Modified extracellular domain of granulocyte colony-stimulating factor receptor (G-CSFR) and cytokine binding thereto

Abstract

Described herein are methods and compositions for selectively activating cells using a variant cytokine receptor and cytokine pair, where the cytokine receptor comprises the extracellular domain (ECD) of granulocyte colony-stimulating factor receptor (G-CSFR). In certain embodiments, the methods and compositions described herein are useful for the exclusive activation of cells for adoptive cell transfer therapy. Accordingly, included herein are methods for producing cells expressing a variant receptor that is selectively activated by a cytokine that does not bind to its native receptor. Also disclosed herein are methods for treating a subject in need of treatment, comprising administering to the subject cells expressing a variant receptor comprising the extracellular domain of G-CSFR and co-administering a variant cytokine that activates the variant receptor. TIFF2022553152000035.tif127170

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 912,318, filed on October 8, 2019, which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing submitted via EFS - Web, which is hereby incorporated by reference in its entirety. The ASCII copy created on October 7, 2020, is named IKE - 068WO_US_SL.txt and is 85,109 bytes in size.

[0003] Field of the Invention The present invention discloses methods and compositions for the selective activation of cells using variant site cytokine receptors and cytokine pairs, wherein the cytokine receptor comprises a variant extracellular domain (ECD) of the granulocyte colony - stimulating factor receptor (G - CSFR). Also disclosed herein are methods of treating a subject by adoptive cell transfer, comprising administering to the subject cells that express a variant receptor and administering a variant cytokine to send a signal to the cells that express the variant receptor. Included in the present disclosure are nucleic acids, expression vectors, and kits for generating cells that express variant cytokines and receptors, and the kit also provides a cytokine for binding to the variant receptor.

Background Art

[0004] Description of Related Art Over the past 20 years, significant progress has been made in the treatment of cancer by adoptive cell therapy (ACT). ACT using endogenous tumor-infiltrating T cells (TIL) now reproducibly achieves an objective clinical response rate of over 50% in advanced melanoma. ACT using T cells engineered to recognize B-cell lineage leukemia (using the CD19-directed chimeric antigen receptor, CD19 CAR) has achieved complete response rates of up to 90%, and the majority of patients achieve durable responses. Stimulated by these remarkable results, several companies have commercialized TIL and CD19 CAR T cell approaches.

[0005] The engraftment, proliferation, and persistence of T cells for ACT are important determinants of clinical safety and efficacy. This is often addressed by systemic administration of IL-2 after ACT transplantation and by growing T cells in vitro with IL-2 prior to transplantation. In addition to the intended immunostimulatory effects, systemic IL-2 treatment can cause severe toxicities such as vascular leak syndrome and needs to be tightly controlled for patient safety. To manage these risks, patients typically need to be hospitalized for 2 - 3 weeks and utilize the intensive care unit as a precautionary measure. Furthermore, administering IL-2 to patients is like stepping on the accelerator and brake pedals simultaneously because IL-2 induces the proliferation of both effector T cells and regulatory (suppressive) T cells (5). CAR T cells pose the opposite problem in that the proliferation and persistence of T cells can exceed safe levels. Moreover, since it eradicates all normal B cells (expressing CD19) without exception, patients remain partially immunocompromised for life. Ideally, it would be desirable to include functions that can precisely control the number of tumor-reactive T cells after adoptive transfer and remove the transferred cells after eradicating cancer. Other cell-based therapies such as stem cell therapy could also benefit from improving control over the proliferation, differentiation, and persistence of the infused cells.

[0006] Human G-CSF is an approved therapeutic agent (Neupogen®, Filgrastim) used to treat neutropenia in cancer patients. G-CSF is a 4-helix bundle (Hill, CP et al. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5167-71 (Non-Patent Document 1)), and the structure of the complex formed by G-CSF and its receptor G-CSFR has been well characterized (Tamada, T et al. Proc Natl Acad Sci U S A. 2006 Feb 28;103(9):3135-40 (Non-Patent Document 2)). The G-CSF:G-CSFR complex is a 2:2 heterodimer. G-CSF has two binding interfaces with G-CSFR. One interface is called Site II, which is the larger interface between G-CSF and the cytokine receptor homology (CRH) domain of G-CSFR. The other interface is called Site III, which is the smaller interface between G-CSF and the N-terminal Ig-like domain of G-CSFR.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

