99% identical to a SEQ ID NO: described herein.

[0081] The term "signaling molecule binding site" refers to a nucleotide or amino acid sequence in the cytokine receptor intracellular domain that is necessary for or enhances binding of the cytokine receptor to a downstream signaling molecule.

[0082] The term "Box1" or "Box2" region refers to a region on the ICD that functions as a binding site for the tyrosine kinases Jak1, Jak2, Jak3, or Tyk2. The Box1 region may comprise a sequence of amino acids that is greater than 50% identical to a Box1 sequence listed in Table 2.

[0083] The term "C-terminal region" refers to the carboxy-terminal region of a cytokine receptor that contains at least one signaling molecule binding site of the chimeric receptor.

[0084] The term "orthogonal" or "orthogonal cytokine-receptor pair" refers to a pair of engineered proteins that (a) do not bind to the native cytokine or cognate receptor, and (b) have been modified by changing amino acids so that they specifically bind to a corresponding engineered (orthogonal) ligand or receptor.

[0085] As used herein, the term "orthogonal receptor" refers to an engineered receptor of an orthogonal cytokine-receptor pair.

[0086] As used herein, the term "orthogonal cytokine" or "orthogonal G-CSF" refers to an engineered cytokine that is an orthogonal cytokine-receptor pair.

[0087] As used herein, "does not bind" or "cannot bind" refers to no detectable binding or little binding, i.e., having a binding affinity much lower than that of the natural ligand.

[0088] A cytokine that can "selectively activate a chimeric receptor" refers to a cytokine that preferentially binds to and activates a chimeric receptor compared to the native (wild-type) cytokine receptor. In certain embodiments, the cytokine selectively activates a chimeric receptor that is the orthogonal counterpart of an orthogonal cytokine-receptor pair. In certain embodiments, the cytokine is a wild-type cytokine and selectively activates a chimeric receptor expressed on a cell, but a native wild-type receptor for the cytokine is not expressed in the cell.

[0089] The term "immune cell" refers to any cell known to have a function in supporting an organism's immune system (including innate and adaptive immune responses), including, but not limited to, lymphocytes (e.g., B cells, plasma cells, and T cells), natural killer cells (NK cells), macrophages, monocytes, dendritic cells, neutrophils, and granulocytes. Immune cells include stem cells, immature immune cells, and differentiated cells. Immune cells also include any cell subpopulation, whether rare or abundant in the body. In certain embodiments, immune cells are identified as such by possessing known markers (e.g., cell surface markers) of immune cell types and subpopulations.

[0090] As used herein, the term "enhanced activity" refers to an increase in the activity of a variant receptor expressed on a cell upon stimulation with a variant cytokine, which activity is the activity observed for the native receptor upon stimulation with the native cytokine.

[0091] The term "T cell" refers to a mammalian immune effector cell that may be characterized by expression of CD3 and / or a T cell antigen receptor, and these cells may be engineered to express orthogonal cytokine receptors. In some embodiments, T cells are naive CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, helper T cells, e.g., T H 2. T H 9. T H 11. T H 22, T FH ;regulatory T cells, e.g. R 1, Natural T Reg , inducible T Reg memory T cells, e.g., selected from central memory T cells, effector memory T cells, NKT cells, and γδT cells;

[0092] Abbreviations used in this application include the following: ECD (extracellular domain), ICD (intracellular domain), TMD or TM (transmembrane domain), G-CSFR (granulocyte colony-stimulating factor receptor), G-CSF (granulocyte colony-stimulating factor), IL-2R (interleukin 2 receptor), IL-12R (interleukin 12 receptor), IL-21R (interleukin 21 receptor), and IL-7R (interleukin 7 receptor), and NK cells (natural killer cells). IL-2Rγ may also be referred to herein as IL-2RG, IL-2Rgc, γc, or IL-2Rγc.

[0093] "JAK" may also be referred to as Janus kinase. JAK is a family of intracellular non-receptor tyrosine kinases that transmit cytokine-mediated signals via the Jak-STAT pathway, and includes JAK1, JAK2, JAK3, and TYK2. Human JAK1 is encoded by the gene having Ensembl identification number: ENSG00000162434. Human JAK1 is encoded by the cDNA sequence corresponding to GeneBank accession number NM_002227. Human JAK2 is encoded by the gene having Ensembl identification number: ENSG00000096968. Human JAK2 is encoded by the cDNA sequence corresponding to GeneBank accession number NM_001322194. Human JAK3 is encoded by the gene having Ensembl identification number: ENSG00000105639. Human JAK3 is encoded by the cDNA sequence corresponding to GeneBank accession number NM_000215. Human TYK2 is encoded by the gene having Ensembl identification number: ENSG00000105397. Human TYK2 is encoded by the cDNA sequence corresponding to GeneBank accession number NM_001385197.

[0094] STATs may also be referred to as signal transducers and activators of transcription. STATs are a family of seven STAT proteins: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. Human STAT1 is encoded by the gene having Ensembl identification number: ENSG00000115415. Human STAT1 is encoded by the cDNA sequence corresponding to GeneBank accession number: NM_007315. Human STAT2 is encoded by the gene having Ensembl identification number: ENSG00000170581. Human STAT2 is encoded by the cDNA sequence corresponding to GeneBank accession number: NM_005419. Human STAT3 is encoded by the gene having Ensembl identification number: ENSG00000168610. Human STAT3 is encoded by the cDNA sequence corresponding to GeneBank accession number: NM_139276. Human STAT4 is encoded by the gene having Ensembl identification number: ENSG00000138378. Human STAT4 is encoded by the cDNA sequence corresponding to GeneBank accession number: NM_003151. Human STAT5A is encoded by the gene having Ensembl identification number: ENSG00000126561. Human STAT5A is encoded by the cDNA sequence corresponding to GeneBank accession number: NM_003152. Human STAT5B is encoded by the gene having Ensembl identification number: ENSG00000173757. Human STAT5B is encoded by the cDNA sequence corresponding to GeneBank accession number: NM_012448. Human STAT6 is encoded by the gene having Ensembl identification number: ENSG00000166888. Human STAT6 is encoded by a cDNA sequence corresponding to GeneBank accession number: NM_003153.

[0095] SHC may also be referred to as Src homology 2 domain-containing transforming protein. Shc is a family of three isoforms, including p66Shc, p52Shc, and p46Shc, SHC1, SHC2, and SHC3. Human SHC1 is encoded by the gene having Ensembl identification number: ENSG00000160691. Human SHC1 is encoded by the cDNA sequence corresponding to GeneBank accession number: NM_183001. Human SHC2 is encoded by the gene having Ensembl identification number: ENSG00000129946. Human SHC2 is encoded by the cDNA sequence corresponding to GeneBank accession number: NM_012435. Human SHC3 is encoded by the gene having Ensembl identification number: ENSG00000148082. Human SHC3 is encoded by a cDNA sequence corresponding to GeneBank accession number: NM_016848.

[0096] SHP-2 may also be referred to as non-receptor protein tyrosine phosphatase 11 (PTPN11) and protein tyrosine phosphatase 1D (PTP-1D). Human SHP-2 is encoded by a gene having Ensembl identification number: ENSG00000179295. Human SHP-2 is encoded by a cDNA sequence corresponding to GeneBank accession number: NM_001330437.

[0097] PI3K may also be referred to as phosphatidylinositol-4,5-bisphosphate 3-kinase. The catalytic subunit of PI3K may be referred to as PIK3CA. Human PIK3CA is encoded by a gene having Ensembl identification number: ENSG00000121879. Human PIK3CA is encoded by a cDNA sequence corresponding to GeneBank accession number: NM_006218.

[0098] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0099] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, is also specifically disclosed, unless the context clearly dictates otherwise. Each smaller range from any stated value or intervening value in a stated range to any other stated value or intervening value in that stated range is included within the invention. The upper and lower limits of these smaller ranges may or may not independently be included within the range, and each range within these smaller ranges, including either one, neither, or both of those limits, is included within the invention, subject to any specifically excluded limits within the stated range. When a stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention.

[0100] Chimeric cytokine receptor design In certain embodiments, described herein are chimeric cytokine receptors comprising the extracellular domain (ECD) of G-CSFR (granulocyte colony-stimulating factor receptor) operably linked to a second domain; the second domain comprises at least a portion of the intracellular domain (ICD) of a multisubunit cytokine receptor, e.g., IL-2R. In certain aspects, the chimeric cytokine receptor comprises a portion of an ICD of Table 1A and Table 1B. In certain aspects, the chimeric cytokine receptor comprises a transmembrane domain selected from Table 1A and Table 1B. In certain aspects, the chimeric cytokine receptor ICD comprises the Box 1 and Box 2 regions of Table 1A, Table 1B, and Table 2. In certain aspects, the chimeric cytokine receptor comprises at least one signaling molecule binding site of Table 1A, Table 1B, and Table 2.

[0101] In certain aspects, the chimeric receptors described herein comprise an ECD domain that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid or nucleic acid sequence identity with an ECD SEQ ID NO: described herein. In certain aspects, the chimeric receptor comprises an ECD of G-CSFR having the amino acid sequence of SEQ ID NO: 5 or the nucleic acid sequence of SEQ ID NO: 6 or 7. In certain aspects, the chimeric receptor comprises an ECD of G-CSFR, wherein the ECD comprises at least one amino acid substitution. In certain aspects, the ECD of G-CSFR comprises at least one amino acid substitution selected from the group consisting of R41E, R141E, and R167D.

[0102] In certain embodiments, the chimeric receptors described herein comprise a transmembrane domain (TMD) that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid or nucleic acid sequence identity to the TMD SEQ ID NOs described herein. In certain embodiments, the chimeric receptor comprises a TMD of gp130 having the amino acid sequence of SEQ ID NO:9 or the nucleic acid sequence of SEQ ID NO:13. In certain embodiments, the chimeric receptor comprises a TMD of G-CSFR having the amino acid sequence of SEQ ID NO:8 or the nucleic acid sequence of SEQ ID NO:12.

[0103] In certain embodiments, the chimeric receptors described herein comprise at least a portion of the ICD of a cytokine receptor that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid or nucleic acid sequence identity to an ICD SEQ ID NO: described herein. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of IL-2Rβ having the amino acid sequence of SEQ ID NO: 16, 19, 21, 29, 31, 33, 35, 37, or 39. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of IL-2Rβ having the nucleic acid sequence of SEQ ID NO: 44, 47, 49, 57, 59, 61, 63, 65, or 67. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 41 or the nucleic acid sequence of SEQ ID NO: 69. In certain embodiments, a chimeric receptor comprises at least a portion of the ICD of IL-7R having the amino acid sequence of SEQ ID NO: 43 or the nucleic acid sequence of SEQ ID NO: 71. In certain embodiments, a chimeric receptor comprises at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO: 35 or 45. In certain embodiments, a chimeric receptor comprises at least a portion of the ICD of IL-21R having the nucleic acid sequence of SEQ ID NO: 25 or 27. In certain embodiments, a chimeric receptor comprises at least a portion of the ICD of IL-12Rβ2 having the amino acid sequence of SEQ ID NO: 23, 32, or 36. In certain embodiments, a chimeric receptor comprises at least a portion of the ICD of IL-12Rβ2 having the nucleic acid sequence of SEQ ID NO: 51, 60, or 64. In certain embodiments, a chimeric receptor comprises at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 34, 40, or 42. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of G-CSFR having the nucleic acid sequence of SEQ ID NO: 48, 50, 52, 54, 56, 58, 62, 68, or 70. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO: 18 or 38. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of gp130 having the nucleic acid sequence of SEQ ID NO: 46 or 66.In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of IL-2Rγ (i.e., IL-2RG, IL-2Rgc, γc, or IL-2Rγc) having the amino acid sequence of SEQ ID NO: 17. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of IL-2Rγ (i.e., IL-2RG, IL-2Rgc, γc, or IL-2Rγc) having the nucleic acid sequence of SEQ ID NO: 45.

[0104] In certain embodiments, at least a portion of the ICD described herein comprises at least one signaling molecule binding site. In certain embodiments, the at least one signaling molecule binding site is a STAT3 binding site for G-CSFR, a STAT3 binding site for gp130, a SHP-2 binding site for gp130, a Shc binding site for IL-2Rβ, a STAT5 binding site for IL-2Rβ, a STAT3 binding site for IL-2Rβ, a STAT1 binding site for IL-2Rβ, a STAT5 binding site for IL-7Rα, a phosphatidylinositol 3-kinase (PI3K) binding site for IL-7Rα, a STAT5 binding site for IL-12Rβ2, a STAT4 binding site for IL-12Rβ2, a STAT3 binding site for IL-12Rβ2, a STAT5 binding site for IL-21R, a STAT3 binding site for IL-21R, and a STAT1 binding site for IL-21R. In certain embodiments, at least one signaling molecule binding site comprises a sequence further comprising an amino acid listed in Table 2.

[0105] In certain aspects, at least a portion of an ICD described herein comprises the Box1 and Box regions of gp130 or G-CSFR. In certain aspects, the Box1 region comprises a sequence of amino acids set forth in Table 2. In certain aspects, the Box1 region comprises an amino acid sequence that is greater than 50% identical to a Box1 sequence set forth in Table 2.

[0106] In certain aspects, the chimeric receptors described herein comprise at least a portion of the G-CSFR ECD domain, transmembrane domain (TMD), and ICD arranged in N-terminal to C-terminal order as shown in the chimeric receptor designs of Figures 1, 4, and 5.

[0107] In certain embodiments, the chimeric receptors described herein comprise the amino acid sequence in N-terminal to C-terminal order of the sequences disclosed in each of Tables 3-6. In certain embodiments, the chimeric receptors described herein comprise the nucleic acid sequence in 5' to 3' order of the sequences disclosed in each of Tables 3-6. In certain embodiments, the chimeric cytokine receptors share at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to the amino acid sequence in N-terminal to C-terminal order of the amino acid sequence disclosed in each of Tables 3-6. In certain embodiments, the chimeric cytokine receptor shares at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleic acid identity to the nucleic acid sequence in 5' to 3' order of the nucleic acid sequences disclosed in each of Tables 3-6.

[0108] Table 1A: Chimeric cytokine receptors TIFF2025163198000002.tif69148

[0109] Table 1B: Chimeric cytokine receptors TIFF2025163198000003.tif112149

[0110] (Table 2) TIFF2025163198000004.tif112168

[0111] Ligands for chimeric cytokine receptors Described herein are ligands that specifically bind to the chimeric receptors described herein. In certain embodiments, the ligand is a wild-type ligand. In certain embodiments, the ligand is an orthogonal cytokine (i.e., a variant cytokine) that binds with higher affinity to the chimeric receptor compared to binding to the wild-type receptor. In certain embodiments, the ligand is wild-type G-CSF. In certain embodiments, the ligand is G-CSF with one or more amino acid substitutions, e.g., one or more amino acid substitutions selected from the group consisting of E46R, L108K, and D112R.

[0112] When a variant cytokine binds to a variant receptor, the variant receptor activates signaling transduced through native cellular components, providing a biological activity that mimics the native response, but is specific to the cell engineered to express the variant receptor. An orthogonal chimeric receptor does not bind to its endogenous counterpart cytokine, including the orthogonal cytokine's native counterpart, while the orthogonal cytokine does not bind to any endogenous receptor, including the chimeric receptor's native counterpart. In certain embodiments, the orthogonal cytokine binds to the native receptor with significantly reduced affinity compared to the binding of the native cytokine to the native cytokine receptor. In certain embodiments, the affinity of the orthogonal cytokine for the native receptor is less than 10-fold, less than 100-fold, less than 1,000-fold, or less than 10,000-fold greater than the affinity of the native cytokine for the native cytokine receptor. In certain embodiments, the orthogonal cytokine binds to a 1×10 -4 Larger than M, 1X10 -5 Larger than M, 1X10 -6 Larger than M; 1X10 -7 Larger than M, 1X10 -8 Larger than M or 1X10 -9 K is bigger than M DIn certain embodiments, the orthogonal cytokine receptor binds to the native cytokine with significantly reduced affinity compared to the binding of the native cytokine receptor to the native cytokine. In certain embodiments, the orthogonal variant cytokine receptor binds the native cytokine less than 10-fold, less than 100-fold, less than 1,000-fold, or less than 10,000-fold that of the native cytokine to the native cytokine receptor. In certain embodiments, the orthogonal cytokine receptor binds to the native cytokine with less than 1×10 -4 Larger than M, 1X10 -5 Larger than M, 1X10 -6 Larger than M, 1X10 -7 Larger than M, 1X10 -8 Larger than M or 1X10 -9 K is bigger than M D In certain embodiments, the affinity of the orthogonal cytokine for the orthogonal chimeric receptor is comparable to the affinity of the native cytokine for the native receptor, e.g., has an affinity that is at least about 1%, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 100% of the affinity of the native cytokine-receptor pair, and can be higher, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more, than the affinity of the native cytokine for the native receptor.

[0113] Affinity can be determined by any number of assays familiar to those skilled in the art. For example, affinity can be determined by competitive binding experiments, which measure receptor binding using a single concentration of labeled ligand in the presence of varying concentrations of unlabeled ligand. Typically, the concentration of unlabeled ligand is varied over at least six orders of magnitude. Competitive binding experiments provide an IC 50 As used herein, "IC 50 " refers to the concentration of unlabeled ligand required for 50% inhibition of the association between the receptor and the labeled ligand. 50 is an indicator of ligand-receptor binding affinity. Low IC50 indicates high affinity, while high IC 50 indicates low affinity.

[0114] Binding of an orthogonal ligand to a chimeric cytokine receptor expressed on the surface of a cell may or may not affect the function of the cytokine receptor (compared to native cytokine receptor activity); native activity is not necessary or desired in all cases. In certain embodiments, binding of an orthogonal ligand to a chimeric cytokine receptor will induce one or more characteristics of native cytokine signaling. In certain embodiments, binding of an orthogonal cytokine to a chimeric cytokine receptor expressed on the surface of a cell causes a cellular response selected from the group consisting of proliferation, survival, and enhanced activity.

[0115] Nucleic acids encoding chimeric cytokine receptors Included in the present disclosure are nucleic acids encoding any one of the chimeric cytokine receptors described herein. Described herein are expression vectors or kits of expression vectors comprising one or more nucleic acid sequence(s) encoding one or more chimeric cytokine receptor(s) described herein.

[0116] The nucleic acid encoding the chimeric cytokine receptor is inserted into a replicable vector for expression. Such a vector can be used to introduce the nucleic acid sequence(s) into host cells to express the chimeric cytokine receptor described herein. Many such vectors are available. Vector components generally include, but are not limited to, one or more of an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Vectors include viral vectors, plasmid vectors, integrating vectors, etc. Vectors can be, for example, retroviral vectors, adenoviral vectors, lentiviral vectors, transposon-based vectors, or synthetic mRNA. Vectors can be capable of transfecting or transducing T cells, NK cells, or any other immune or non-immune cells.

[0117] Chimeric cytokine receptors can be recombinantly produced not only directly but also as fusion polypeptides with heterologous polypeptides, such as a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. Generally, the signal sequence may be a component of the vector or may be part of a coding sequence that is inserted into the vector. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, the native signal sequence may be used, or other mammalian signal sequences, such as signal sequences from secreted polypeptides of the same or closely related species, and viral secretory leaders, may be suitable. In a specific embodiment, the signal sequence is the nucleic acid sequence of SEQ ID NO:6 or the amino acid sequence of SEQ ID NO:1.

[0118] Expression vectors encoding chimeric cytokine receptors Expression vectors usually contain a selection gene, also called a selectable marker. A selectable marker gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with a vector containing a selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement an auxotrophic deficiency, or (c) supply a critical nutrient unavailable from complex media.

[0119] Expression vectors contain a promoter recognized by a host organism and operably linked to an orthogonal protein-coding sequence. Promoters are untranslated sequences (generally within about 100-1000 bp) located upstream (5') of the start codon of a structural gene that control the transcription and translation of the specific nucleic acid sequence to which they are operably linked. Such promoters are typically divided into two classes: inducible promoters and constitutive promoters. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Numerous promoters recognized by a variety of potential host cells are well known.

[0120] Transcription from vectors in mammalian host cells can be controlled by promoters derived from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus (e.g., murine stem cell virus), hepatitis B virus, and most preferably, simian virus 40 (SV40), heterologous mammalian promoters such as the actin promoter, PGK (phosphoglycerate kinase), or immunoglobulin promoter, or heat shock promoters, provided that such promoters are compatible with the host cell system. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.

[0121] Transcription in higher eukaryotes is often increased by inserting an enhancer sequence into a vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, that act on a promoter to promote its transcription. Enhancers are relatively orientation- and position-independent and are found 5' and 3' to transcription units, within introns, and within the coding sequence itself. Many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin). However, enhancers from eukaryotic viruses are typically used. Examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Enhancers may be spliced ​​into expression vectors at either a 5' or 3' position from the coding sequence, but are preferably located at a 5' site from the promoter.

[0122] Expression vectors used in eukaryotic host cells also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. Construction of suitable vectors containing one or more of the above components employs standard techniques.

[0123] In certain aspects, disclosed herein are lentiviral vectors encoding the chimeric receptors disclosed herein. In certain aspects, the lentiviral vector comprises an HIV-1 5'LTR and 3'LTR. In certain aspects, the lentiviral vector comprises an EF1a promoter. In certain aspects, the lentiviral vector comprises an SV40 polyterminator sequence. In certain aspects, the lentiviral vector is the vector of Figure 6. In certain aspects, the vector is psPAX2, Addgene® 12260, pCMV-VSV-G, or Addgene® 8454.

[0124] Described herein are nucleic acid and polypeptide sequences. In some embodiments, nucleic acid and polypeptide sequences having high sequence identity to the sequences described herein, e.g., 95, 96, 97, 98, 99% or greater, are also described. The term percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotide or amino acid residues that are identical when compared and aligned for maximum correspondence, as determined using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those of skill in the art) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequences being compared, e.g., over a functional domain, or over the entire length of the two sequences being compared.

[0125] For sequence comparison, typically one sequence serves as a reference sequence to which test sequences are compared. When a sequence comparison algorithm is used, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0126] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0127] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0128] Cells expressing chimeric receptors Further described herein are cells that express the chimeric receptor. Host cells, including engineered immune cells, can be transfected or transduced with the above-described expression vectors for cytokine or chimeric cytokine receptor expression.

[0129] The present invention provides cells comprising one or more chimeric cytokine receptors. The cells may comprise a nucleic acid or vector encoding the chimeric cytokine receptor described herein. The present disclosure also provides methods for producing cells expressing a chimeric cytokine receptor. In certain embodiments, the cells are produced by introducing a nucleic acid or expression vector described herein into the cells. The nucleic acid or expression vector can be introduced into the cells by any process, including, but not limited to, transfection, viral vector transduction, transposition, or gene editing. Any gene editing technology known in the art can be used, including, but not limited to, technologies consisting of clustered regularly interspaced short batch repeats (CRISPR-Cas) systems, zinc finger nucleases, transcription activator-like effector-based nucleases, and meganucleases.

[0130] The host cell can be any cell in the body. In certain embodiments, the cell is an immune cell. In some embodiments, the cell is a T cell, including naive CD8+ T cells, cytotoxic CD8+ T cells, and the like. + T cells, naive CD4 + T cells, helper T cells, e.g., T H 1. T H 2. T H 9. T H 11. T H 22, T FH ;regulatory T cells, e.g. R 1, Natural T Reg , inducible T Regmemory T cells, including, but not limited to, central memory T cells, effector memory T cells, NKT cells, γδT cells, etc. In certain embodiments, the cell is a B cell, including, but not limited to, naive B cells, germinal center B cells, memory B cells, cytotoxic B cells, cytokine-producing B cells, regulatory B cells (Bregs), centroblasts, centrocytes, antibody-secreting cells, plasma cells, etc. In certain embodiments, the cell is an innate lymphoid cell, including, but not limited to, an NK cell, etc. In certain embodiments, the cell is a myeloid cell, including, but not limited to, a macrophage, a dendritic cell, a myeloid-derived suppressor cell, etc.

[0131] In certain embodiments, the cells are stem cells, including but not limited to hematopoietic stem cells, mesenchymal stem cells, neural stem cells, and the like.

[0132] In some embodiments, the cells are genetically modified in an ex vivo procedure before being transplanted into a subject. The cells may be provided in a unit dose for treatment and can be allogeneic, autologous, etc. with respect to the intended recipient.

[0133] T cells or T lymphocytes are a type of lymphocyte that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of T cell receptors (TCRs) on their cell surface. There are various types of T cells, as summarized below.

[0134] In certain aspects, cells expressing the chimeric cytokine receptors described herein are helper T cells (Th cells). Th cells assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. Th cells typically express CD4 on their surface. Th cells become activated when peptide antigens are presented by MHC class II molecules on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes, including Th1, Th2, Th17, Th9, or Tfh, which secrete different cytokines to promote different types of immune responses.

[0135] In certain embodiments, the cells expressing the chimeric cytokine receptors described herein are cytolytic T cells (TC cells or CTLs). CTLs destroy virus-infected cells and tumor cells and are also involved in transplant rejection. CTLs typically express CD8 on their surface. These cells recognize targets by binding to antigens associated with MHC class I (present on the surface of all healthy nucleated cells).

[0136] In certain embodiments, the cells expressing the chimeric cytokine receptors described herein are memory T cells. Memory T cells are a subset of antigen-specific T cells that persist long-term, even after resolution of an infection. Upon re-exposure to their cognate antigen, memory T cells rapidly expand into large numbers of effector T cells, providing the immune system with "memory" against past infections. Memory T cells include at least three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Typically, memory T cells express the cell surface protein CD45RO.

[0137] In certain embodiments, the cells expressing the chimeric cytokine receptors described herein are regulatory T cells (Treg cells). Treg cells, formerly known as suppressor T cells, are essential for maintaining immune tolerance. Their primary role is to shut down T cell-mediated immunity toward the end of an immune response and suppress autoreactive T cells that have escaped the negative selection process in the thymus. Two major classifications of CD4+ Treg cells have been described: endogenous Treg cells and adaptive (or inducible) Treg cells. Endogenous Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and are associated with interactions between both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells activated by TSLP and developing T cells. Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3.

[0138] In certain aspects, the cells expressing the chimeric cytokine receptors described herein are tumor-infiltrating lymphocytes (TILs) or tumor-associated lymphocytes (TALs). In certain aspects, TILs / TALs include CD4+ T cells, CD8+ T cells, natural killer (NK) cells, and combinations thereof.

[0139] In certain embodiments, the T cells described herein are chimeric antigen receptor T cells (CAR-T cells) genetically engineered to produce an artificial T cell receptor for use in immunotherapy. In certain aspects, the CAR-T cells are derived from T cells in the patient's own blood (i.e., autologous). In certain aspects, the CAR-T cells are derived from T cells of a donor (i.e., allogeneic).

[0140] In certain embodiments, the T cells described herein are engineered T cell receptor (eTCR-T cells) that have been genetically engineered to produce a specific T cell receptor for use in immunotherapy. In certain aspects, the eTCR-T cells are derived from T cells in the patient's own blood (i.e., autologous). In certain aspects, the eTCR-T cells are derived from T cells of a donor (i.e., allogeneic).

[0141] In certain embodiments, the cells expressing the chimeric cytokine receptors described herein are natural killer cells (NK cells). NK cells form part of the innate immune system. NK cells provide a rapid response to innate immune signals from virus-infected cells in an MHC-independent manner. NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGLs) and constitute a third type of cell differentiated from a common lymphoid progenitor cell that generates B lymphocytes and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, from where they then enter the circulatory system.

[0142] In certain aspects, the cells expressing the chimeric cytokine receptors described herein are B cells, including, but not limited to, naive B cells, germinal center B cells, memory B cells, cytotoxic B cells, cytokine-producing B cells, regulatory B cells (Bregs), centroblasts, centrocytes, antibody-secreting cells, and plasma cells.

[0143] In certain aspects, the cells expressing the chimeric cytokine receptors described herein are myeloid cells, including but not limited to macrophages, dendritic cells, myeloid-derived suppressor cells, and the like.

[0144] Cells expressing the variant receptors or variant cytokines described herein can be of any cell type. In certain aspects, cells expressing the chimeric receptors or cytokines described herein are cells of the hematopoietic system. Immune cells (e.g., T cells or NK cells) can be derived ex vivo from the patient's own peripheral blood (first party), in the setting of hematopoietic stem cell transplantation from donor peripheral blood (second party), or from peripheral blood from an unrelated donor (third party). Alternatively, immune cells can be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells into immune cells. Alternatively, immortalized immune cell lines that retain effector function and can act as therapeutics can be used (e.g., T cell or NK cell lines that retain lytic function, plasma cell lines that retain antibody-producing function, or dendritic cell lines or macrophages that retain phagocytic and antigen-presenting functions, etc.). In all of these embodiments, cells expressing the chimeric cytokine receptors are generated by introducing DNA or RNA encoding each chimeric cytokine receptor(s) by one of a number of means, including transduction with a viral vector or transfection with DNA or RNA.

[0145] The cells may be immune cells derived from a subject and engineered ex vivo to express the chimeric cytokine receptor and / or cytokine. The immune cells may be derived from a peripheral blood mononuclear cell (PBMC) sample. The immune cells may be activated and / or expanded, for example, by treatment with an anti-CD3 monoclonal antibody, before being transduced with a nucleic acid encoding a molecule providing a chimeric cytokine receptor according to the first aspect of the invention. The immune cells of the invention may be produced by (i) isolating a sample comprising the cells from a subject or other source as described above, and (ii) transducing or transfecting the immune cells with one or more nucleic acid sequence(s) encoding the chimeric cytokine receptor(s).

[0146] Cells can be cultured in conventional nutrient media, modified as needed for inducing promoters, selecting transformants, or amplifying the gene encoding the desired sequence. Mammalian host cells can be cultured in a variety of media. Commercially available media, such as Ham's F10 (Sigma), minimal essential medium (MEM, Sigma), RPMI 1640 (Sigma), and Dulbecco's modified Eagle's medium (DMEM, Sigma), are suitable for culturing host cells. Any of these media may be supplemented, as needed, with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those of skill in the art. Culture conditions, such as temperature and pH, will be those previously used for the host cell selected for expression and will be apparent to those of skill in the art.

[0147] The immune cells can then be purified, for example, by selection based on expression of the antigen-binding domain of an antibody. In certain embodiments, the cells are selected by expression of a selectable marker (e.g., a protein, fluorescent marker, or epitope tag) or by any method known in the art for cell selection, isolation, and / or purification.

[0148] kit The present disclosure also describes a kit for producing cells expressing at least one of the chimeric cytokine receptors described herein. In certain embodiments, the kit includes at least one expression vector encoding at least one chimeric cytokine receptor and instructions for use. In certain aspects, the kit further includes an expression vector encoding at least one cytokine in a pharmaceutical formulation, or a cytokine that binds to at least one of the chimeric cytokine receptors described herein. In certain embodiments, the kit includes cells containing an expression vector encoding a chimeric receptor described herein.

[0149] In certain embodiments, the kit comprises cells comprising an expression vector encoding a variant receptor described herein. In certain embodiments, the kit comprises cells comprising an expression vector encoding a chimeric antigen receptor (CAR) / engineered T cell receptor (eTCR), etc. (e.g., an engineered non-native TCR receptor). In certain embodiments, the kit comprises an expression vector encoding a chimeric antigen receptor (CAR) / engineered T cell receptor (eTCR), etc. In certain embodiments, the kit comprises an expression vector encoding a variant receptor and a chimeric antigen receptor (CAR) / engineered T cell receptor (eTCR), etc. described herein.

[0150] In certain aspects, the kits described herein further comprise an orthogonal cytokine. In certain aspects, the kits further comprise at least one cytokine in a pharmaceutical formulation. In certain embodiments, the kits further comprise at least one additional cytokine. In certain embodiments, the components are provided in dosage forms, in liquid or solid form, in any convenient packaging.

[0151] Additional reagents can be provided for the growth, selection, and preparation of the cells provided or produced as described herein. For example, the kits can include components for cell culture, growth factors, differentiation agents, reagents for transfection or transduction, etc.

[0152] In certain embodiments, in addition to the above components, the kit can also include instructions for use. The instructions can be provided in any convenient form. For example, the instructions can be provided as printed information on the kit packaging, package insert, etc. The instructions can also be provided as a computer-readable medium having the information recorded thereon. Additionally, the instructions can be provided as a website address that can be used to access the information.

[0153] Methods for selective activation of chimeric receptors The present disclosure provides a method for selectively activating a chimeric cytokine receptor expressed on the surface of a cell, the method comprising contacting the chimeric cytokine receptor described herein with a cytokine that selectively activates the chimeric receptor. In certain embodiments, the cytokine that selectively activates the chimeric receptor is G-CSF. The G-CSF can be wild-type G-CSF or G-CSF that contains one or more mutations that result in preferential binding and activation of G-CSF to the chimeric receptor compared to the native (wild-type) cytokine receptor.

[0154] In certain aspects, selective activation of the chimeric receptor by cytokine binding to the chimeric receptor results in receptor homodimerization, receptor heterodimerization, or a combination thereof. In certain aspects, activation of the chimeric cytokine receptor results in activation of downstream signaling molecules. In certain aspects, activation of downstream signaling molecules includes activation of cell signaling pathways that stimulate cell cycle progression, proliferation, survival, and / or functional activity of the cell. In certain aspects, the signaling pathways or molecules activated include, but are not limited to, Jak1, Jak2, Jak3, STAT1, STAT2, STAT3, Shc, ERK1 / 2, and Akt. In certain aspects, activation of the chimeric cytokine receptor results in increased cell proliferation following administration of a cytokine that binds to the receptor. In certain embodiments, the degree of proliferation is 1 to 1,000-fold, 1 to 100-fold, 1 to 50-fold, 1 to 10-fold, 1 to 5-fold, 1 to 2-fold, 1 to 1.5-fold, or 0.1 to 10-fold the proliferation observed when the cells are stimulated with IL-2.

[0155] Methods of using stem cells expressing variant cytokine receptors The present invention provides methods for treating and / or preventing a condition or disease comprising administering stem cells expressing a chimeric cytokine receptor and / or an orthogonal cytokine described herein. In certain embodiments, the stem cells expressing the variant cytokine receptors and / or variant cytokines described herein are used in regenerative medicine, cell / tissue / organ transplantation, tissue reconstruction, or tissue repair.

[0156] Adoptive cell transfer methods The present invention provides methods for treating and / or preventing disease, comprising administering to a subject cells expressing a chimeric cytokine receptor described herein (e.g., a pharmaceutical composition described below).

[0157] Methods for treating and / or preventing disease involve the therapeutic use of the cells described herein, e.g., T cells, NK cells, or any other immune or non-immune cells expressing a chimeric cytokine receptor. The cells can be administered to a subject with an existing disease or condition to alleviate, reduce, or ameliorate at least one symptom associated with the disease and / or to slow, reduce, or prevent the progression of the disease. Methods for preventing disease involve the prophylactic use of the cells of the invention. Such cells can be administered to a subject who does not yet suffer from the disease and / or does not exhibit symptoms of the disease to prevent or impair the cause of the disease or to reduce or prevent the occurrence of at least one symptom associated with the disease. The subject may be considered to be predisposed to or at risk of developing the disease.

[0158] In some embodiments, the subject compositions, methods, and kits are used to enhance an immune response, in some embodiments, against conditions where depletion or modulation of target cells, such as cancer cells, infected cells, immune cells involved in autoimmune diseases, etc., is desirable, by systemic administration (e.g., intramuscular, intraperitoneal, intravenous, etc.) of cytokines.

[0159] In certain aspects, a method for treating and / or preventing a disease may include the steps of (i) isolating a sample containing immune cells, (ii) transducing or transfecting such cells with a nucleic acid sequence or vector expressing, for example, a chimeric cytokine receptor, (iii) administering or injecting the cells of (ii) into a subject, and (iv) administering a cytokine that stimulates the infused cells. In certain aspects, the subject has undergone immunodepleting therapy before administering or injecting the cells into the subject. In certain aspects, the subject has not undergone immunodepleting therapy before administering or injecting the cells into the subject. In certain aspects, the subject has undergone immunodepleting therapy with reduced severity without using a chimeric receptor as described herein before administering or injecting the cells into the subject.

[0160] Samples containing immune cells can be isolated from a subject or other sources, for example, as described above. Immune cells can be isolated from the subject's own peripheral blood (first party), in the setting of hematopoietic stem cell transplantation from donor peripheral blood (second party), or from peripheral blood from an unrelated donor (third party). Immune cells can also be isolated from tumor tissue or other tissues in the body.

[0161] In some embodiments, immune cells are contacted with the orthogonal cytokine in vivo, i.e., the immune cells are transferred to a recipient and an effective amount of the orthogonal cytokine is administered to the recipient to contact the immune cells in their native location, e.g., a lymph node. In some embodiments, the contacting is performed in vitro. When cells are contacted with the orthogonal cytokine in vitro, the cytokine is added to the cells at a dose and for a duration sufficient to activate signaling from the receptor, which can utilize features of the native intracellular machinery, e.g., accessory proteins, co-receptors, etc. The activated cells can be used for any purpose, including, but not limited to, determining antigen specificity, cytokine profiling, and experimental purposes related to in vivo delivery.

[0162] In certain aspects, a therapeutically effective number of cells are administered to a subject. In certain aspects, a subject is administered or infused with cells expressing a chimeric cytokine receptor on multiple separate occasions. In certain embodiments, at least 1 x 10 6 Cells / kg, at least 1x10 7 Cells / kg, at least 1x10 8 Cells / kg, at least 1x10 9 Cells / kg, at least 1x10 10 Cells / kg, or more, may be administered, which may be limited by the number of cells, e.g., T cells, available during harvest. The transfected cells can be injected into the subject in any physiologically acceptable medium, usually intravascularly, but may also be introduced at any other convenient site where the cells can find a suitable site for proliferation.

[0163] In certain embodiments, the methods described herein comprise administering a therapeutically effective amount of a cytokine to a subject. In certain embodiments, the subject is administered the cytokine on multiple separate occasions. In certain embodiments, the amount of cytokine administered is sufficient to achieve a therapeutically desirable result (e.g., alleviate symptoms of a disease in the subject). In certain embodiments, the amount of cytokine administered is sufficient to stimulate cell cycle progression, proliferation, survival, and / or functional activity of cells expressing a chimeric cytokine receptor described herein. In certain embodiments, the cytokine is administered at a dose and / or for a duration required to achieve a therapeutically desirable result. In certain embodiments, the cytokine is administered at a dose and / or for a duration sufficient to stimulate cell cycle progression, proliferation, survival, and / or functional activity of cells expressing a chimeric cytokine receptor described herein. The dosage and frequency may vary depending on the agent, the method of administration, the nature of the cytokine, etc. It will be understood by those skilled in the art that such guidelines will be tailored to individual circumstances. Dosages can also vary for local administration (eg, intranasal administration, inhalation, etc.) and for systemic administration (eg, intramuscular, intraperitoneal, intravascular, etc.).