[0008] In certain embodiments, disclosed herein is a receptor comprising a variant extracellular domain (ECD) of the granulocyte colony-stimulating factor receptor (G-CSFR), wherein the variant ECD of G-CSFR comprises at least one mutation in the Site II interface region, at least one mutation in the Site III interface region, or a combination thereof. In certain aspects, at least one mutation in the Site II interface region is located at an amino acid position of the G-CSFR ECD selected from the group consisting of amino acid positions 141, 167, 168, 171, 172, 173, 174, 197, 199, 200, 202, and 288 of SEQ ID NO: 2. In certain aspects, at least one mutation in the Site II interface region is selected from the group of G-CSFR ECD mutations consisting of R141E, R167D, K168D, K168E, L171E, L172E, Y173K, Q174E, D197K, D197R, M199D, D200K, D200R, V202D, R288D, and R288E. In certain aspects, at least one mutation in the Site III interface region is selected from the group of G-CSFR ECD mutations consisting of amino acid positions 30, 41, 73, 75, 79, 86, 87, 88, 89, 91, and 93 of SEQ ID NO: 2. In certain aspects, at least one mutation in the Site III interface region is selected from the group of G-CSFR ECD mutations consisting of S30D, R41E, Q73W, F75KF, S79D, L86D, Q87D, I88E, L89A, Q91D, Q91K, and E93K. In certain aspects, the G-CSFR ECD comprises a combination of mutations of the design numbers in Table 6, and the mutations correspond to the amino acid positions of SEQ ID NO: 2. In certain aspects, the G-CSFR ECD comprises mutations that are R41E, R141E, and R167D.

[0009] In certain embodiments, the receptor disclosed herein is a chimeric receptor. In certain embodiments, the receptor is expressed on the cell surface. In certain embodiments, the receptor is expressed on an immune cell. In certain embodiments, the immune cell is 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, optionally, the cell is a primary cell, and optionally, the cell is a human cell.

[0010] In certain embodiments, activation of the receptor by mutant G-CSF elicits a cellular response selected from the group consisting of proliferation, survival, and enhanced activity of the cells expressing the receptor.

[0011] In certain embodiments, disclosed herein is a nucleic acid encoding any of the receptors disclosed herein. In certain aspects, described herein is an expression vector comprising the nucleic acid. In certain embodiments, described herein is a cell engineered to express a receptor disclosed herein. In certain aspects, the cell is an immune cell. In certain embodiments, the immune cell is 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, optionally, the cell is a primary cell, and optionally, the cell is a human cell. In certain embodiments, disclosed herein is a variant granulocyte colony-stimulating factor (G-CSF), wherein the variant G-CSF comprises at least one mutation in the site II interface region, at least one mutation in the site III interface region, or a combination thereof. In certain aspects, at least one mutation in the site II interface region of the variant G-CSF is located at an amino acid position selected from the group consisting of amino acid positions 12, 16, 19, 20, 104, 108, 109, 112, 115, 116, 118, 119, 122, and 123 of SEQ ID NO: 1. In certain aspects, at least one mutation in the site II interface region of the variant G-CSF is selected from the group of mutations consisting of S12E, S12K, S12R, K16D, L18F, E19K, Q20E, D104K, D104R, L108K, L108R, D109R, D112R, D112K, T115E, T115K, T116D, Q119E, Q119R, E122K, E122R, and E123R. In certain aspects, at least one mutation in the site III interface region of the variant G-CSF is selected from the group of mutations consisting of amino acid positions 38, 39, 40, 41, 46, 47, 48, 49, and 147 of SEQ ID NO: 1.In certain embodiments, at least one mutation in the site III interface region of the variant G-CSF is selected from the group of mutations consisting of T38R, Y39E, K40D, K40F, L41D, L41E, L41K, E46R, L47D, V48K, V48R, L49K, and R147E. In certain embodiments, the variant G-CSF comprises a combination of mutations of the design numbers in Table 6, and the mutations correspond to the amino acid positions of SEQ ID NO: 1. In certain embodiments, the variant G-CSF comprises the mutations E46R, L108K, and D112R. In certain embodiments, the variant G-CSF selectively binds to the receptor disclosed herein. In certain embodiments, the receptor is expressed on the cell surface. In certain embodiments, the cell is an immune cell. In certain embodiments, the immune cell is 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, optionally, the cell is a primary cell, and optionally, the cell is a human cell. In certain embodiments, the selective binding of the variant G-CSF to the receptor elicits a cell response selected from the group consisting of proliferation, survival, and enhanced activity of the immune cell.

[0012] In certain embodiments, disclosed herein is a nucleic acid encoding a variant G-CSF. In certain embodiments, disclosed herein is an expression vector comprising the nucleic acid. In certain embodiments, disclosed herein is a cell engineered to express a variant G-CSF. In certain embodiments, the cell is an immune cell.