[0164] Indications for Adoptive Cell Transfer The present disclosure provides cells expressing a chimeric cytokine receptor described herein for use in the treatment and / or prevention of disease. The present invention also relates to the use of cells expressing a chimeric cytokine receptor described herein in the manufacture of a medicament for the treatment and / or prevention of disease.

[0165] The disease treated and / or prevented by the methods of the present invention may be a cancerous disease such as, but not limited to, bile duct cancer, bladder cancer, breast cancer, cervical cancer, ovarian cancer, colon cancer, endometrial cancer, hematological malignancies, kidney cancer (renal cell), leukemia, lymphoma, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, sarcoma, and thyroid cancer.

[0166] The disease to be treated and / or prevented may be an autoimmune disease. Autoimmune diseases are characterized by T and B lymphocytes or other immune cell types aberrantly targeting self-proteins, polypeptides, peptides, and / or other self-molecules, causing damage and / or dysfunction of organs, tissues, or cell types within the body (e.g., the pancreas, brain, thyroid, or gastrointestinal tract), resulting in clinical symptoms of the disease. Some autoimmune diseases affect specific tissues, while others can affect multiple tissues, depending in part on whether the response is directed against antigens restricted to specific tissues or against antigens that are widely distributed throughout the body. Autoimmune diseases include, but are not limited to, type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, autoimmune thyroid disease, and Graves' disease.

[0167] The disease to be treated and / or prevented may be an inflammatory disease such as cardiac fibrosis. Generally, inflammatory conditions or disorders typically involve the immune system attacking the body's own cells or tissues, potentially causing abnormal inflammation and resulting in chronic pain, redness, swelling, stiffness, and damage to normal tissue. Inflammatory diseases are characterized by or caused by inflammation, and include, but are not limited to, celiac disease, vasculitis, lupus, chronic obstructive pulmonary disease (COPD), irritable bowel disease, arteriosclerosis, arthritis, myositis, scleroderma, gout, Sjogren's syndrome, ankylosing spondylitis, antiphospholipid syndrome, and psoriasis.

[0168] In certain embodiments, the chimeric cytokine receptors described herein are used to prevent and treat transplant rejection. In certain aspects, the disease to be treated and / or prevented is allograft rejection. In certain aspects, the allograft rejection is acute allograft rejection.

[0169] In certain embodiments, the method is used to treat an infection.

[0170] The disease to be treated and / or prevented may involve transplantation of cells, tissues, organs, or other anatomical structures into an affected individual. The cells, tissues, organs, or other anatomical structures may be derived from the same individual (autologous or "autologous" transplant) or a different individual (allogeneic or "allogeneic" transplant). The cells, tissues, organs, or other anatomical structures may also be produced using in vitro methods, including cell cloning, induced cell differentiation, or production from synthetic biomaterials.

[0171] Therapy may be combined with other active agents, such as, but not limited to, antibiotics, anti-cancer agents, anti-viral agents, and other immunomodulatory agents (e.g., antibodies against the programmed cell death protein-1 [PD-1] pathway or antibodies against cytotoxic T lymphocyte antigen-4 [CTLA-4]), and may include additional cytokines, such as interferon-gamma, tumor necrosis factor-alpha, interleukin-12, and the like.

[0172] Pharmaceutical compositions of the present invention The present invention also relates to pharmaceutical compositions containing a plurality of cells expressing the chimeric cytokine receptor(s) and / or cytokine(s) described herein. The cells and cytokines of the present invention can be formulated into pharmaceutical compositions. These compositions can contain, in addition to one or more cytokines or cells expressing the chimeric cytokine receptor(s), pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The pharmaceutical composition may optionally contain one or more additional pharmaceutically active polypeptides and / or compounds. Such formulations can be in a form suitable for intravenous infusion, for example.

[0173] In the case of cytokines and cells expressing the chimeric receptors described herein administered to an individual, the administration is preferably in a "therapeutically effective amount" sufficient to show benefit to the individual. A "prophylactically effective amount," if sufficient to show benefit to the individual, can also be administered. The actual amount of cytokine or number of cells administered, as well as the rate and time course of administration, will depend on the nature and severity of the disease being treated. Treatment methods, such as determining dosage, are within the responsibility of general practitioners and other physicians and will usually take into account the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to practitioners. Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0174] The compositions may be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated. [Example]

[0175] Below are examples of specific embodiments for carrying out the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0176] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, cell culture, adoptive cell transfer, and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry, 3rd Ed. (Plenum Press), Vols. A and B (1992).

[0177] method Primary cells and cell lines: The lentivirus packaging cell line HEK293T / 17 (ATCC) was cultured in DMEM containing 10% fetal bovine serum and penicillin / streptomycin. BAF3-IL-2Rβ cells were previously generated by stable transfection of the BAF3 cell line with the human IL-2Rβ subunit and grown in RPMI-1640 containing 10% fetal bovine serum, penicillin, streptomycin, and 100 IU / ml of human IL-2 (hIL-2) (PROLEUKIN®, Novartis Pharmaceuticals Canada). The 32D-IL-2Rβ cell line was previously generated by stable transfection of the 32D cell line with the human IL-2Rβ subunit and grown in RPMI-1640 containing 10% fetal bovine serum, penicillin, streptomycin, and 300 IU / ml of hIL-2 or other cytokines as indicated. Human PBMC-derived T cells (Hemacare) were expanded in TexMACS™ medium (Miltenyi Biotec, 130-097-196) containing 3% human AB serum (Sigma-Aldrich, H4522) and 300 IU / ml hIL-2 or other cytokines as indicated. Human tumor-associated lymphocytes (TAL) were generated by culturing primary ascites samples for 14 days in T cell medium (50:50 mixture of the following): 1) RPMI-1640 containing 10% fetal bovine serum, 50 μM β-mercaptoethanol, 10 mM HEPES, 2 mM L-glutamine, penicillin, and streptomycin; and 2) AIM V™ medium (ThermoFisher, 12055083) containing hIL-2 at a final concentration of 3000 IU / ml. After this high-dose IL-2 expansion, TALs were cultured in T cell medium containing 300 IU / ml of hIL-2 or other cytokines as indicated. The retroviral packaging cell line Platinum-E (Cell Biolabs, RV-101) was cultured in DMEM containing 10% FBS, penicillin / streptomycin, puromycin (1 mcg / ml), and blasticidin (10 mcg / ml).

[0178] Generation of lentivirus and transduction of 32D-IL-2Rβ cells: The chimeric receptor construct was cloned into a lentiviral transfer plasmid, and the resulting sequence was confirmed by Sanger sequencing. The transfer plasmid and lentiviral packaging plasmid were co-transfected into HEK293T / 17 cells using the calcium phosphate transfection method as follows: cells were plated overnight and the medium was changed 2–4 h before transfection. Plasmid DNA and water were mixed in a polypropylene tube, and CaCl2 (0.25 M) was added dropwise. After incubation for 2–5 min, the DNA was precipitated by mixing 1:1 with 2x HEPES-buffered saline (0.28 M NaCl, 1.5 mM Na2HPO4, 0.1 M HEPES). The precipitated DNA mixture was added to the cells and incubated overnight at 37°C, 5% CO2. The next day, the HEK293T / 17 medium was changed, and the cells were incubated for an additional 24 h. The following morning, cell supernatants were harvested from the plates, briefly centrifuged to remove debris, and filtered through a 0.45 micron filter. The supernatant was spun at 25,000 rpm for 90 minutes using the SW-32Ti rotor in a Beckman Optima L-XP ultracentrifuge. The supernatant was removed, and the pellet was resuspended in an appropriate volume of Opti-MEM medium. Viral titers were determined by adding serial dilutions of virus to BAF3-IL-2Rβ cells. Forty-eight to 72 hours after transduction, cells were incubated with anti-human G-CSFR APC-conjugated antibody (1:50; Miltenyi Biotec, 130-097-308) and Fixable Viability Dye eFluor™ 450 (1:1000; eBioscience™, 65-0863-14) for 15 minutes at 4°C, washed, and analyzed using a Cytek Aurora or BD FACS Calibur flow cytometer. Using the estimated titer determined by this method, the 32D-IL-2Rβ cell line was transduced with lentiviral supernatant encoding the chimeric receptor construct at a multiplicity of infection (MOI) of 0.5. Transduction was performed by adding the appropriate amount of viral supernatant to the cells, incubating for 24 hours, and then replacing the medium.Three to four days after transduction, expression of human G-CSFR was measured by flow cytometry as described above.

[0179] Lentiviral transduction of human primary T cells: For transduction of PBMC-derived T cells and TALs, cells were thawed and plated in the presence of Human T Cell TransAct™ (Miltenyi Biotec, 130-111-160) according to the manufacturer's guidelines. Twenty-four hours after activation, lentiviral supernatant was added at an MOI of 0.125–0.5. Forty-eight hours after activation, cells were split into fresh medium to remove residual virus and activation reagents. Two to four days after transduction, transduction efficiency was measured by flow cytometry as described above. In experiments measuring the transduction efficiency of the CD4+ and CD8+ fractions separately, antibodies against human G-CSFR were utilized: CD4 (1:50, Alexa Fluor® 700 conjugate, BioLegend, 300526), ​​CD8 (1:50, PerCP conjugate, BioLegend, 301030), CD3 (1:50, Brilliant Violet 510™ conjugate, BioLegend, 300448), and CD56 (1:50, Brilliant Violet 711™ conjugate, BioLegend, 318336), along with Fixable Viability Dye eFluor™ 450 (1:1000).

[0180] Human T cell and 32D-IL-2Rβ expansion assay: Human primary T cells or 32D-IL-2Rβ cells expressing the indicated chimeric receptor constructs, as generated above, were washed three times in PBS and replated with fresh medium, or the medium was gradually replaced as indicated. Complete medium was replaced to contain either wild-type human G-CSF (autologous or NEUPOGEN®, Amgen Canada), mutant G-CSF (autologous), hIL-2, or no cytokine. Cell viability and density were measured by trypan blue exclusion every 3–5 days, and fold expansion relative to the initial cell number was calculated. G-CSFR expression was assessed by flow cytometry as described above.

[0181] CD4+ and CD8+ human TAL expansion assay: To examine the expansion of the CD4+ and CD8+ fractions of TALs, ex vivo ascites samples were thawed, and the CD4+ and CD8+ fractions were enriched using a Human CD4+ T Cell Isolation Kit (Miltenyi Biotec, 130-096-533) and a Human CD8+ T Cell Isolation Kit (Miltenyi Biotec, 130-096-495), respectively. After expansion in cytokine-containing medium, the immunophenotype of the cells was assessed by flow cytometry using antibodies against human G-CSFR, CD4 (1:50, Alexa Fluor® 700 conjugate, BioLegend, 300526), ​​CD8 (1:50, PerCP conjugate, BioLegend, 301030), CD3 (1:50, Brilliant Violet 510™ conjugate, BioLegend, 300448), and CD56 (1:50, Brilliant Violet 711™ conjugate, BioLegend, 318336), together with Fixable Viability Dye eFluor™ 450 (1:1000).

[0182] Primary human T cell immunophenotyping assay: After expansion in cytokine-containing medium, T cells were immunophenotyped using antibodies against human G-CSFR, CD4 (1:100, Alexa Fluor® 700 conjugate, BioLegend, 300526 or PE conjugate, eBioscience™, 12-0048-42, or Brilliant Violet 570™ conjugate, BioLegend, 317445), CD8 (1:100, PerCP conjugate, BioLegend, 301030), CD3 (1:100, Brilliant Violet 510™ or Brilliant Violet 750™ conjugate, BioLegend, 300448 or 344845), CD56 (1:100, Brilliant Violet 570™ conjugate, BioLegend, 300448 or 344845), and CD8 (1:100, PerCP conjugate, BioLegend, 301030). Fixable Viability Dye (FvD) was used to detect CCR7 (1:50, APC / Fire™ 711 conjugate, BioLegend, 318336), CCR7 (1:50, APC / Fire™ 750 conjugate, BioLegend, 353246), CD62L (1:33, PE / Dazzle™ 594 conjugate, BioLegend, 304842), CD45RA (1:33, FITC conjugate, BioLegend, 304148), CD45RO (1:25, PerCP-eFluor® 710 conjugate, eBioscience™, 46-0457-42), and CD95 (1:33, PE-Cyanine 7 conjugate, eBioscience™, 25-0959-42). Evaluated by flow cytometry with eFluor™ 450 or 5106 (1:1000).

[0183] Retroviral transduction: The pMIG transfer plasmid (plasmid #9044, Addgene) was modified by restriction endonuclease cloning to remove the IRES-GFP (BglII to PacI sites) and introduce annealing primers encoding a custom multiple cloning site. The chimeric receptor construct was cloned into the customized transfer plasmid, and the resulting sequence was confirmed by Sanger sequencing. The transfer plasmid was transfected into Platinum-E cells using the calcium phosphate transfection method, as described above. 24 hours after transfection, the medium was replaced with 5 ml of fresh complete medium. 48 hours after transfection, the cell supernatant was harvested from the plate and filtered through a 0.45 micron filter. Hexadimethrine bromide (1.6 mcg / ml, Sigma-Aldrich) and mouse IL-2 (2 ng / ml, Peprotech) were added to the supernatant. This purified retroviral supernatant was used to transduce mouse lymphocytes as described below.

[0184] Forty-eight hours before collecting retroviral supernatants, 24-well adherent plates were coated with unconjugated anti-mouse CD3 (5 mcg / ml, BD Biosciences, 553058) and anti-mouse CD28 (1 mcg / ml, BD Biosciences, 553294) antibodies diluted in PBS and stored at 4°C. Twenty-four hours before collecting retroviral supernatants, C57Bl / 6J mice (autologous) were euthanized under an approved animal protocol administered by the University of Victoria Animal Care and Use Committee. Spleens were harvested, and mouse T cells were isolated as follows: the spleens were manually removed and filtered through a 100-micron filter. Erythrocytes were lysed by incubation in ACK lysis buffer (Gibco, A1049201) at room temperature for 5 minutes, followed by one wash with serum-containing medium. CD8a-positive cells or Pan-T cells were isolated using specific bead-based isolation kits (Miltenyi Biotec, 130-104-075 or 130-095-130, respectively). The cells were added to plates coated with anti-CD3 and anti-CD28 antibodies or 300 IU / mL human IL-2 (Proleukin) in mouse T cell expansion medium (RPMI-1640 containing 10% FBS, penicillin / streptomycin, 0.05 mM β-mercaptoethanol, and 2 ng / mL mouse IL-2 (Peprotech, 212-12)) and incubated at 37°C and 5% CO for 24 hours. On the day of transduction, approximately half of the medium was replaced with the retroviral supernatant prepared above. The cells were spinfected with this retroviral supernatant at 1000 x g for 90 minutes at 30°C. The plates were returned to the incubator for 0-4 hours, after which approximately half of the medium was replaced with fresh T cell expansion medium. Retroviral transduction was repeated 24 hours later, as described above, for a total of two transductions. Twenty-four hours after the last transduction, T cells were split into 6-well plates and removed from antibody stimulation.

[0185] Forty-eight to 72 hours after transduction, transduction efficiency was assessed by flow cytometry detecting human G-CSFR, CD4 (Alexa Fluor 532 conjugate, eBioscience™, 58-0042-82), CD8a (PerCP-eFluor 710 conjugate, eBioscience™, 46-0081-82), and Fixable Viability Dye eFluor™ 450 (1:1000 dilution), as described above.

[0186] BrdU incorporation assay: Human primary T cells, 32D-IL-2Rβ cells, or mouse primary T cells generated as described above were washed three times with PBS and replated for 48 hours in fresh medium containing the relevant assay cytokine: no cytokine, hIL-2 (300 IU / ml), or wild-type or engineered G-CSF (at the concentrations indicated in individual experiments). The BrdU assay procedure was performed according to the instructions for the BD Pharmingen™ APC BrdU Flow Kit (BD Biosciences, 557892), with the following addition: cells were co-incubated with BrdU and Fixable Viability Dye eFluor™ 450 (1:5000) for 30 minutes to 4 hours at 37°C. Flow cytometry was performed using a Cytek Aurora instrument. To specifically assess proliferation of mouse T cells expressing chimeric receptors, additional staining for human G-CSFR (1:20 dilution), CD4 (1:50 dilution), and CD8 (1:50 dilution) was performed on ice for 15 min before fixation.

[0187] Western blot: Human primary T cells, 32D-IL-2Rβ cells, or mouse primary T cells generated as described above were washed three times in PBS and incubated in cytokine-free medium for 16–20 h. Cells were incubated with no cytokine, IL-2 (300 IU / ml), wild-type G-CSF (at the concentrations indicated in individual experiments), or G-CSF. 137The cells were stimulated with ATP (30 ng / ml) for 20 minutes at 37°C. The cells were washed once with a buffer containing 10 mM HEPES, pH 7.9, 1 mM MgCl2, 0.05 mM EGTA, 0.5 mM EDTA, pH 8.0, 1 mM DTT, and 1x Pierce Protease and Phosphatase Inhibitor Minitablets (A32961). The cells were lysed in the above wash buffer, and 0.2% NP-40 (Sigma) was added for 10 minutes on ice. The lysate was centrifuged at 13,000 rpm for 10 minutes at 4°C, and the supernatant (cytoplasmic fraction) was collected. The pellet (containing nuclear proteins) was resuspended in the above wash buffer supplemented with 0.42 M NaCl and 20% glycerol. Nuclei were incubated on ice for 30 minutes with frequent vortexing, then centrifuged at 13,000 rpm at 4°C for 20 minutes, after which the supernatant (nuclear fraction) was collected. The cytoplasmic and nuclear fractions were reduced (70°C) for 10 minutes and run on a NuPAGE™ 4-12% Bis-Tris protein gel. The gel was transferred to a nitrocellulose membrane (60 minutes at 20V in a Trans-Blot™ SD semi-dry transfer cell), dried, and blocked for 1 hour with Odyssey™ Blocking Buffer (927-50000) in TBS. The blot was incubated overnight with primary antibody (1:1,000) in Odyssey™ Blocking Buffer in TBS containing 0.1% Tween 20 at 4°C.Primary antibodies used were obtained from Cell Signaling Technologies: Phospho-Jak1 (Tyr1034 / 1035) (D7N4Z) rabbit mAb #74129, Phospho-Jak2 (Tyr1007 / 1008) #3771, Phospho-Jak3 (Tyr980 / 981) (D44E3) rabbit mAb #5031, Phospho-p70 S6 kinase (Thr421 / Ser424) antibody #9204, Phospho-Shc (Tyr239 / 240) antibody #2434, Phospho-Akt (Ser473) (D9E) XP® rabbit mAb #4060, Phospho-S6 ribosomal protein (Ser235 / 236) antibody #2211, Phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) antibody #9101, β-actin (13E5) rabbit mAb #4970, Phospho-STAT1 (Tyr701) (58D6) rabbit mAb #9167, Phospho-STAT3 (Tyr705) (D3A7) XP® rabbit mAb #9145, Phospho-STAT4 (Tyr693) antibody #5267, Phospho-STAT5 (Tyr694) (C11C5) rabbit mAb #9359, and Histone H3 (96C10) mouse mAb #3638. Blots were washed three times with TBS containing 0.1% Tween 20 and incubated with secondary antibodies (1:10,000) in TBS buffer containing 0.1% Tween 20 for 30–60 min at room temperature. Secondary antibodies obtained from Cell Signaling Technologies were anti-mouse IgG (H+L) (DyLight™ 800 4X PEG conjugate) #5257 and anti-rabbit IgG (H+L) (DyLight™ 800 4X PEG conjugate) #5151. Blots were washed and exposed on a LI-COR Odyssey imager.

[0188] Flow cytometry to detect phosphorylated proteins: Human primary T cells, 32D-IL-2Rβ cells, or mouse primary T cells generated as described above were washed three times in PBS and incubated for 16–20 hours in cytokine-free medium. Cells were stimulated with no cytokines, IL-2 (300 IU / ml), or wild-type G-CSF (100 ng / ml) for 20 minutes at 37°C in the presence of Fixable Viability Dye eFluor™ 450 (1:1000), and anti-G-CSFR (1:20), anti-CD4 (1:50), and anti-CD8a (1:50), as indicated. Cells were pelleted and fixed for 15 minutes at room temperature using BD Phosflow™ Fixation Buffer I (BD Biosciences, 557870). After washing, cells were permeabilized for 15 minutes on ice using BD Phosflow™ Perm Buffer III (BD Biosciences, 558050). Cells were washed twice and resuspended in buffer containing 20 μl of BD Phosflow™ PE Mouse Anti-Stat3 (pY705) (BD Biosciences, 612569) or PE Mouse IgG2a, Kappa Isotope Control (BD Biosciences, 558595). Cells were washed and flow cytometry was performed using a Cytek Aurora instrument.

[0189] Example 1: Expansion of human T cells expressing G2R-1, G-CSFR / IL-2R β subunit only, MYC-tagged G-CSFR / γC subunit only, or full-length G-CSFR. PBMC-derived T cells or tumor-associated lymphocytes (TALs) were transduced with lentivirus encoding the chimeric receptor constructs shown in Figure 1. After washing, the cells were replated with the indicated cytokines. Cells were counted every 3–4 days. G / γc was tagged with a Myc epitope at its N-terminus (Myc / G / γc), and G / IL-2Rβ was tagged with a Flag epitope at its N-terminus (Flag / G / IL-2Rβ); these epitope tags aid in detection by flow cytometry and do not affect receptor function. As expected, all T cell cultures showed proliferation in response to the positive control cytokine, IL-2 (300 IU / ml). After stimulation with G-CSF (100 ng / ml), proliferation was observed only for PBMC-derived T cells and TALs expressing the G2R-1 chimeric cytokine receptor (Figure 3). Note that lentiviral transduction efficiency was less than 100%, with less than 100% of T cells expressing the indicated chimeric cytokine receptors. This likely accounts for the lower proliferation rate mediated by G2R-1 compared to IL-2. Similarly, increased proliferation was observed in 32D-IL-2Rβ cells (stably expressing the human IL-2Rβ subunit) expressing the G-CSF chimeric receptor subunits G2R-1 and G2R-2 and stimulated with G-CSF (Figure 2). In contrast to T cells, 32D-IL-2Rβ cells expressing only the G / IL-2Rβ chimeric receptor subunit proliferated in response to G-CSF (Figure 2); G-CSF-induced proliferation was not observed in 32D-IL-2Rβ cells expressing only the G / γc chimeric receptor subunit (Figure 2).

[0190] These results indicated that G-CSF could stimulate the proliferation and survival of PMBC-derived T cells and TALs expressing the G2R-1 chimeric receptor, as well as 32D-IL-2Rβ cells expressing the G / IL-2Rβ, G2R-1, and G2R-2 chimeric receptors.

[0191] Example 2: G-CSFR ECD is expressed on the surface of G / IL-2Rβ, G2R-1, and G2R-2 transduced cells. Flow cytometry was performed on the 32D-IL-2Rβ cell line, PBMC-derived human T cells, and human tumor-associated lymphocytes after transduction with a lentiviral vector encoding the G2R-2 chimeric cytokine receptor (schematically shown in Figures 4 and 6) to determine whether the cells expressed the G-CSFR ECD on the cell surface. G-CSFR-positive cells were detected in all transduced cell types (Figure 7). In a separate experiment, 32D-IL-2Rβ cells expressing the G / IL-2Rβ, G2R-1, and G2R-2 chimeric receptors were positive for the G-CSFR ECD by flow cytometry (bottom panel of Figures 2B–D).

[0192] These results demonstrate that the G / IL-2Rβ, G2R-1, and G2R-2 chimeric receptors are expressed on the cell surface.

[0193] Example 3: Expansion of G2R-2-expressing cells compared to non-transduced cells Human PBMC-derived T cells and human tumor-associated lymphocytes were lentivirally transduced with the G2R-2 receptor construct (Figures 4 and 6), washed, and replated with the indicated cytokines. In some experiments, T cells were also periodically reactivated by stimulation with TransAct reagent. Viable cells were counted every 3–4 days. Proliferation of PBMC-derived T cells (Figure 8A) and tumor-associated lymphocytes (Figures 8B and C, two independent experiments) was observed after stimulation with G-CSF (100 ng / ml) in cells expressing the G2R-2 chimeric receptor, but not in untransduced cells.

[0194] These results indicate that G-CSF-induced activation of the G2R-2 chimeric receptor is sufficient to induce immune cell proliferation and survival.

[0195] Example 4: Expansion and immunophenotype of CD4 or CD8 selected human tumor-associated lymphocytes expressing G2R-2 compared to non-transduced cells CD4- and CD8-selected human T cells were transduced with a lentiviral vector encoding G2R-2 (Figure 6) or left untransduced where indicated. Cells were washed, replated with the indicated cytokines, and counted every 3–4 days. Proliferation of CD4- or CD8-selected T cells expressing G2R-2 was observed after stimulation with G-CSF (100 ng / ml) or IL-2 (300 IU / ml), but not in the absence of added cytokines (media only) (Figures 9 and 10). In Figure 9, each line represents results from one of five patient samples.

[0196] Immunophenotyping by flow cytometry revealed that T cells cultured with G-CSF or IL-2 retained CD4+ or CD8+ characteristics under these culture conditions (Figure ​(Figure11A),11A), lacked the NK cell phenotype (CD3-CD56+) (Figure ​(Figure11A),11B), and exhibited CD45RA-CCR7- T effector memory (T EM ) phenotype (Figure 11B).

[0197] To confirm that G-CSF stimulation promotes cell cycle progression of G2R-2-expressing T cells, a BrdU assay was performed (Figure 12). T cells were selected by culture with IL-2 or G-CSF, as indicated, prior to the assay. Both tumor-associated lymphocytes (Figure 12A) and PBMC-derived T cells (Figure 12B) were evaluated.

[0198] These results demonstrate that G-CSF can selectively activate cell cycle progression and long-term expansion of primary human TAL cells through activation of the chimeric cytokine receptor G2R-2. These results also demonstrate that activation of the G2R-2 chimeric receptor by homodimerization is sufficient to activate cytokine-like signaling and proliferation in TAL cells. Furthermore, G2R-2-expressing TAL cells maintain cytokine dependence, undergoing cell death upon G-CSF withdrawal, similar to the response to IL-2 withdrawal. TAL cells cultured with G-CSF maintain a similar immunophenotype to TAL cells cultured with IL-2.

[0199] Example 5: Proliferation of primary murine T cells expressing G2R-2 in response to G-CSF. BrdU incorporation assays were performed to assess the proliferation of primary murine T cells expressing G2R-2 or single-chain G / IL-2Rβ (a component of G2R-1) versus mock-transduced cells upon stimulation with G-CSF. All cells were expanded with IL-2 for 3 days before the assay. Cell surface expression of G2R-2 or G / IL-2Rβ was confirmed by flow cytometry (Figure 13A). As indicated, cells were then plated with IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. Enhanced cell cycle progression after G-CSF stimulation was observed in cells expressing G2R-2 compared with untransduced cells or cells expressing single-chain G / IL-2Rβ (Figure 13B,C). Panels B and C show the results for total live cells or G-CSFR+ cells, respectively.

[0200] These results indicate that in response to G-CSF-induced homodimerization, the G2R-2 chimeric receptor is more efficient than the single-chain G / IL-2Rβ receptor in activating cytokine-like signaling and proliferation in mouse T cells.

[0201] Example 6: Activation of cytokine-associated intracellular signaling events in G2R-2-expressing human primary T cells in response to G-CSF or IL-2 To confirm that the chimeric cytokine receptors could indeed activate cytokine signaling similar to that of IL-2, we assessed the ability of the cytokine receptors to activate various signaling molecules. G2R-2-expressing tumor-associated lymphocytes and PBMC-derived T cells were pre-expanded in G-CSF, whereas untransduced cells were pre-expanded in IL-2. Cells were washed and then stimulated with IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine, and cell lysates were Western blotted with antibodies against the indicated signaling molecules (Figure 14). Panels A and B show the results for TAL, and panel C shows the results for PBMC-derived T cells. G2R-2-expressing T cells stimulated with G-CSF activated IL-2-associated signaling molecules to a similar extent as that seen after IL-2 stimulation of untransduced or transduced cells (with the expected exception that G-CSF induced Jak2 phosphorylation, whereas IL-2 induced Jak3 phosphorylation).

[0202] These results confirm that the G2R-2 chimeric receptor is capable of activating IL-2 receptor-like cytokine receptor signaling upon stimulation with G-CSF.

[0203] Example 7: Cytokine signaling is activated in response to G-CSF in primary murine T cells expressing G2R-2. To assess whether the chimeric cytokine receptor G2R-2 or the single-chain G / IL-2Rβ (derived from G2R-1) can activate cytokine signaling, the ability of these cytokine receptors to activate various signaling molecules was assessed by Western blot of cell lysates from mouse primary T cells expressing G2R-2 or G / IL-2Rβ versus mock-transduced cells. All cells were expanded with IL-2 for 3 days before the assay. Cells were then washed and stimulated with IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. G2R-2-expressing T cells stimulated with G-CSF activated IL-2-associated signaling molecules to a similar extent as seen after IL-2 stimulation of untransduced or transduced cells (with the expected exception that G-CSF induced Jak2 phosphorylation, whereas IL-2 induced Jak3 phosphorylation) (Figure 15). In contrast, G / IL-2Rβ did not activate cytokine signaling upon exposure to G-CSF.

[0204] These results confirmed in primary murine T cells that the G2R-2 chimeric receptor can activate IL-2 receptor-like cytokine receptor signaling through homodimerization upon G-CSF stimulation, whereas the single-chain G / IL-2Rβ alone is unable to activate cytokine signaling through homodimerization in response to G-CSF. Example 8: Expression of chimeric receptors results in proliferation of 32D-IL-2Rβ cells and primary murine T cells after stimulation with orthogonal G-CSF. To determine whether cells expressing chimeric cytokine receptors could be selectively activated in response to orthogonal G-CSF, 32D-IL-2Rβ cells or primary murine T cells were transduced with chimeric receptors G2R-1 and G2R-2, which contain the wild-type G-CSFR ECD (G2R-1 WT ECD, G2R-2 WT ECD) and G2R-1 and G2R-2, which contain the G-CSFR ECD with the amino acid substitutions R41E, R141E, and R167D (G2R-1 134 ECD, G2R-2 134 ECD). Cells were stimulated with either IL-2, wild-type G-CSF, or an orthogonal G-CSF (130 G-CSF) that can bind to the G2R-1 134 ECD and G2R-2 134 ECD but has significantly reduced binding to wild-type G-CSFR. BrdU incorporation assays were performed to assess the ability of cytokines to promote cell cycle progression (Figure 16). 32D-IL-2Rβ cells expressing the G2R-2 134 ECD showed cell cycle progression when stimulated with 130 G-CSF (harboring amino acid substitutions E46R, L108K, and D112R; 30 ng / ml), but not with wild-type G-CSF (30 ng / ml). The orthogonality of the engineered cytokine:receptor ECD pair was further demonstrated by stimulating primary murine T cells in a "criss-cross" proliferation assay, in which cells expressing G2R-3 (Figure 4) with WT, 130, 134, 304, or 307 ECDs were stimulated with WT, 130, 304, or 307 cytokines (100 ng / ml) (Figure 17). The 130 ECD contains the amino acid substitutions R41E and R167D. The 304 ECD has amino acid substitutions: R41E, E93K, and R167D, and the 304 cytokine has amino acid substitutions: E46R, L108K, D112R, and R147E. The 307 ECD has amino acid substitutions: R41E, D197K, D200K, and R288E, and the 307 cytokine has amino acid substitutions: S12E, K16D, E19K, and E46R. Panels A and B of Figure 17 represent independent replicate experiments.

[0205] These results demonstrate that cells expressing orthogonal chimeric cytokine receptors are capable of selective activation and cell cycle progression upon stimulation with orthogonal G-CSF.

[0206] Example 9: Intracellular signaling is activated in 32D-IL2Rβ cells and primary human T cells expressing orthogonal chimeric cytokine receptors and stimulated with orthogonal G-CSF. To determine whether cells expressing chimeric cytokine receptors could selectively activate intracellular cytokine signaling events in response to orthogonal G-CSF, 32D-IL-2Rβ cells were transduced with chimeric receptors G2R-1 and G2R-2 containing the wild-type G-CSFR ECD (G2R-1 WT ECD and G2R-2 WT ECD) and chimeric receptors G2R-1 and G2R-2 containing the G-CSFR ECD with the amino acid substitutions R41E, R141E, and R167D (G2R-1 134 ECD, G2R-2 134 ECD). Cells were stimulated with either IL-2 (300 IU / ml), wild-type G-CSF (30 ng / ml), or an orthogonal G-CSF (130 G-CSF-E46R_L108K_D112R; 30 ng / ml) that can bind to the G2R-1 134 ECD and G2R-2 134 ECD but has significantly reduced binding to wild-type G-CSFR. To assess the cells' ability to activate cytokine signaling upon exposure to cytokines, Western blots were performed on cell lysates (Figure 18). Cells expressing the G2R-2 134 ECD showed evidence of cytokine signaling upon stimulation with 130 G-CSF but not wild-type G-CSF. Furthermore, cells expressing the G2R-2 WT ECD failed to activate cytokine signaling when stimulated with 130 G-CSF.

[0207] The orthogonality of the engineered cytokine:receptor pair was further demonstrated by Western blot analysis of primary murine T cells. In this analysis, cells expressing G2R-3 with WT, 134, or 304 ECD (R41E_E93K_R167D) were stimulated with WT, 130, or 304 G-CSF (E46R_L108K_D112R_R147E; 100 ng / ml) and the indicated signaling events were measured (Figure 19A). IL-2 (300 IU / ml) and IL-12 (10 ng / ml) served as control cytokines. Cells expressing the G2R-3 WT ECD showed evidence of cytokine signaling upon stimulation with IL-2, IL-12, or WT G-CSF. Cells expressing the G2R-3 134 ECD showed evidence of cytokine signaling upon stimulation with IL-2, IL-12, or 130 G-CSF. Cells expressing the G2R-3 304 ECD showed evidence of cytokine signaling upon stimulation with IL-2, IL-12, or 304 G-CSF.

[0208] Cell surface expression of the three ECD variants of G2R-3 was confirmed by flow cytometry (Fig. 19B).

[0209] These results demonstrate that cells expressing orthogonal chimeric cytokine receptors can selectively activate intracellular cytokine signaling events upon stimulation with orthogonal G-CSF.

[0210] Example 10: Expression of G2R-3 leads to expansion, cell cycle progression, and cytokine-related intracellular signaling and immune phenotype in primary human T cells To determine whether the G2R-3 chimeric receptor could promote cytokine signaling-related events in primary human T cells upon stimulation with G-CSF, TALs were transduced with a lentiviral vector encoding G2R-3. T cell expansion assays were performed to examine cell proliferation upon stimulation with IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. Viable cells were counted every 3–4 days. In contrast to their non-transduced counterparts, primary TALs expressing G2R-3 expanded in culture in response to G-CSF (Figure 21A).

[0211] To determine whether cytokine signaling events were activated upon stimulation with G-CSF, cell lysates were subjected to Western blot analysis to assess intracellular signaling. Cells were harvested from the expansion assay, washed, and then stimulated with IL-2 (300 IU / ml) or wild-type G-CSF (100 ng / ml). Primary TALs expressing G2R-3 demonstrated IL-2-related signaling events in response to G-CSF, with the expected exception that G-CSF induced Jak2 phosphorylation, whereas IL-2 induced Jak3 phosphorylation (Figure 21B).

[0212] To assess cell cycle progression upon stimulation with G-CSF, a BrdU incorporation assay was performed. Cells were harvested from the expansion assay, washed, and then replated in IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. Primary TALs expressing G2R-3 exhibited cell cycle progression in response to G-CSF (Figure 21C).

[0213] G-CSF-induced expansion of G2R-3-expressing cells was also demonstrated using human T cells derived from primary PBMCs (Figure 22). Cells expressing the G2R-3 WT ECD expanded in response to WT G-CSF but not media alone (Figure 22A). To demonstrate the cells' continued dependence on exogenous cytokines, on day 21 of culture, cells from G-CSF expansion conditions were washed and replated with WT G-CSF (100 ng / mL), IL-7 (20 ng / mL) + IL-15 (20 ng / mL), or media alone. Only cells replated in the presence of G-CSF or IL-7 + IL-15 maintained viability over time.

[0214] Expression of G-CSFR ECD, as assessed by flow cytometry, remained stable between days 21 and 42 of expansion in both CD4+ and CD8+ T cells (FIG. 22B).

[0215] By Western blot, primary PBMC-derived T cells expressing G2R-3 displayed IL-2-associated signaling events in response to G-CSF (FIG. 23A).

[0216] Flow cytometry-based immunophenotyping was performed on primary PBMC-derived T cells expanded for 42 days in WT G-CSF versus IL-7+IL-15. Cells expressing the G2R-3 WT ECD and cultured in G-CSF retained a similar phenotype to untransduced cells cultured in IL-7+IL-15, demonstrating a stem cell-like memory T cell phenotype (T SCM The CD62L+, CD45RO+ phenotype, which indicates central memory (T) (Fig. 23B, C). CM ), Effector Molly (T EM ), and terminal differentiation (T TE ) T cell fractions were also similar.

[0217] These results confirm that the G2R-3 chimeric cytokine receptor can activate cytokine signaling events and promote cell cycle progression and expansion in primary cells. The immunophenotype of G2R-3-expressing T cells expanded long-term in G-CSF is similar to that of untransduced cells expanded in IL-7 plus IL-15.

[0218] Example 11: Orthogonal G-CSF induces expansion and proliferation in primary human T cells expressing G2R-3 with orthogonal ECD We evaluated whether the chimeric cytokine receptor G2R-3, harboring the 304 (R41E_E93K_R167D) or 307 (R41E_D197K_D200K_R288E) ECD, could induce proliferation and expansion in response to stimulation with the orthogonal ligands 130, 304, or 307 (S12E_K16D_E19K_E46R) G-CSF. Primary PBMC-derived human T cells were transduced with lentiviral vectors encoding the G2R-3 304 ECD or the G2R-3 307 (R41E_D197K_D200K_R288E) ECD. T cell proliferation assays were performed to assess the fold expansion of cells when cultured with IL-2 (300 IU / ml), 304 G-CSF (100 ng / ml), 307 G-CSF (100 ng / ml), or no cytokines. Viable cells were counted every 3–4 days. T cells expressing the G2R-3 304 ECD expanded in culture in response to IL-2 or 304 G-CSF (Figure 24A). T cells expressing the G2R-3 307 ECD expanded in culture in response to IL-2 or 307 G-CSF (Figure 24B). Untransduced T cells alone expanded in response to IL-2 (Figure 24C).