[0013] In certain embodiments, disclosed herein is a system for selectively activating a receptor expressed on the cell surface, the system comprising a receptor and a variant G-CSF, the receptor comprising at least one mutation in the Site II interface region, the Site III interface region, or a combination thereof, the variant G-CSF comprising at least one mutation in the amino acid sequence of G-CSF that binds to the Site II interface region of the receptor, the Site III interface region of the receptor, or a combination thereof; the variant G-CSF binding preferentially to the receptor over wild-type G-CSFR ECD, and the receptor binding preferentially to the variant G-CSF over wild-type G-CSF. In certain aspects, the receptor and the variant G-CSF comprise a combination of mutations at the Site II interface of the design numbers in Table 2, the mutations in the receptor corresponding to the amino acid positions of SEQ ID NO: 2, and the mutations in the variant G-CSF corresponding to the amino acid positions of SEQ ID NO: 1. In certain aspects, the receptor and the variant G-CSF comprise a combination of mutations at the Site III interface of the design numbers in Table 4, the mutations in the receptor corresponding to the amino acid positions of SEQ ID NO: 2, and the mutations in the variant G-CSF corresponding to the amino acid positions of SEQ ID NO: 1. In certain aspects, the receptor and the variant G-CSF comprise a combination of mutations at the Site II interface and the Site III interface of the design numbers in Table 6, the mutations in the receptor corresponding to the amino acid positions of SEQ ID NO: 2, and the mutations in the variant G-CSF corresponding to the amino acid positions of SEQ ID NO: 1. In certain aspects, the combination of mutations comprises the mutations of design number 106, the variant G-CSF comprising the E46R and D104K mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor comprising the R41E and K168D mutations corresponding to the amino acid positions of SEQ ID NO: 2. In certain aspects, the combination of mutations comprises the mutations of design number 117, the variant G-CSF comprising the E46R, E122R, and E123R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor comprising the R41E and R141E mutations corresponding to the amino acid positions of SEQ ID NO: 2.In certain embodiments, the combination of mutations includes the mutation of Design No. 130, and the variant G-CSF includes the E46R, L108K, and D112R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E and R167D mutations corresponding to the amino acid positions of SEQ ID NO: 2. In certain embodiments, the combination of mutations includes the mutation of Design No. 134, and the variant G-CSF includes the E46R, L108K, D112R, E122R, and E123R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, R141E, and R167D mutations corresponding to the amino acid positions of SEQ ID NO: 2. In certain embodiments, the combination of mutations includes the mutation of Design No. 135, and the variant G-CSF includes the E46R, T115K, E122R, and E123R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, R141E, L171E, and Q174E mutations corresponding to the amino acid positions of SEQ ID NO: 2. In certain embodiments, the combination of mutations includes the mutation of Design No. 137, and the variant G-CSF includes the E46R, L108K, and D112R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, R141E, and R167D mutations corresponding to the amino acid positions of SEQ ID NO: 2. In certain embodiments, the combination of mutations includes the mutation of Design No. 300, and the variant G-CSF includes the K40D, L41D, L108K, and D112R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the F75K, Q91K, and R167D mutations corresponding to the amino acid positions of SEQ ID NO: 2. In certain embodiments, the combination of mutations includes the mutation of Design No. 301, and the variant G-CSF includes the T38R, E46R, L108K, and D112R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, Q73E, and R167D mutations corresponding to the amino acid positions of SEQ ID NO: 2. In certain embodiments, the combination of mutations includes the mutation of Design No. 302, and the variant G-CSF includes the E46R, L108K, and D112R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, L86D, and R167D mutations corresponding to the amino acid positions of SEQ ID NO: 2.In certain embodiments, the combination of mutations includes the mutation of Design No. 303, and the variant G-CSF includes the L108K, D112R, and R147E mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the E93K and R167D mutations corresponding to the amino acid positions of SEQ ID NO: 2. In certain embodiments, the combination of mutations includes the mutation of Design No. 304, and the variant G-CSF includes the E46R, L108K, D112R, and R147E mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, E93K, and R167D mutations corresponding to the amino acid positions of SEQ ID NO: 2. In certain embodiments, the combination of mutations includes the mutation of Design No. 305, and the variant G-CSF includes the E19K, E46R, L108K, and D112R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, R167D, and R288E mutations corresponding to the amino acid positions of SEQ ID NO: 2. In certain embodiments, the combination of mutations includes the mutation of Design No. 307, and the variant G-CSF includes the S12E, K16D, E19K, and E46R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, D197K, D200K, and R288E mutations corresponding to the amino acid positions of SEQ ID NO: 2. In certain embodiments, the combination of mutations includes the mutation of Design No. 308, and the variant G-CSF includes the E19R, E46R, D112K mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, R167D, V202D and R288E mutations corresponding to the amino acid positions of SEQ ID NO: 2. In certain embodiments, the combination of mutations includes the mutation of Design No. 400, and the variant G-CSF includes the E19K, E46R, D109R, and D112R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, R167D, M199D, and R288D mutations corresponding to the amino acid positions of SEQ ID NO: 2. In certain embodiments, the combination of mutations includes the mutation of Design No. 401, and the variant G-CSF includes the E19K, E46R, L108K, and D112R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, R167D, and R288D mutations corresponding to the amino acid positions of SEQ ID NO: 2.In certain embodiments, the combination of mutations includes the mutation of Design No. 402, and the variant G-CSF includes the E19K, E46R, D112K, and T115K mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, R167E, Q174E, and R288E mutations corresponding to the amino acid positions of SEQ ID NO: 2. In certain embodiments, the combination of mutations includes the mutation of Design No. 403, and the variant G-CSF includes the E19R, E46R, and D112K mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, R167D, and R288E mutations corresponding to the amino acid positions of SEQ ID NO: 2.