[0219] BrdU incorporation assays were performed in a crisscross design to assess cell cycle progression upon stimulation with 130, 304, and 307 G-CSF. Cells were harvested from the expansion assay, washed, and then replated with IL-2 (300 IU / ml), 130 G-CSF (100 ng / ml), 304 G-CSF (100 ng / ml), 307 G-CSF (100 ng / ml), or no cytokine. Primary human T cells expressing the G2R-3 304 ECD progressed through the cell cycle in response to 130 or 304 G-CSF, but not to 307 G-CSF (Figure 25). T cells expressing the G2R-3 307 ECD progressed through the cell cycle in response to 307 G-CSF, but not to 130 or 304 G-CSF. All T cells progressed through the cell cycle in response to IL-2.

[0220] These results demonstrate that the chimeric receptors G2R-3 304 ECD and G2R-3 307 ECD can induce selective cell cycle progression and expansion of primary human CD4+ and CD8+ T cells upon stimulation with orthogonal 304 or 307 G-CSF, respectively. Furthermore, 130 G-CSF can stimulate proliferation of cells expressing G2R-3 304 ECD, but not G2R-3 307 ECD.

[0221] Example 12: G-CSFR ECD is expressed on the surface of primary human tumor-associated lymphocytes (TAL) transduced with G21R-1, G21R-2, G12R-1, and G2R-3 chimeric receptor constructs To assess whether chimeric cytokine receptor constructs could be expressed on the surface of primary human tumor-associated lymphocytes (TALs), TALs were transduced with lentiviral vectors encoding the G21R-1, G21R-2, G12R-1, and G2R-3 chimeric receptors, and the cells were examined for G-CSFR ECD expression on the cell surface by flow cytometry (Figure 20). G-CSFR ECD-positive cells were detected for all four chimeric cytokine receptor designs.

[0222] These results demonstrate that the G21R-1, G21R-2, G12R-1, and G2R-3 chimeric receptors can be expressed on the surface of primary cells. These results also demonstrate that the G-CSFR ECD chimeric receptor design is expressed on the surface of primary cells.

[0223] Example 13: G-CSFR ECD is expressed on the surface of primary murine T cells transduced with G12R-1 and G21R-1 chimeric receptor constructs To determine whether the G12R-1 and G21R-1 chimeric receptors can be expressed on the surface of primary T cells, primary murine T cells were transduced with retroviral vectors encoding the G12R-1 and G21R-1 chimeric receptors and analyzed by flow cytometry (Figure 26).

[0224] The results demonstrate that the G-CSFR ECD is expressed on the surface of primary murine CD4+ and CD8+ T cells transduced with retroviral vectors encoding G12R-1 and G21R-1.

[0225] Example 14: G-CSF induces cytokine signaling events in primary PBMC-derived human T cells expressing G21R-1 or G21R-2 To determine whether the G21R-1 and G21R-2 constructs could induce cytokine signaling events in primary cells, primary PBMC-derived human T cells were transduced with lentiviral vectors encoding the G21R-1 or G21R-2 chimeric cytokine receptor. Cells were intracellularly stained with a phospho-STAT3 (p-STAT3)-specific antibody and evaluated by flow cytometry to measure the degree of STAT3 phosphorylation, a measure of STAT3 activation (Figure 27). Stimulation with G-CSF (100 ng / ml) increased the number of cells expressing phosphorylated STAT3 in the subset of G-CSFR-positive cells transduced with either G21R-1 or G21R-2. In contrast, G-CSFR-negative (i.e., non-expressing) cells did not show an increase in phosphorylated STAT3 upon stimulation with G-CSF, but did increase it upon stimulation with IL-21.

[0226] These results demonstrate that the G21R-1 and G21R-2 chimeric cytokine receptors can activate IL-21-associated cytokine signaling events upon G-CSF stimulation in primary human T cells.

[0227] Example 15: G-CSF induces intracellular signaling events in primary murine T cells expressing G21R-1 or G-12R-1 To determine whether the chimeric cytokine receptor G21R-1 can activate cytokine signaling events, primary murine T cells were transduced with a retroviral vector encoding G21R-1 and assessed by flow cytometry to detect phosphorylated STAT3 upon stimulation with G-CSF. Live cells were gated on CD8 or CD4, and the percentage of cells staining positive for phospho-STAT3 was measured for both CD8 and CD4 cell populations after stimulation with no cytokine, IL-21 (1 ng / ml), or G-CSF (100 ng / ml). Upon stimulation with G-CSF, cells expressing G21R-1 (but not untransduced cells) showed increased amounts of phosphorylated STAT3 (Figures 28A and 28B).

[0228] Western blots were performed to assess intracellular cytokine signaling in cells expressing G21R-1 or G12R-1 upon stimulation with G-CSF. As expected, cells expressing G21R-1 stimulated with G-CSF showed increased phosphorylation of STAT3 and slight increases in phospho-STAT4 and phospho-STAT5 (Figure 28C). Also as expected, cells expressing G12R-1 showed strong phosphorylation of STAT4 in response to G-CSF. G-CSF did not induce signaling events in mock-transduced cells. (Note that in the G12R-1 group, the positive control (hIL-12 10 ng / ml) did not appear to induce signaling events; this may be due to insufficient binding of human IL-12 to the mouse IL-12R.)

[0229] The results show that G21R-1 and G12R-1 can induce cytokine signaling events in primary murine T cells upon stimulation with G-CSF.

[0230] Example 16: G-CSF induces proliferation and intracellular signaling events in primary murine T cells expressing G2R-2, G2R-3, G7R-1, G21 / 7R-1, G27 / 2R-1, G21 / 2R-1, G12 / 2R-1, or G21 / 12 / 2R-1 To assess cytokine signaling events and cell proliferation mediated by chimeric cytokine receptors, primary murine T cells were transduced with retroviral vectors encoding G2R-2, G2R-3, G7R-1, G21 / 7R-1, G27 / 2R-1, G21 / 2R-1, G12 / 2R-1, or G21 / 12 / 2R-1. BrdU incorporation assays were performed to assess cell cycle progression upon stimulation with G-CSF. Cells were harvested, washed, and then replated in IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. G-CSF-induced cell cycle progression was observed in primary murine T cells expressing G2R-2, G2R-3, G7R-1, G21 / 7R-1, or G27 / 2R-1 (Figures 29A and 29B), or G21 / 2R-1, G12 / 2R-1, or G21 / 12 / 2R-1 (Figures 30A and 30B). Expression of the G-CSFR ECD was also detectable by flow cytometry (Figures 29C and 30C).

[0231] By Western blot, multiple cytokine signaling events were observed in response to G-CSF (100 ng / ml) in cells expressing the indicated chimeric cytokine receptors but not in mock-transduced cells (Figures 29D and 30D). Overall, the observed cytokine signaling events were as expected based on the signaling domains incorporated into the various ICD designs (Figures 4 and 5). As one example, the G7R-1 chimeric receptor induced STAT5 phosphorylation (Figure 29D), which is expected due to the incorporation of the STAT5 binding site from IL-7Rα (Figure 4). As a second example, the G21 / 2R-1 chimeric receptor induced STAT3 phosphorylation (Figure 30D), which is expected due to the incorporation of the STAT3 binding site from G-CSFR (Figure 5). As a third example, the G12 / 2R-1 chimeric receptor induced STAT4 phosphorylation, which would be expected to result from the incorporation of the STAT4 binding site from IL-12Rβ2 (Fig. 5). Other chimeric cytokine receptors exhibited other patterns of intracellular signaling events.

[0232] The results show that G2R-2, G2R-3, G7R-1, G21 / 7R-1, G27 / 2R-1, G21 / 2R-1, G12 / 2R-1, and G21 / 12 / 2R-1 can induce cytokine signaling events and proliferation in primary murine T cells upon stimulation with G-CSF. Furthermore, by incorporating different signaling domains into the ICD of the chimeric receptors, distinct patterns of intracellular signaling events can be generated.

[0233] Example 17: Orthogonal G-CSF induces expansion, proliferation, cytokine-associated intracellular signaling, and immune phenotype in primary human T cells expressing G12 / 2R-1 with orthogonal ECD To determine whether the chimeric cytokine receptor G12 / 2R-1 bearing the 134 ECD could induce proliferation and expansion in response to stimulation with the orthogonal ligand G-CSF, primary PBMC-derived human T cells were transduced with a lentiviral vector encoding the G12 / 2R-1 134 ECD. T cell proliferation assays were performed to assess the fold expansion of cells when cultured with IL-2 (300 IU / ml), G-CSF (100 ng / ml), or no cytokine. Viable cells were counted every 4–5 days. Primary human T cells expressing the G12 / 2R-1 134 ECD expanded in culture in response to IL-2 or G-CSF (Figure 31A), but showed limited, transient expansion in culture medium alone.

[0234] On day 19 of this experiment, T cells expanded with 130 G-CSF or IL-2 were washed three times and replated with IL-2, 130 G-CSF, or medium alone. With medium alone, T cells exhibited reduced viability and decreased numbers (Figure 31B). In contrast, T cells replated with IL-2 or G-CSF 130 exhibited continued viability and stable numbers.

[0235] Expression of G12 / 2R-1 134 ECD, detected by flow cytometry using an antibody against the G-CSF receptor, increased from day 4 to day 16 in both CD4+ and CD8+ T cells expanded by stimulation with 130 G-CSF (Figure 31C). To assess cell cycle progression upon stimulation with 130 G-CSF, a BrdU incorporation assay was performed.

[0236] To assess cell cycle progression by BrdU assay, cells were harvested from the expansion assay, washed, and replated with IL-2 (300 IU / ml), IL-2 and IL-12 (10 ng / ml), G-CSF (300 ng / ml), or no cytokines. Primary human T cells expressing the G12 / 2R-1 ECD exhibited cell cycle progression in response to G-CSF, IL-2, or IL-2 + IL-12, whereas untransduced cells responded only to IL-2 or IL-2 + IL-12 (Figure 32A).

[0237] After a 16-day culture period, immunophenotyping was performed by flow cytometry using antibodies against CD62L and CD45RO to compare G12 / 2R-1 ECD-expressing T cells expanded in G-CSF with untransduced cells expanded in IL-2. The two T cell populations were characterized as stem cell-like memory (T SCM ), Central Memory (T CM ), Effector Memory (T EM ), and terminal differentiation (T TE ) showed similar rates of phenotypes (Figure 32B,C).

[0238] Similar experiments were performed using the chimeric cytokine receptor G12 / 2R-1, which has the 304 ECD (rather than the 134 ECD). Primary PBMC-derived human T cells were transduced with a lentiviral vector encoding the G12 / 2R-1 304 ECD. T cell proliferation assays were performed to assess the fold expansion of cells when cultured with IL-2 (300 IU / ml), 130 G-CSF (100 ng / ml), 304 G-CSF (100 ng / ml), or medium alone. Viable cells were counted every 4–5 days. T cells expressing G12 / 2R-1 with the 304 ECD could expand in the presence of IL-2, 130 G-CSF, or 304 G-CSF, but not in medium alone, whereas untransduced cells could expand in response to IL-2 alone (Figure 33A).

[0239] To assess cell cycle progression by BrdU assay, T cells expressing G12 / 2R-1 304 ECD, previously expanded with either G-CSF or G-CSF, were harvested from the expansion assay, washed, and replated with IL-2 (300 IU / ml), G-CSF (100 ng / ml), G-CSF (100 ng / ml), G-CSF (100 ng / ml), or medium alone. G12 / 2R-1 304 ECD-expressing T cells exhibited cell cycle progression in response to G-CSF or G-CSF, but not to G-CSF or medium alone (Figure 33B).

[0240] These results demonstrate that G12 / 2R-1 134 ECD can induce cell cycle progression and expansion of primary human CD4+ and CD8+ T cells upon stimulation with 130 G-CSF. The T cell memory phenotype of cells expressing G12 / 2R-1 134 ECD and expanded with 130 G-CSF is similar to that of untransduced cells expanded with IL-2. Furthermore, G12 / 2R-1 304 ECD can induce selective cell cycle progression and expansion of T cells upon stimulation with 130 or 304 G-CSF, but not in response to 307 G-CSF.

[0241] Example 18: Orthogonal G-CSF induces distinct intracellular signaling events in primary human T cells expressing G2R-3 or G12 / 2R-1 with orthogonal ECDs To assess intracellular signaling events, primary PBMC-derived human T cells were transduced with lentiviral vectors encoding G2R-3 ECD or G12 / 2R-1 ECD. Western blots were performed to assess intracellular cytokine signaling in cells expressing G2R-3 ECD or G12 / 2R-1 ECD or non-transduced cells upon stimulation with G-CSF (100 ng / mL), IL-2 (300 IU / mL), IL-2, and IL-12 (10 ng / mL), or medium alone. In both transduced and non-transduced T cells, robust phosphorylation of STAT5 was detected in response to stimulation with either IL-2 + IL-12 or IL-2 alone (Figure 34). Strong phosphorylation of STAT4 was detected in response to stimulation with both IL-2 and IL-12, but only weak phosphorylation of STAT4 was detected in response to stimulation with IL-2 alone. In cells expressing the G2R-3 304 ECD, weak phosphorylation of STAT4 and strong phosphorylation of STAT5 were detected in response to stimulation with 304 G-CSF, a pattern similar to that seen in response to IL-2 alone. In cells expressing the G12 / 2R-1 304 ECD, strong phosphorylation of STAT4 and STAT5 was detected in response to stimulation with 304 G-CSF, a pattern similar to that seen in response to IL-2 and IL-12. Untransduced T cells showed no response to 304 G-CSF. These results indicate that G12 / 2R-1, which has the 304 ECD, can induce cytokine signaling events, including strong phosphorylation of STAT4 and STAT5, in response to stimulation with 304 G-CSF. A different pattern of signaling events was observed in cells expressing the G2R-3 304 ECD after stimulation with 304 G-CSF, including strong phosphorylation of STAT5 but not STAT4.

[0242] While the present invention has been particularly shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and detail can be made therein without departing from the spirit and scope of the invention.

[0243] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

[0244] (Table 3) Signal peptides. Signal peptides consist of one of the following: TIFF2025163198000005.tif97158

[0245] Table 4. Wild-type G-CSFR extracellular domain (ECD): The G-CSFR ECD is composed of one of the following: TIFF2025163198000006.tif87158TIFF2025163198000007.tif214158TIFF2025163198000008.tif201158

[0246] Table 5. Transmembrane domains (TM). A TM consists of one of the following: TIFF2025163198000009.tif120158

[0247] (Table 6) Intracellular domain (ICD). The ICD consists of one of the following: TIFF2025163198000010.tif84159TIFF2025163198000011.tif217159TIFF2025163198000012.tif219159TIFF2025163198000013.tif169159TIFF2025163198000014.tif202159TIFF2025163198000015.tif213159TIFF2025163198000016.tif214159TIFF2025163198000017.tif224159TIFF2025163198000018.tif95159

[0248] (Table 7) G-CSF Sequence TIFF2025163198000019.tif84158TIFF2025163198000020.tif158158

[0249] Sequence Information SEQUENCE LISTING <110> UVIC INDUSTRY PARTNERSHIPS INC. PROVINCIAL HEALTH SERVICES AUTHORITY <120> CHIMERIC CYTOKINE RECEPTORS <150> US 62 / 912,223 <151> 2019-10-08 <160> 80 <170> PatentIn version 3.5 <210> 1 <211> 24 <212> PRT <213> Homo sapiens <400> 1 Met Ala Arg Leu Gly Asn Cys Ser Leu Thr Trp Ala Ala Leu Ile Ile 1 5 10 15 Leu Leu Leu Pro Gly Ser Leu Glu 20 <210> 2 <211> 22 <212> PRT <213> Homo sapiens <400> 2 Put Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro 20 <210> 3 <211> 72 <212> DNA <213> Homo sapiens <400> 3 atggcaaggc tgggaaactg cagcctgact tgggctgccc tgatcatcct gctgctcccc 60 ggaagtctgg ag 72 <210> 4 <211> 66 <212> DNA <213> Mus musculus <400> 4 atgctgctgc tagtgacctc cctgctgctc tgtgagctgc ctcacccggc gttcctgctg 60 attcct 66 <210> 5 <211> 603 <212> PRT <213> Homo sapiens <400> 5 Glu Cys Gly His Ile Ser Val Ser Ala Pro Ile Val His Leu Gly Asp 1 5 10 15 Pro Ile Thr Ala Ser Cys Ile Ile Lys Gln Asn Cys Ser His Leu Asp 20 25 30 Pro Glu Pro Gln Ile Leu Trp Arg Leu Gly Ala Glu Leu Gln Pro Gly 35 40 45 Gly Arg Gln Gln Arg Leu Ser Asp Gly Thr Gln Glu Ser Ile Ile Thr 50 55 60 Leu Pro His Leu Asn His Thr Gln Ala Phe Leu Ser Cys Cys Leu Asn 65 70 75 80 Trp Gly Asn Ser Leu Gln Ile Leu Asp Gln Val Glu Leu Arg Ala Gly 85 90 95 Tyr Pro Pro Ala Ile Pro His Asn Leu Ser Cys Leu Met Asn Leu Thr 100 105 110 Thr Ser Ser Leu Ile Cys Gln Trp Glu Pro Gly Pro Glu Thr His Leu 115 120 125 Pro Thr Ser Phe Thr Leu Lys Ser Phe Lys Ser Arg Gly Asn Cys Gln 130 135 140 Thr Gln Gly Asp Ser Ile Leu Asp Cys Val Pro Lys Asp Gly Gln Ser 145 150 155 160 His Cys Cys Ile Pro Arg Lys His Leu Leu Leu Tyr Gln Asn Met Gly 165 170 175 Ile Trp Val Gln Ala Glu Asn Ala Leu Gly Thr Ser Met Ser Pro Gln 180 185 190 Leu Cys Leu Asp Pro Met Asp Val Val Lys Leu Glu Pro Pro Met Leu 195 200 205 Arg Thr Met Asp Pro Ser Pro Glu Ala Ala Pro Pro Gln Ala Gly Cys 210 215 220 Leu Gln Leu Cys Trp Glu Pro Trp Gln Pro Gly Leu His Ile Asn Gln 225 230 235 240 Lys Cys Glu Leu Arg His Lys Pro Gln Arg Gly Glu Ala Ser Trp Ala 245 250 255 Leu Val Gly Pro Leu Pro Leu Glu Ala Leu Gln Tyr Glu Leu Cys Gly 260 265 270 Leu Leu Pro Ala Thr Ala Tyr Thr Leu Gln Ile Arg Cys Ile Arg Trp 275 280 285 Pro Leu Pro Gly His Trp Ser Asp Trp Ser Pro Ser Leu Glu Leu Arg 290 295 300 Thr Thr Glu Arg Ala Pro Thr Val Arg Leu Asp Thr Trp Trp Arg Gln 305 310 315 320 Arg Gln Leu Asp Pro Arg Thr Val Gln Leu Phe Trp Lys Pro Val Pro 325 330 335 Leu Glu Glu Asp Ser Gly Arg Ile Gln Gly Tyr Val Val Ser Trp Arg 340 345 350 Pro Ser Gly Gln Ala Gly Ala Ile Leu Pro Leu Cys Asn Thr Thr Glu 355 360 365 Leu Ser Cys Thr Phe His Leu Pro Ser Glu Ala Gln Glu Val Ala Leu 370 375 380 Val Ala Tyr Asn Ser Ala Gly Thr Ser Arg Pro Thr Pro Val Val Phe 385 390 395 400 Ser Glu Ser Arg Gly Pro Ala Leu Thr Arg Leu His Ala Met Ala Arg 405 410 415 Asp Pro His Ser Leu Trp Val Gly Trp Glu Pro Pro Asn Pro Trp Pro 420 425 430 Gln Gly Tyr Val Ile Glu Trp Gly Leu Gly Pro Pro Ser Ala Ser Asn 435 440 445 Ser Asn Lys Thr Trp Arg Met Glu Gln Asn Gly Arg Ala Thr Gly Phe 450 455 460 Leu Leu Lys Glu Asn Ile Arg Pro Phe Gln Leu Tyr Glu Ile Ile Val 465 470 475 480 Thr Pro Leu Tyr Gln Asp Thr Met Gly Pro Ser Gln His Val Tyr Ala 485 490 495 Tyr Ser Gln Glu Met Ala Pro Ser His Ala Pro Glu Leu His Leu Lys 500 505 510 His Ile Gly Lys Thr Trp Ala Gln Leu Glu Trp Val Pro Glu Pro Pro 515 520 525 Glu Leu Gly Lys Ser Pro Leu Thr His Tyr Thr Ile Phe Trp Thr Asn 530 535 540 Ala Gln Asn Gln Ser Phe Ser Ala Ile Leu Asn Ala Ser Ser Arg Gly 545 550 555 560 Phe Val Leu His Gly Leu Glu Pro Ala Ser Leu Tyr His Ile His Leu 565 570 575 Met Ala Ala Ser Gln Ala Gly Ala Thr Asn Ser Thr Val Leu Thr Leu 580 585 590 Met Thr Leu Thr Pro Glu Gly Ser Glu Leu His 595 600 <210> 6 <211> 1809 <212> DNA <213> Homo sapiens <400> 6 gagtgcgggc acatcagtgt ctcagccccc atcgtccacc tgggggatcc catcacagcc 60 tcctgcatca tcaagcagaa ctgcagccat ctggacccgg agccacagat tctgtggaga 120 ctgggagcag agcttcagcc cggggcagg cagcagcgtc tgtctgatgg gacccaggaa 180 tctatcatca ccctgcccca cctcaaccac actcaggcct ttctctcctg ctgcctgaac 240 tggggcaaca gcctgcagat cctggaccag gttgagctgc gcgcaggcta ccctccagcc 300 ataccccaca acctctcctg cctcatgaac ctcacaacca gcagcctcat ctgccagtgg 360 gagccaggac ctgagaccca cctacccacc agcttcactc tgaagagttt caagagccgg 420 ggcaactgtc agacccaagg ggactccatc ctggactgcg tgcccaagga cgggcagagc 480 cactgctgca tcccacgcaa acacctgctg ttgtaccaga atatgggcat ctgggtgcag 540 gcagagaatg cgctggggac cagcatgtcc ccacaactgt gtcttgatcc catggatgtt 600 gtgaaactgg agccccccat gctgcggacc atggacccca gccctgaagc ggcccctccc 660 caggcaggct gcctacagct gtgctgggag ccatggcagc caggcctgca cataaatcag 720 aagtgtgagc tgcgccacaa gccgcagcgt ggagaagcca gctgggcact ggtgggcccc 780 ctccccttgg aggcccttca gtatgagctc tgcgggctcc tcccagccac ggcctacacc 840 ctgcagatac gctgcatccg ctggcccctg cctggccact ggagcgactg gagccccagc 900 ctggagctga gaactaccga acgggccccc actgtcagac tggacacatg gtggcggcag 960 aggcagctgg accccaggac agtgcagctg ttctggaagc cagtgcccct ggaggaagac 1020 agcggacgga tccaaggtta tgtggtttct tggagaccct caggccaggc tggggccatc 1080 ctgcccctct gcaacaccac agagctcagc tgcaccttcc acctgccttc agaagcccag 1140 gaggtggccc ttgtggccta taactcagcc gggacctctc gtcccactcc ggtggtcttc 1200 tcagaaagca gaggcccagc tctgaccaga ctccatgcca tggcccgaga ccctcacagc 1260 ctctgggtag gctgggagcc ccccaatcca tggcctcagg gctatgtgat tgagtggggc 1320 ctgggccccc ccagcgcgag caatagcaac aagacctgga ggatggaaca gaatgggaga 1380 gccacggggt ttctgctgaa ggagaacatc aggccctttc agctctatga gatcatcgtg 1440 actcccttgt accaggacac catgggaccc tcccagcatg tctatgccta ctctcaagaa 1500 atggctccct cccatgcccc agagctgcat ctaaagcaca ttggcaagac ctgggcacag 1560 ctggagtggg tgcctgagcc ccctgagctg gggaagagcc cccttaccca ctacaccatc 1620 ttctggacca acgctcagaa ccagtccttc tccgccatcc tgaatgcctc ctcccgtggc 1680 tttgtcctcc atggcctgga gcccgccagt ctgtatcaca tccacctcat ggctgccagc 1740 caggctgggg ccaccaacag tacagtcctc accctgatga ccttgacccc agaggggtcg 1800 gagctacac 1809 <210> 7 <211> 1809 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 7 gagtgcgggc acatcagtgt ctcagccccc atcgtccacc tgggggatcc catcacagcc 60 tcctgcatca tcaagcagaa ctgcagccat ctggacccgg agccacagat tctgtggaga 120 ctgggagcag agcttcagcc cgggggcagg cagcagcgtc tgtctgatgg gacccaggaa 180 tctatcatca ccctgcccca cctcaaccac actcaggcct ttctctcctg ctgcctgaac 240 tggggcaaca gcctgcagat cctggaccag gttgagctgc gcgcaggcta ccctccagcc 300 ataccccaca acctctcctg cctcatgaac ctcacaacca gcagcctcat ctgccagtgg 360 gagccaggac ctgagaccca cctacccacc agcttcactc tgaagagttt caagagccgg 420 ggcaactgtc agacccaagg ggactccatc ctggactgcg tgcccaagga cgggcagagc 480 cactgctgca tcccacgcaa acacctgctg ttgtaccaga atatgggcat ctgggtgcag 540 gcagagaatg cgctggggac cagcatgtcc ccacaactgt gtcttgatcc catggatgtt 600 gtgaaactgg agccccccat gctgcggacc atggacccca gccctgaagc ggcccctccc 660 caggcaggct gcctacagct gtgctgggag ccatggcagc caggcctgca cataaatcag 720 aagtgtgagc tgcgccacaa gccgcagcgt ggagaagcca gctgggcact ggtgggcccc 780 ctccccttgg aggcccttca gtatgagctc tgcgggctcc tcccagccac ggcctacacc 840 ctgcagatac gctgcatccg ctggcccctg cctggccact ggagcgactg gagccccagc 900 ctggagctga gaactaccga acgggccccc actgtcagac tggacacatg gtggcggcag 960 aggcagctgg accccaggac agtgcagctg ttctggaagc cagtgcccct ggaggaagac 1020 agcggacgca tccaaggtta tgtggtttct tggagaccct caggccaggc tggggccatc 1080 ctgcccctct gcaacaccac agagctcagc tgcaccttcc acctgccttc agaagcccag 1140 gaggtggccc ttgtggccta taactcagcc gggacctctc gccccacccc ggtggtcttc 1200 tcagaaagca gaggcccagc tctgaccaga ctccatgcca tggcccgaga ccctcacagc 1260 ctctgggtag gctgggagcc ccccaatcca tggcctcagg gctatgtgat tgagtggggc 1320 ctgggccccc ccagcgcgag caatagcaac aagacctgga ggatggaaca gaatgggaga 1380 gccacggggt ttctgctgaa ggagaacatc aggccctttc agctctatga gatcatcgtg 1440 actcccttgt accaggacac catgggaccc tcccagcatg tctatgccta ctctcaagaa 1500 atggctccct cccatgcccc agagctgcat ctaaagcaca ttggcaagac ctgggcacag 1560 ctggagtggg tgcctgagcc ccctgagctg gggaagagcc cccttaccca ctacaccatc 1620 ttctggacca acgctcagaa ccagtccttc tccgccatcc tgaatgcatc ctcccgtggc 1680 tttgtcctcc atggcctgga gcccgccagt ctgtatcaca tccacctcat ggctgccagc 1740 caggctgggg ccaccaacag tacagtcctc accctgatga ccttgacccc agaggggtcg 1800 gagctacac 1809 <210> 8 <211> 23 <212> PRT <213> Homo sapiens <400> 8 Ile Ile Leu Gly Leu Phe Gly Leu Leu Leu Leu Leu Thr Cys Leu Cys 1 5 10 15 Gly Thr Ala Trp Leu Cys Cys 20 <210> 9 <211> 22 <212> PRT <213> Homo sapiens <400> 9 Path I Val Val Pro Val Cys Leu Path Phe Leu Leu Thr Thr Leu Leu 1 5 10 15 Gly Val Leu Phe Cys Phe 20 <210> 10 <211> 25 <212> PRT <213> Homo sapiens <400> 10 Ile Pro Trp Leu Gly His Leu Leu Val Gly Leu Ser Gly Ala Phe Gly 1 5 10 15 Phe Ile Ile Leu Val Tyr Leu Leu Ile 20 25 <210> 11 <211> 21 <212> PRT <213> Homo sapiens <400> 11 Val Val Ile Ser Val Gly Ser Met Gly Leu Ile Ile Ser Leu Leu Cys 1 5 10 15 Val Tyr Phe Trp Leu 20 <210> 12 <211> 69 <212> DNA <213> Homo sapiens <400> 12 atcatcctgg gcctgttcgg cctcctgctg ttgctcacct gcctctgtgg aactgcctgg 60 ctctgtgc 69 <210> 13 <211> 66 <212> DNA <213> Homo sapiens <400> 13 gccatagtcg tgcctgtttg cttagcattc ctattgacaa ctcttctggg agtgctgttc 60 tgcttt 66 <210> 14 <211> 75 <212> DNA <213> Homo sapiens <400> 14 attccgtggc tcggccacct cctcgtgggt ctcagcgggg cttttgctt catcatctta 60 gtgtacttgc tgatc 75 <210> 15 <211> 63 <212> DNA <213> Homo sapiens <400> 15 gtggttatct ctgttggctc catgggattg attatcagcc ttctctgtgt gtatttctgg 60 ctg 63 <210> 16 <211> 286 <212> PRT <213> Homo sapiens <400> 16 Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn 1 5 10 15 Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly 20 25 30 Gly Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe 35 40 45 Ser Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu 50 55 60 Arg Asp Lys Val Thr Gln Leu Leu Leu Gln Gln Asp Lys Val Pro Glu 65 70 75 80 Pro Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn 85 90 95 Gln Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala 100 105 110 Cys Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp 115 120 125 Glu Gly Val Ala Gly Ala Pro Thr Gly Ser Ser Pro Gln Pro Leu Gln 130 135 140 Pro Leu Ser Gly Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp 145 150 155 160 Asp Leu Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro 165 170 175 Ser Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro 180 185 190 Ser Leu Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly 195 200 205 Pro Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro 210 215 220 Glu Leu Val Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly Pro 225 230 235 240 Arg Glu Gly Val Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly Glu 245 250 255 Phe Arg Ala Leu Asn Ala Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu 260 265 270 Ser Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu Val 275 280 285 <210> 17 <211> 86 <212> PRT <213> Homo sapiens <400> 17 Glu Arg Thr Met Pro Arg Ile Pro Thr Leu Lys Asn Leu Glu Asp Leu 1 5 10 15 Val Thr Glu Tyr His Gly Asn Phe Ser Ala Trp Ser Gly Val Ser Lys 20 25 30 Gly Leu Ala Glu Ser Leu Gln Pro Asp Tyr Ser Glu Arg Leu Cys Leu 35 40 45 Val Ser Glu Ile Pro Pro Lys Gly Gly Ala Leu Gly Glu Gly Pro Gly 50 55 60 Ala Ser Pro Cys Asn Gln His Ser Pro Tyr Trp Ala Pro Pro Cys Tyr 65 70 75 80 Thr Leu Lys Pro Glu Thr 85 <210> 18 <211> 101 <212> PRT <213> Homo sapiens <400> 18 Asn Lys Arg Asp Leu Ile Lys Lys His Ile Trp Pro Asn Val Pro Asp 1 5 10 15 Pro Ser Lys Ser His Ile Ala Gln Trp Ser Pro His Thr Pro Pro Arg 20 25 30 His Asn Phe Asn Ser Lys Asp Gln Met Tyr Ser Asp Gly Asn Phe Thr 35 40 45 Asp Val Ser Val Val Glu Ile Glu Ala Asn Asp Lys Lys Pro Phe Pro 50 55 60 Glu Asp Leu Lys Ser Leu Asp Leu Phe Lys Lys Glu Lys Ile Asn Thr 65 70 75 80 Glu Gly His Ser Ser Gly Ile Gly Gly Ser Ser Cys Met Ser Ser Ser 85 90 95 Arg Pro Ser Ile Ser 100 <210> 19 <211> 205 <212> PRT <213> Homo sapiens <400> 19 Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn Gln 1 5 10 15 Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys 20 25 30 Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu 35 40 45 Gly Val Ala Gly Ala Pro Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro 50 55 60 Leu Ser Gly Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp 65 70 75 80 Leu Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser 85 90 95 Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro Ser 100 105 110 Leu Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro 115 120 125 Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro Glu 130 135 140 Leu Val Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly Pro Arg 145 150 155 160 Glu Gly Val Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe 165 170 175 Arg Ala Leu Asn Ala Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser 180 185 190 Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu Val 195 200 205 <210> 20 <211> 62 <212> PRT <213> Homo sapiens <400> 20 Ser Pro Asn Arg Lys Asn Pro Leu Trp Pro Ser Val Pro Asp Pro Ala 1 5 10 15 His Ser Ser Leu Gly Ser Trp Val Pro Thr Ile Met Glu Glu Asp Ala 20 25 30 Phe Gln Leu Pro Gly Leu Gly Thr Pro Pro Ile Thr Lys Leu Thr Val 35 40 45 Leu Glu Glu Asp Glu Lys Lys Pro Val Pro Trp Glu Ser His 50 55 60 <210> 21 <211> 202 <212> PRT <213> Homo sapiens <400> 21 Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn Gln Gly Tyr Phe 1 5 10 15 Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys Gln Val Tyr 20 25 30 Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu Gly Val Ala 35 40 45 Gly Ala Pro Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro Leu Ser Gly 50 55 60 Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp Leu Leu Leu 65 70 75 80 Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser Thr Ala Pro 85 90 95 Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro Ser Leu Gln Glu 100 105 110 Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro Pro Thr Pro 115 120 125 Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro Glu Leu Val Leu 130 135 140 Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly Pro Arg Glu Gly Val 145 150 155 160 Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe Arg Ala Leu 165 170 175 Asn Ala Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser Leu Gln Glu 180 185 190 Leu Gln Gly Gln Asp Pro Thr His Leu Val 195 200 <210> 22 <211> 67 <212> PRT <213> Homo sapiens <400> 22 Ser Pro Asn Arg Lys Asn Pro Leu Trp Pro Ser Val Pro Asp Pro Ala 1 5 10 15 His Ser Ser Leu Gly Ser Trp Val Pro Thr Ile Met Glu Glu Asp Ala 20 25 30 Phe Gln Leu Pro Gly Leu Gly Thr Pro Pro Ile Thr Lys Leu Thr Val 35 40 45 Leu Glu Glu Asp Glu Lys Lys Pro Val Pro Trp Glu Ser His Asn Ser 50 55 60 Serum Glu Thr 65 <210> 23 <211> 72 <212> PRT <213> Homo sapiens <400> 23 Ala Gly Asp Leu Pro Thr His Asp Gly Tyr Leu Pro Ser Asn Ile Asp 1 5 10 15 Asp Leu Pro Ser His Glu Ala Pro Leu Ala Asp Ser Leu Glu Glu Leu 20 25 30 Glu Pro Gln His Ile Ser Leu Ser Val Phe Pro Ser Ser Ser Leu His 35 40 45 Pro Leu Thr Phe Ser Cys Gly Asp Lys Leu Thr Leu Asp Gln Leu Lys 50 55 60 Met Arg Cys Asp Ser Leu Met Leu 65 70 <210> 24 <211> 67 <212> PRT <213> Homo sapiens <400> 24 Ser Pro Asn Arg Lys Asn Pro Leu Trp Pro Ser Val Pro Asp Pro Ala 1 5 10 15 His Ser Ser Leu Gly Ser Trp Val Pro Thr Ile Met Glu Glu Asp Ala 20 25 30 Phe Gln Leu Pro Gly Leu Gly Thr Pro Pro Ile Thr Lys Leu Thr Val 35 40 45 Leu Glu Glu Asp Glu Lys Lys Pro Val Pro Trp Glu Ser His Asn Ser 50 55 60 Ser Glu Thr 65 <210> 25 <211> 30 <212> PRT <213> Homo sapiens <400> 25 Ser Pro Gly Asp Glu Gly Pro Pro Arg Ser Tyr Leu Arg Gln Trp Val 1 5 10 15 Val Ile Pro Pro Pro Leu Ser Ser Pro Gly Pro Gln Ala Ser 20 25 30 <210> 26 <211> 63 <212> PRT <213> Homo sapiens <400> 26 Ser Pro Asn Arg Lys Asn Pro Leu Trp Pro Ser Val Pro Asp Pro Ala 1 5 10 15 His Ser Ser Leu Gly Ser Trp Val Pro Thr Ile Met Glu Glu Asp Ala 20 25 30 Phe Gln Leu Pro Gly Leu Gly Thr Pro Pro Ile Thr Lys Leu Thr Val 35 40 45 Leu Glu Glu Asp Glu Lys Lys Pro Val Pro Trp Glu Ser His Asn 50 55 60 <210> 27 <211> 183 <212> PRT <213> Homo sapiens <400> 27 Gln Asn Ser Gly Gly Ser Ala Tyr Ser Glu Glu Arg Asp Arg Pro Tyr 1 5 10 15 Gly Leu Val Ser Ile Asp Thr Val Thr Val Leu Asp Ala Glu Gly Pro 20 25 30 Cys Thr Trp Pro Cys Ser Cys Glu Asp Asp Gly Tyr Pro Ala Leu Asp 35 40 45 Leu Asp Ala Gly Leu Glu Pro Ser Pro Gly Leu Glu Asp Pro Leu Leu 50 55 60 Asp Ala Gly Thr Thr Val Leu Ser Cys Gly Cys Val Ser Ala Gly Ser 65 70 75 80 Pro Gly Leu Gly Gly Pro Leu Gly Ser Leu Leu Asp Arg Leu Lys Pro 85 90 95 Pro Leu Ala Asp Gly Glu Asp Trp Ala Gly Gly Leu Pro Trp Gly Gly 100 105 110 Arg Ser Pro Gly Gly Val Ser Glu Ser Glu Ala Gly Ser Pro Leu Ala 115 120 125 Gly Leu Asp Met Asp Thr Phe Asp Ser Gly Phe Val Gly Ser Asp Cys 130 135 140 Ser Ser Pro Val Glu Cys Asp Phe Thr Ser Pro Gly Asp Glu Gly Pro 145 150 155 160 Pro Arg Ser Tyr Leu Arg Gln Trp Val Val Ile Pro Pro Pro Leu Ser 165 170 175 Ser Pro Gly Pro Gln Ala Ser 180 <210> 28 <211> 93 <212> PRT <213> Homo sapiens <400> 28 Ser Pro Asn Arg Lys Asn Pro Leu Trp Pro Ser Val Pro Asp Pro Ala 1 5 10 15 His Ser Ser Leu Gly Ser Trp Val Pro Thr Ile Met Glu Glu Asp Ala 20 25 30 Phe Gln Leu Pro Gly Leu Gly Thr Pro Pro Ile Thr Lys Leu Thr Val 35 40 45 Leu Glu Glu Asp Glu Lys Lys Pro Val Pro Trp Glu Ser His Asn Ser 50 55 60 Ser Glu Thr Cys Gly Leu Pro Thr Leu Val Gln Thr Tyr Val Leu Gln 65 70 75 80 Gly Asp Pro Arg Ala Val Ser Thr Gln Pro Gln Ser Gln 85 90 <210> 29 <211> 202 <212> PRT <213> Homo sapiens <400> 29 Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn Gln Gly Tyr Phe 1 5 10 15 Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys Gln Val Tyr 20 25 30 Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu Gly Val Ala 35 40 45 Gly Ala Pro Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro Leu Ser Gly 50 55 60 Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp Leu Leu Leu 65 70 75 80 Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser Thr Ala Pro 85 90 95 Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro Ser Leu Gln Glu 100 105 110 Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro Pro Thr Pro 115 120 125 Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro Glu Leu Val Leu 130 135 140 Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly Pro Arg Glu Gly Val 145 150 155 160 Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe Arg Ala Leu 165 170 175 Asn Ala Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser Leu Gln Glu 180 185 190 Leu Gln Gly Gln Asp Pro Thr His Leu Val 195 200 <210> 30 <211> 62 <212> PRT <213> Homo sapiens <400> 30 Ser Pro Asn Arg Lys Asn Pro Leu Trp Pro Ser Val Pro Asp Pro Ala 1 5 10 15 His Ser Ser Leu Gly Ser Trp Val Pro Thr Ile Met Glu Glu Asp Ala 20 25 30 Phe Gln Leu Pro Gly Leu Gly Thr Pro Pro Ile Thr Lys Leu Thr Val 35 40 45 Leu Glu Glu Asp Glu Lys Lys Pro Val Pro Trp Glu Ser His 50 55 60 <210> 31 <211> 43 <212> PRT <213> Homo sapiens <400> 31 Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn Gln Gly Tyr Phe 1 5 10 15 Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys Gln Val Tyr 20 25 30 Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro 35 40 <210> 32 <211> 20 <212> PRT <213> Homo sapiens <400> 32 Ala Gly Asp Leu Pro Thr His Asp Gly Tyr Leu Pro Ser Asn Ile Asp 1 5 10 15 Asp Leu Pro Ser 20 <210> 33 <211> 116 <212> PRT <213> Homo sapiens <400> 33 Gly Gly Pro Ser Pro Pro Ser Thr Ala Pro Gly Gly Ser Gly Ala Gly 1 5 10 15 Glu Glu Arg Met Pro Pro Ser Leu Gln Glu Arg Val Pro Arg Asp Trp 20 25 30 Asp Pro Gln Pro Leu Gly Pro Pro Thr Pro Gly Val Pro Asp Leu Val 35 40 45 Asp Phe Gln Pro Pro Pro Glu Leu Val Leu Arg Glu Ala Gly Glu Glu 50 55 60 Val Pro Asp Ala Gly Pro Arg Glu Gly Val Ser Phe Pro Trp Ser Arg 65 70 75 80 Pro Pro Gly Gln Gly Glu Phe Arg Ala Leu Asn Ala Arg Leu Pro Leu 85 90 95 Asn Thr Asp Ala Tyr Leu Ser Leu Gln Glu Leu Gln Gly Gln Asp Pro 100 105 110 Thr His Leu Val 115 <210> 34 <211> 93 <212> PRT <213> Homo sapiens <400> 34 Ser Pro Asn Arg Lys Asn Pro Leu Trp Pro Ser Val Pro Asp Pro Ala 1 5 10 15 His Ser Ser Leu Gly Ser Trp Val Pro Thr Ile Met Glu Glu Asp Ala 20 25 30 Phe Gln Leu Pro Gly Leu Gly Thr Pro Pro Ile Thr Lys Leu Thr Val 35 40 45 Leu Glu Glu Asp Glu Lys Lys Pro Val Pro Trp Glu Ser His Asn Ser 50 55 60 Ser Glu Thr Cys Gly Leu Pro Thr Leu Val Gln Thr Tyr Val Leu Gln 65 70 75 80 Gly Asp Pro Arg Ala Val Ser Thr Gln Pro Gln Ser Gln 85 90 <210> 35 <211> 43 <212> PRT <213> Homo sapiens <400> 35 Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn Gln Gly Tyr Phe 1 5 10 15 Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys Gln Val Tyr 20 25 30 Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro 35 40 <210> 36 <211> 20 <212> PRT <213> Homo sapiens <400> 36 Ala Gly Asp Leu Pro Thr His Asp Gly Tyr Leu Pro Ser Asn Ile Asp 1 5 10 15 Asp Leu Pro Ser 20 <210> 37 <211> 116 <212> PRT <213> Homo sapiens <400> 37 Gly Gly Pro Ser Pro Pro Ser Thr Ala Pro Gly Gly Ser Gly Ala Gly 1 5 10 15 Glu Glu Arg Met Pro Pro Ser Leu Gln Glu Arg Val Pro Arg Asp Trp 20 25 30 Asp Pro Gln Pro Leu Gly Pro Pro Thr Pro Gly Val Pro Asp Leu Val 35 40 45 Asp Phe Gln Pro Pro Pro Glu Leu Val Leu Arg Glu Ala Gly Glu Glu 50 55 60 Val Pro Asp Ala Gly Pro Arg Glu Gly Val Ser Phe Pro Trp Ser Arg 65 70 75 80 Pro Pro Gly Gln Gly Glu Phe Arg Ala Leu Asn Ala Arg Leu Pro Leu 85 90 95 Asn Thr Asp Ala Tyr Leu Ser Leu Gln Glu Leu Gln Gly Gln Asp Pro 100 105 110 Thr His Leu Val 115 <210> 38 <211> 138 <212> PRT <213> Homo sapiens <400> 38 Asn Lys Arg Asp Leu Ile Lys Lys His Ile Trp Pro Asn Val Pro Asp 1 5 10 15 Pro Ser Lys Ser His Ile Ala Gln Trp Ser Pro His Thr Pro Pro Arg 20 25 30 His Asn Phe Asn Ser Lys Asp Gln Met Tyr Ser Asp Gly Asn Phe Thr 35 40 45 Asp Val Ser Val Val Glu Ile Glu Ala Asn Asp Lys Lys Pro Phe Pro 50 55 60 Glu Asp Leu Lys Ser Leu Asp Leu Phe Lys Lys Glu Lys Ile Asn Thr 65 70 75 80 Glu Gly His Ser Ser Gly Ile Gly Gly Ser Ser Cys Met Ser Ser Ser 85 90 95 Arg Pro Ser Ile Ser Ser Ser Asp Glu Asn Glu Ser Ser Gln Asn Thr 100 105 110 Ser Ser Thr Val Gln Tyr Ser Thr Val Val His Ser Gly Tyr Arg His 115 120 125 Gln Val Pro Ser Val Gln Val Phe Ser Arg 130 135 <210> 39 <211> 205 <212> PRT <213> Homo sapiens <400> 39 Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn Gln 1 5 10 15 Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys 20 25 30 Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu 35 40 45 Gly Val Ala Gly Ala Pro Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro 50 55 60 Leu Ser Gly Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp 65 70 75 80 Leu Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser 85 90 95 Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro Ser 100 105 110 Leu Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro 115 120 125 Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro Glu 130 135 140 Leu Val Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly Pro Arg 145 150 155 160 Glu Gly Val Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe 165 170 175 Arg Ala Leu Asn Ala Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser 180 185 190 Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu Val 195 200 205 <210> 40 <211> 62 <212> PRT <213> Homo sapiens <400> 40 Ser Pro Asn Arg Lys Asn Pro Leu Trp Pro Ser Val Pro Asp Pro Ala 1 5 10 15 His Ser Ser Leu Gly Ser Trp Val Pro Thr Ile Met Glu Glu Asp Ala 20 25 30 Phe Gln Leu Pro Gly Leu Gly Thr Pro Pro Ile Thr Lys Leu Thr Val 35 40 45 Leu Glu Glu Asp Glu Lys Lys Pro Val Pro Trp Glu Ser His 50 55 60 <210> 41 <211> 67 <212> PRT <213> Homo sapiens <400> 41 Ser Gly Lys Asn Gly Pro His Val Tyr Gln Asp Leu Leu Leu Ser Leu 1 5 10 15 Gly Thr Thr Asn Ser Thr Leu Pro Pro Pro Phe Ser Leu Gln Ser Gly 20 25 30 Ile Leu Thr Leu Asn Pro Val Ala Gln Gly Gln Pro Ile Leu Thr Ser 35 40 45 Leu Gly Ser Asn Gln Glu Glu Ala Tyr Val Thr Met Ser Ser Phe Tyr 50 55 60 Gln Asn Gln 65 <210> 42 <211> 93 <212> PRT <213> Homo sapiens <400> 42 Ser Pro Asn Arg Lys Asn Pro Leu Trp Pro Ser Val Pro Asp Pro Ala 1 5 10 15 His Ser Ser Leu Gly Ser Trp Val Pro Thr Ile Met Glu Glu Asp Ala 20 25 30 Phe Gln Leu Pro Gly Leu Gly Thr Pro Pro Ile Thr Lys Leu Thr Val 35 40 45 Leu Glu Glu Asp Glu Lys Lys Pro Val Pro Trp Glu Ser His Asn Ser 50 55 60 Ser Glu Thr Cys Gly Leu Pro Thr Leu Val Gln Thr Tyr Val Leu Gln 65 70 75 80 Gly Asp Pro Arg Ala Val Ser Thr Gln Pro Gln Ser Gln 85 90 <210> 43 <211> 77 <212> PRT <213> Homo sapiens <400> 43 Ser Ser Ser Arg Ser Leu Asp Cys Arg Glu Ser Gly Lys Asn Gly Pro 1 5 10 15 His Val Tyr Gln Asp Leu Leu Leu Ser Leu Gly Thr Thr Asn Ser Thr 20 25 30 Leu Pro Pro Pro Phe Ser Leu Gln Ser Gly Ile Leu Thr Leu Asn Pro 35 40 45 Val Ala Gln Gly Gln Pro Ile Leu Thr Ser Leu Gly Ser Asn Gln Glu 50 55 60 Glu Ala Tyr Val Thr Met Ser Ser Phe Tyr Gln Asn Gln 65 70 75 <210> 44 <211> 861 <212> DNA <213> Homo sapiens <400> 44 aactgcagga acaccgggcc atggctgaag aaggtcctga agtgtaacac cccagacccc 60 tcgaagttct tttcccagct gagctcagag catggaggag acgtccagaa gtggctctct 120 tcgcccttcc cctcatcgtc cttcagccct ggcggcctgg cacctgagat ctcgccacta 180 gaagtgctgg agagggacaa ggtgacgcag ctgctcctgc agcaggacaa ggtgcctgag 240 cccgcatcct taagcagcaa ccactcgctg accagctgct tcaccaacca gggttacttc 300 ttcttccacc tcccggatgc cttggagata gaggcctgcc aggtgtactt tacttacgac 360 ccctactcag aggaagaccc tgatgagggt gtggccgggg cacccacagg gtcttccccc 420 caacccctgc agcctctgtc aggggaggac gacgcctact gcaccttccc ctccagggat 480 gacctgctgc tcttctcccc cagtctcctc ggtggcccca gccccccaag cactgcccct 540 gggggcagtg gggccggtga agagaggatg cccccttctt tgcaagaaag agtccccaga 600 gactgggacc cccagcccct ggggcctccc accccaggag tcccagacct ggtggatttt 660 cagccacccc ctgagctggt gctgcgagag gctggggagg aggtccctga cgctggcccc 720 agggagggag tcagtttccc ctggtccagg cctcctgggc agggggagtt cagggccctt 780 aatgctcgcc tgcccctgaa cactgatgcc tacttgtccc tccaagaact ccagggtcag 840 gacccaactc acttggtgta g 861 <210> 45 <211> 261 <212> DNA <213> Homo sapiens <400> 45 gaacggacga tgccccgaat tcccaccctg aagaacctag aggatcttgt tactgaatac 60 cacgggaact tttcggcctg gagtggtgtg tctaagggac tggctgagag tctgcagcca 120 gactacagtg aacgactctg cctcgtcagt gagattcccc caaaaggagg ggcccttggg 180 gaggggcctg gggcctcccc atgcaaccag catagcccct actgggcccc cccatgttac 240 accctaaagc ctgaaacctg a 261 <210> 46 <211> 303 <212> DNA <213> Homo sapiens <400> 46 aataagcgag acctaattaa aaaacacatc tggcctaatg ttccagatcc ttcaaagagt 60 catattgccc agtggtcacc tcacactcct ccaaggcaca atttaattc aaaagatcaa 120 atgtattcag atggcaattt cactgatgta agtgttgtgg aatagaagc aaatgacaaa 180 aagccttttc cagaagatct gaaatcattg gacctgttca aaaaggaaaa aattaatact 240 gaaggacaca gcagtggtat tggggggtct tcatgtatgt catcttctag gccaagcatt 300 tct 303 <210> 47 <211> 618 <212> DNA <213> Homo sapiens <400> 47 gcatccttaa gcagcaacca ctcgctgacc agctgcttca ccaaccaggg ttacttcttc 60 ttccacctcc cggatgcctt ggagatagag gcctgccagg tgtactttac ttacgacccc 120 tactcagagg aagaccctga tgagggtgtg gccggggcac ccacagggtc ttccccccaa 180 cccctgcagc ctctgtcagg ggaggacgac gcctactgca cttcccctc cagggatgac 240 ctgctgctct tctcccccag tctcctcggt ggccccagcc ccccaagcac tgcccctggg 300 ggcagtgggg ccggtgaaga gaggatgccc ccttctttgc aagaaagagt ccccagagac 360 tgggaccccc agcccctggg gcctcccacc ccaggagtcc cagacctggt ggattttcag 420 ccaccccctg agctggtgct gcgagaggct ggggaggagg tccctgacgc tggccccagg 480 gagggagtca gtttcccctg gtccaggcct cctgggcagg gggagttcag ggcccttaat 540 gctcgcctgc ccctgaacac tgatgcctac ttgtccctcc aagaactcca gggtcaggac 600 ccaactcact tggtgtag 618 <210> 48 <211> 186 <212> DNA <213> Homo sapiens <400> 48 agccccaaca ggaagaatcc cctctggcca agtgtcccag acccagctca cagcagcctg 60 ggctcctggg tgcccacaat catggaggag gatgccttcc agctgcccgg ccttggcacg 120 ccacccatca ccaagctcac agtgctggag gaggatgaaa agaagccggt gccctgggag 180 tcccat 186 <210> 49 <211> 609 <212> DNA <213> Homo sapiens <400> 49 agcagcaacc actcgctgac cagctgcttc accaaccagg gttacttctt cttccacctc 60 ccggatgcct tggagataga ggcctgccag gtgtacttta cttacgaccc ctactcagag 120 gaagaccctg atgagggtgt ggccggggca cccacagggt cttcccccca acccctgcag 180 cctctgtcag gggaggacga cgcctactgc accttcccct ccagggatga cctgctgctc 240 ttctccccca gtctcctcgg tggccccagc cccccaagca ctgcccctgg gggcagtggg 300 gccggtgaag agaggatgcc cccttctttg caagaaagag tccccagaga ctgggacccc 360 cagcccctgg ggcctcccac cccaggagtc ccagacctgg tggattttca gccaccccct 420 gagctggtgc tgcgagaggc tggggaggag gtccctgacg ctggccccag ggaggggagtc 480 agttcccct ggtccaggcc tcctgggcag ggggagttca gggcccttaa tgctcgcctg 540 cccctgaaca ctgatgccta cttgtccctc caagaactcc agggtcagga cccaactcac 600 ttgggtgtag 609 <210> 50 <211> 201 <212> DNA <213> Homo sapiens <400> 50 agccccaaca ggaagaatcc cctctggcca agtgtcccag acccagctca cagcagcctg 60 ggctcctggg tgcccacaat catggaggag gatgccttcc agctgcccgg ccttggcacg 120 ccacccatca ccaagctcac agtgctggag gaggatgaaa agaagccggt gccctgggag 180 tcccataaca gctcagagac c 201 <210> 51 <211> 219 <212> DNA <213> Homo sapiens <400> 51 gcaggtgacc ttcccaccca tgatggctac ttaccctcca acatagatga cctcccctca 60 catgaggcac ctctcgctga ctctctggaa gaactggagc ctcagcacat ctccctttct 120 gttttcccct caagttctct tcacccactc accttctcct gtggtgataa gctgactctg 180 gatcagttaa agatgaggtg tgactccctc atgctctga 219 <210> 52 <211> 201 <212> DNA <213> Homo sapiens <400> 52 agccccaaca ggaagaatcc cctctggcca agtgtcccag acccagctca cagcagcctg 60 ggctcctggg tgcccacaat catggaggag gatgccttcc agctgcccgg ccttggcacg 120 ccacccatca ccaagctcac agtgctggag gaggatgaaa agaagccggt gccctgggag 180 tcccataaca gctcagagac c 201 <210> 53 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 53 agccctgggg acgaaggacc cccccggagc tacctccgcc agtgggtggt cattcctccg 60 ccactttcga gccctggacc ccaggccagc taa 93 <210> 54 <211> 189 <212> DNA <213> Homo sapiens <400> 54 agccccaaca ggaagaatcc cctctggcca agtgtcccag acccagctca cagcagcctg 60 ggctcctggg tgcccacaat catggaggag gatgccttcc agctgcccgg ccttggcacg 120 ccacccatca ccaagctcac agtgctggag gaggatgaaa agaagccggt gccctgggag 180 tcccataac 189 <210> 55 <211> 549 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 55 cagaactcgg ggggctcagc ttacagtgag gagagggatc ggccatacgg cctggtgtcc 60 attgacacag tgactgtgct agatgcagag gggccatgca cctggccctg cagctgtgag 120 gatgacggct acccagccct ggacctggat gctggcctgg agcccagccc aggcctagag 180 gacccactct tggatgcagg gaccacagtc ctgtcctgtg gctgtgtctc agctggcagc 240 cctgggctag gagggcccct gggaagcctc ctggacagac taaagccacc ccttgcagat 300 ggggaggact gggctggggg actgccctgg ggtggccggt cacctggagg ggtctcagag 360 agtgaggcgg gctcacccct ggccggcctg gatatggaca cgtttgacag tggctttgtg 420 ggctctgact gcagcagccc tgtggagtgt gacttcacca gccctgggga cgaaggaccc 480 ccccggagct acctccgcca gtgggtggtc attcctccgc cactttcgag ccctggaccc 540 caggccagc 549 <210> 56 <211> 279 <212> DNA <213> Homo sapiens <400> 56 agccccaaca ggaagaatcc cctctggcca agtgtcccag acccagctca cagcagcctg 60 ggctcctggg tgcccacaat catggaggag gatgccttcc agctgcccgg ccttggcacg 120 ccacccatca ccaagctcac agtgctggag gaggatgaaa agaagccggt gccctgggag 180 tcccataaca gctcagagac ctgtggcctc cccactctgg tccagaccta tgtgctccag 240 ggggacccaa gagcagtttc cacccagccc caatcccag 279 <210> 57 <211> 609 <212> DNA <213> Homo sapiens <400> 57 agcagcaacc actcgctgac cagctgcttc accaaccagg gttacttctt cttccacctc 60 ccggatgcct tggagataga ggcctgccag gtgtacttta cttacgaccc ctactcagag 120 gaagaccctg atgagggtgt ggccggggca cccacagggt cttcccccca acccctgcag 180 cctctgtcag gggaggacga cgcctactgc accttcccct ccagggatga cctgctgctc 240 ttctccccca gtctcctcgg tggccccagc cccccaagca ctgcccctgg gggcagtggg 300 gccggtgaag agaggatgcc cccttctttg caagaaagag tccccagaga ctgggacccc 360 cagcccctgg ggcctcccac cccaggagtc ccagacctgg tggattttca gccaccccct 420 gagctggtgc tgcgagaggc tggggaggag gtccctgacg ctggccccag ggaggggagtc 480 agttcccct ggtccaggcc tcctgggcag ggggagttca gggcccttaa tgctcgcctg 540 cccctgaaca ctgatgccta cttgtccctc caagaactcc agggtcagga cccaactcac 600 ttgggtgtag 609 <210> 58 <211> 186 <212> DNA <213> Homo sapiens <400> 58 agccccaaca ggaagaatcc cctctggcca agtgtcccag acccagctca cagcagcctg 60 ggctcctggg tgcccacaat catggaggag gatgccttcc agctgcccgg ccttggcacg 120 ccacccatca ccaagctcac agtgctggag gaggatgaaa agaagccggt gccctgggag 180 tcccat 186 <210> 59 <211> 129 <212> DNA <213> Homo sapiens <400> 59 agcagcaacc actcgctgac cagctgcttc accaaccagg gttacttctt cttccacctc 60 ccggatgcct tggagataga ggcctgccag gtgtacttta cttacgaccc ctactcagag 120 gaagaccct 129 <210> 60 <211> 60 <212> DNA <213> Homo sapiens <400> 60 gcaggtgacc ttcccaccca tgatggctac ttaccctcca acatagatga cctcccctca 60 <210> 61 <211> 351 <212> DNA <213> Homo sapiens <400> 61 ggtggcccca gccccccaag cactgcccct gggggcagtg gggccggtga agagaggatg 60 cccccttctt tgcaagaaag agtccccaga gactgggacc cccagcccct ggggcctccc 120 accccaggag tcccagacct ggtggatttt cagccacccc ctgagctggt gctgcgagag 180 gctggggagg aggtccctga cgctggcccc agggagggag tcagtttccc ctggtccagg 240 cctcctgggc agggggagtt cagggccctt aatgctcgcc tgcccctgaa cactgatgcc 300 tacttgtccc tccaagaact ccagggtcag gacccaactc acttggtgta g 351 <210> 62 <211> 279 <212> DNA <213> Homo sapiens <400> 62 agccccaaca ggaagaatcc cctctggcca agtgtcccag acccagctca cagcagcctg 60 ggctcctggg tgcccacaat catggaggag gatgccttcc agctgcccgg ccttggcacg 120 ccacccatca ccaagctcac agtgctggag gaggatgaaa agaagccggt gccctgggag 180 tcccataaca gctcagagac ctgtggcctc cccactctgg tccagaccta tgtgctccag 240 ggggacccaa gagcagtttc cacccagccc caatcccag 279 <210> 63 <211> 129 <212> DNA <213> Homo sapiens <400> 63 agcagcaacc actcgctgac cagctgcttc accaaccagg gttacttctt cttccacctc 60 ccggatgcct tggagataga ggcctgccag gtgtacttta cttacgaccc ctactcagag 120 gaagaccct 129 <210> 64 <211> 60 <212> DNA <213> Homo sapiens <400> 64 gcaggtgacc ttcccaccca tgatggctac ttaccctcca acatagatga cctcccctca 60 <210> 65 <211> 351 <212> DNA <213> Homo sapiens <400> 65 ggtggcccca gccccccaag cactgcccct gggggcagtg gggccggtga agagaggatg 60 cccccttctt tgcaagaaag agtccccaga gactgggacc cccagcccct ggggcctccc 120 accccaggag tcccagacct ggtggatttt cagccacccc ctgagctggt gctgcgagag 180 gctggggagg aggtccctga cgctggcccc agggagggag tcagtttccc ctggtccagg 240 cctcctgggc agggggagtt cagggccctt aatgctcgcc tgcccctgaa cactgatgcc 300 tacttgtccc tccaagaact ccagggtcag gacccaactc acttggtgta g 351 <210> 66 <211> 414 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 66 aataagcgag acctaattaa aaaacacatc tggcctaatg ttccagatcc ttcaaagagt 60 catattgccc agtggtcacc tcacactcct ccaaggcaca atttcaattc aaaggatcaa 120 atgtattcag atggcaattt cactgatgta agtgttgtgg aatagaagc aaatgacaaa 180 aagccttttc cagaagatct gaaatcattg gacctgttca aaaaggaaaa aattaatact 240 gaaggacaca gcagtggtat tggggggtct tcatgtatgt catcttctag gccaagcatt 300 tctagcagtg atgaaaatga atcttcacaa aacacttcga gcactgtcca gtattctacc 360 gtggtacaca gtggctacag acaccaagtt ccgtcagtcc aagtcttctc aaga 414 <210> 67 <211> 618 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 67 gcatccttaa gcagcaacca ctcgctgacc agctgcttca ccaaccaggg ttacttcttc 60 ttccacctcc cggatgcctt ggagatagag gcctgccagg tgtactttac ttacgacccc 120 tactcagagg aagaccctga tgagggtgtg gccggggcac ccacagggtc ttccccccaa 180 cccctgcagc ctctgtcagg ggaggacgac gcctactgca cttcccctc cagggatgac 240 ctgctgctct tctcccccag tctctcggt ggccccagcc ccccaagcac tgcccctggg 300 ggcagtgggg ccggtgaaga gaggatgccc ccttctttgc aagaaagagt ccccagagac 360 tgggaccccc agcccctggg gcctcccacc ccaggagtcc cagacctggt ggattttcag 420 ccaccccctg agctggtgct gcgagaggct ggggaggagg tccctgacgc tggccccagg 480 gagggagtca gtttcccctg gtccaggcct cctgggcagg gggagttcag ggcccttaat 540 gctcgcctgc ccctgaacac tgatgcctac ttgtccctcc aagaactcca gggtcaggac 600 ccaactcact tggtgtag 618 <210> 68 <211> 186 <212> DNA <213> Homo sapiens <400> 68 agccccaaca ggaagaatcc cctctggcca agtgtcccag acccagctca cagcagcctg 60 ggctcctggg tgcccacaat catggaggag gatgccttcc agctgcccgg ccttggcacg 120 ccacccatca ccaagctcac agtgctggag gaggatgaaa agaagccggt gccctgggag 180 tcccat 186 <210> 69 <211> 204 <212> DNA <213> Homo sapiens <400> 69 agtggcaaga atgggcctca tgtgtaccag gacctcctgc ttagccttgg gactacaaac 60 agcacgctgc cccctccatt ttctctccaa tctggaatcc tgacattgaa cccagttgct 120 cagggtcagc ccattcttac ttccctggga tcaaatcaag aagaagcata tgtcaccatg 180 tccagcttct accaaaacca gtga 204 <210> 70 <211> 279 <212> DNA <213> Homo sapiens <400> 70 agccccaaca ggaagaatcc cctctggcca agtgtcccag acccagctca cagcagcctg 60 ggctcctggg tgcccacaat catggaggag gatgccttcc agctgcccgg ccttggcacg 120 ccacccatca ccaagctcac agtgctggag gaggatgaaa agaagccggt gccctgggag 180 tcccataaca gctcagagac ctgtggcctc cccactctgg tccagaccta tgtgctccag 240 ggggaccca gagcagtttc cacccagccc caatcccag 279 <210> 71 <211> 234 <212> DNA <213> Homo sapiens <400> 71 tcctcttcca gtccctaga ctgcagggag agtggcaaga atgggcctca tgtgtaccag 60 gacctcctgc ttagccttgg gactacaaac agcacgctgc cccctccatt ttctctccaa 120 tctggaatcc tgacattga cccagttgct cagggtcagc ccattcttac ttccctggga 180 tcaatcaag agaagcata tgtcaccatg tccagctct accaaacca gtga 234 <210> 72 <211> 174 <212> PRT <213> Homo sapiens <400> 72 Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Lys 1 5 10 15 Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 20 25 30 Glu Lys Cys Ala Thr Tyr Lys Cys His Pro Glu Glu Leu Val 35 40 45 Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys 50 55 60 Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser 65 70 75 80 Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser 85 90 95 Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp 100 105 110 Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro 115 120 125 Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 130 135 140 Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe 145 150 155 160 Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 165 170 <210> 73 <211> 1405 <212> Ms <213> Homo sapiens <400> 73 agcccggagc ctgcagccca gccccaccca gacccatggc tggacctgcc acccagagcc 60 ccatgaagct gatggccctg cagctgctgc tgtggcacag tgcactctgg acagtgcagg 120 aagccacccc cctgggccct gccagctccc tgccccagag cttcctgctc aagtgcttag 180 agcaagtgag gaagatccag ggcgatggcg cagcgctcca ggagaagctg gtgagtgagg 240 caggctgctt gagccaactc catagcggcc ttttcctcta ccaggggctc ctgcaggccc 300 tggaagggat ctcccccgag ttgggtccca ccttggacac actgcagctg gacgtcgccg 360 actttgccac caccatctgg cagcagatgg aagaactggg aatggcccct gccctgcagc 420 ccacccaggg tgccatgccg gccttcgcct ctgctttcca gcgccgggca ggaggggtcc 480 tggttgcctc ccatctgcag agcttcctgg aggtgtcgta ccgcgttcta cgccaccttg 540 cccagccctg agccaagccc tccccatccc atgtatttat ctctatttaa tatttatgtc 600 tatttaagcc tcatatttaa agacagggaa gagcagaacg gagccccagg cctctgtgtc 660 cttccctgca tttctgagtt tcattctcct gcctgtagca gtgagaaaaa gctcctgtcc 720 tcccatcccc tggactggga ggtagatagg taaataccaa gtatttatta ctatgactgc 780 tccccagccc tggctctgca atgggcactg ggatgagccg ctgtgagccc ctggtcctga 840 gggtccccac ctgggaccct tgagagtatc aggtctccca cgtgggagac aagaaatccc 900. tgtttaat ttaaacagca gtgttcccca tctgggtcct tgcacccctc actctggcct cagccgactg cacagcggcc cctgcatccc cttggctgtg aggcccctgg acaagcagag 1020 gtggccagag ctggggaggca tggccctggg gtcccacgaa tttgctgggg aatctcgttt 1080 ttcttcttaa gacttttggg acatggtttg actcccgaac atcaccgacg cgtctcctgt ttttctgggt ggcctcggga cacctgccct gcccccacga gggtcaggac tgtgactctt 1200 tttagggcca ggcaggtgcc tggacatttg ccttgctgga cggggactgg ggatgtgggga 1260 gggagcagac aggaggatc atgtcaggcc tgtgtgtgaa aggagctcc actgtcaccc tccacctctt caccccccac tcaccagtgt cccctccact gtcacattgt aactgaactt 1380 afterwards aagtgtttgc ctcca <210> 74 <211> 9 <212> PRT <213> Homo sapiens <400> 74 Leu Trp Pro Ser Val Pro Asp Pro Ala 1 5 <210> 75 <211> 9 <212> PRT <213> Homo sapiens <400> 75 Ile Trp Pro Asn Val Pro Asp Pro Ser 1 5 <210> 76 <211> 9 <212> PRT <213> Homo sapiens <400> 76 Leu Lys Cys Asn Thr Pro Asp Pro Ser 1 5 <210> 77 <211> 9 <212> PRT <213> Homo sapiens <400> 77 Lys Ile Trp Ala Val Pro Ser Pro Glu 1 5 <210> 78 <211> 9 <212> PRT <213> Homo sapiens <400> 78 Cys Ser Arg Glu Ile Pro Asp Pro Ala 1 5 <210> 79 <211> 9 <212> PRT <213> Homo sapiens <400> 79 Val Trp Pro Ser Leu Pro Asp His Lys 1 5 <210> 80 <211> 175 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 80 Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu 1 5 10 15 Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu 20 25 30 Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 35 40 45 Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser 50 55 60 Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His 65 70 75 80 Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile 85 90 95 Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala 100 105 110 Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala 115 120 125 Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala 130 135 140 Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser 145 150 155 160 Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 165 170 175

Claims

1. A chimeric receptor comprising: (a) the extracellular domain (ECD) of G-CSFR (granulocyte colony-stimulating factor receptor) operably linked to a second domain; (b) i) at least a portion of the intracellular domain (ICD) of IL-2R (interleukin-2 receptor); ii) the Box 1 and Box 2 regions of a protein selected from the group consisting of G-CSFR and gp130; and iii) at least a portion of the transmembrane domain of a protein selected from the group consisting of G-CSFR, gp130 (glycoprotein 130), and IL-2Rβ. the second domain comprising: The chimeric receptor comprising:

2. A chimeric receptor comprising: comprising the ECD of G-CSFR operably linked to a second domain; The second domain is (i) (a) the transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-2Rβ, or (ii) (a) the transmembrane domain of gp130; (b) the Box 1 and Box 2 regions of gp130, and (c) the C-terminal region of IL-2Rβ, or (iii) (a) the transmembrane domain of IL-2Rβ+γc; (b) the Box 1 and Box 2 regions of IL-2Rβ+γc, and (c) C-terminal region of IL-2Rβ+γc The chimeric receptor comprising:

3. The chimeric receptor of claim 1 or 2, wherein the activated chimeric receptor forms a homodimer, and the chimeric receptor is activated by contact with G-CSF.

4. The chimeric receptor of claim 3, wherein activation of the chimeric receptor elicits a cellular response selected from the group consisting of proliferation, survival, and enhanced activity of cells expressing the chimeric receptor.

5. The chimeric receptor of claim 3, wherein the G-CSF is a wild-type G-CSF or the extracellular domain of the G-CSFR is a wild-type extracellular domain.

6. 4. The chimeric receptor of claim 3, wherein the G-CSF is wild-type G-CSF and the extracellular domain of the G-CSFR is a wild-type extracellular domain.

7. The chimeric receptor of claim 1 or 2, wherein the chimeric receptor is expressed in a cell.

8. The chimeric receptor of claim 7 , wherein the cell is an immune cell.

9. The chimeric receptor of claim 8 , wherein the immune cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a B cell, a plasma cell, a macrophage, and a dendritic cell.

10. The chimeric receptor of claim 7 , wherein the cell is a stem cell, a primary cell, or a human cell.

11. The ICD (a) at least a portion of the ICD of IL-2Rβ having the amino acid sequence of SEQ ID NO: 16, 19, 21, 29, 31, 33, 35, 37, or 39; or (b) at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 34, 40, or 42; or (c) at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO: 18 or 38 The chimeric receptor of claim 1 or 2, comprising:

12. the transmembrane domain is (a) SEQ ID NO: 8, or (b) SEQ ID NO: 9, or (c) SEQ ID NO: 10, or (d) SEQ ID NO: 11 3. The chimeric receptor of claim 1 or 2, comprising the sequence set forth in

13. A nucleic acid encoding a chimeric receptor, said chimeric receptor comprising: (a) the extracellular domain (ECD) of G-CSFR (granulocyte colony-stimulating factor receptor) operably linked to a second domain; (b) i) at least a portion of the intracellular domain (ICD) of IL-2R (interleukin-2 receptor); ii) the Box 1 and Box 2 regions of a protein selected from the group consisting of G-CSFR and gp130; and iii) at least a portion of the transmembrane domain of a protein selected from the group consisting of G-CSFR, gp130 (glycoprotein 130), and IL-2Rβ. the second domain comprising: Including, A nucleic acid encoding the chimeric receptor.

14. A nucleic acid encoding a chimeric receptor, the chimeric receptor comprises an ECD of G-CSFR operably linked to a second domain; The second domain is (i) (a) the transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-2Rβ, or (ii) (a) the transmembrane domain of gp130; (b) the Box 1 and Box 2 regions of gp130, and (c) the C-terminal region of IL-2Rβ, or (iii) (a) the transmembrane domain of IL-2Rβ+γc; (b) the Box 1 and Box 2 regions of IL-2Rβ+γc, and (c) C-terminal region of IL-2Rβ+γc A nucleic acid encoding the chimeric receptor comprising:

15. 15. The nucleic acid of claim 14, wherein the ECD of the G-CSFR is encoded by the nucleic acid sequence set forth in SEQ ID NO: 5 or 6.

16. (a) a sequence encoding at least a portion of the ICD of IL-2Rβ having the amino acid sequence of SEQ ID NO: 16, 19, 21, 29, 31, 33, 35, 37, or 39; or (b) a sequence encoding at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 34, 40, or 42; or (c) a sequence encoding at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO: 18 or 38; 16. The nucleic acid of claim 14 or 15, comprising:

17. An expression vector comprising the nucleic acid of claim 14 or 15.

18. 18. The expression vector of claim 17, which is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and a plasmid.

19. A cell comprising the nucleic acid of claim 14 or 15.

20. 1. A method for selectively activating a chimeric receptor expressed on the surface of a cell, comprising: The method comprises contacting the chimeric receptor of claim 1 or 2 with G-CSF, which selectively activates the chimeric receptor.

21. 1. A method for producing a chimeric receptor in a cell, comprising:

18. The method of claim 17, further comprising introducing into said cell the nucleic acid of claim 14 or 15, or the expression vector of claim 17. The method.

22. 1. A pharmaceutical composition for treating a subject in need thereof, comprising a cell expressing a chimeric receptor and a cytokine that binds to said chimeric receptor, The chimeric receptor (a) the extracellular domain (ECD) of G-CSFR (granulocyte colony-stimulating factor receptor) operably linked to a second domain; (b) i) at least a portion of the intracellular domain (ICD) of IL-2R (interleukin-2 receptor); ii) the Box 1 and Box 2 regions of a protein selected from the group consisting of G-CSFR and gp130; and iii) at least a portion of the transmembrane domain of a protein selected from the group consisting of G-CSFR, gp130 (glycoprotein 130), and IL-2Rβ. A second domain including The pharmaceutical composition comprising:

23. 1. A pharmaceutical composition for treating a subject in need thereof, comprising a cell expressing a chimeric receptor and a cytokine that binds to said chimeric receptor, the chimeric receptor comprises an ECD of G-CSFR operably linked to a second domain; The second domain is (i) (a) the transmembrane domain of G-CSFR; (b) the Box 1 and Box 2 regions of G-CSFR, and (c) the C-terminal region of IL-2Rβ, or (ii) (a) the transmembrane domain of gp130; (b) the Box 1 and Box 2 regions of gp130, and (c) the C-terminal region of IL-2Rβ, or (iii) (a) the transmembrane domain of IL-2Rβ+γc; (b) the Box 1 and Box 2 regions of IL-2Rβ+γc, and (c) C-terminal region of IL-2Rβ+γc Including, The pharmaceutical composition.

24. 24. The pharmaceutical composition of claim 22 or 23, wherein the activated chimeric receptor forms a homodimer.

25. 24. The pharmaceutical composition of claim 22 or 23, for use in treating cancer, an autoimmune disease, an inflammatory condition, transplant rejection, or an infectious disease.

26. 24. The pharmaceutical composition of claim 22 or 23, wherein the cells expressing the chimeric antigen receptor are prepared by a process comprising: (i) isolating a sample containing immune cells; and (ii) transducing or transfecting the immune cells with a nucleic acid sequence encoding the chimeric cytokine receptor.

27. A cell comprising the chimeric receptor of claim 1 or 2; Instructions for use and Including, kit.

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