[0014] In certain embodiments, disclosed herein is a method of selectively activating a receptor expressed on the surface of a cell, the method comprising contacting the receptor with a variant G-CSF. In certain embodiments, the receptor is expressed on a cell. In certain embodiments, the receptor is expressed on an immune cell. In certain embodiments, the receptor is expressed on a T cell or an NK cell. In certain embodiments, selective activation of the immune cell elicits a cell response selected from the group consisting of proliferation, survival, and enhanced activity of the immune cell.

[0015] In certain embodiments, disclosed herein is a method of generating a cell that expresses a receptor disclosed herein, the method comprising introducing a nucleic acid or expression vector disclosed herein into the cell.

[0016] In certain embodiments, disclosed herein is a method of treating a subject in need of treatment, the method comprising injecting into the subject a cell (e.g., an immune cell) disclosed herein. In certain embodiments, the method further comprises administering a variant G-CSF to the subject.

[0017] In certain embodiments, disclosed herein is a kit for creating a system that selectively activates a receptor expressed on the cell surface, the kit comprising a nucleic acid or expression vector disclosed herein, a variant G-CSF disclosed herein, and instructions for use.

[0018] In certain embodiments, described herein is a chimeric receptor, wherein the chimeric receptor comprises: (a) an extracellular domain (ECD) of G-CSFR (granulocyte colony-stimulating factor receptor) operably linked to a second domain; the second domain comprises (b) at least a portion of the intracellular domain (ICD) of a multi-subunit 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, 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 ICD of the cytokine receptor comprises at least one signal transduction 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 the STAT3 binding site of G-CSFR; the STAT3 binding site of gp130; the SHP-2 binding site of gp130; the SHC binding site of IL-2Rβ; the STAT5 binding site of IL-2Rβ; the STAT3 binding site of IL-2Rβ; the STAT1 binding site of IL-2Rβ; the 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 Box1 region and the 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 the 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, which is a chimeric receptor.

[0019] In certain embodiments, described herein is a chimeric receptor comprising an ECD of G-CSFR operably linked to a second domain, wherein the second domain is (i) (a) the transmembrane domain of gp130, and (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, and (b) the Box1 and Box2 regions of G-CSFR, and (c) the C-terminal region of IL-2Rβ, or (iii) (a) the transmembrane domain of G-CSFR, and (b) the Box1 and Box2 regions of G-CSFR, and (c) the C-terminal region of IL-12Rβ2, or (iv) (a) the transmembrane domain of G-CSFR, and (b) the Box1 and Box2 regions of G-CSFR, and (c) the C-terminal region of IL-21R, or (v) (a) the transmembrane domain of IL-2Rβ+γc, and (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, and (b) the Box1 and Box2 regions of G-CSFR, and (c) the C-terminal region of IL-7Rα and is a chimeric receptor.

[0020] In certain embodiments, the activating 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 cells 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 the G-CSFR is a wild-type extracellular domain. In certain embodiments, the activating 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 cells 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 the G-CSFR is a wild-type extracellular domain.

[0021] In certain embodiments, 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, optionally the cell is a stem cell, optionally the cell is a primary cell, and optionally the cell is a human cell.

[0022] In certain embodiments, the ICD 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; 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.

[0023] In certain embodiments, 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.

[0024] In certain aspects, described herein are cells comprising a nucleic acid encoding a chimeric receptor, wherein the chimeric receptor comprises: (a) an extracellular domain (ECD) of G-CSFR (granulocyte colony-stimulating factor receptor) operably linked to a second domain; the second domain comprises (b) 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 signal transduction molecule binding site of the intracellular domain of the cytokine receptor, optionally wherein the ICD comprises a STAT3 binding site of G-CSFR; a STAT3 binding site of gp130; an SHP-2 binding site of gp130; an 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 wherein the ICD comprises a Box1 region and a Box2 region of a protein selected from the group consisting of G-CSFR and gp130; optionally wherein the chimeric receptor comprises a third domain comprising 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, and optionally wherein 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, and

[0025] In certain embodiments, the present disclosure describes an expression vector comprising a nucleic acid encoding the chimeric receptor described herein. In certain embodiments, the vector is selected from the group consisting of retroviral vectors, lentiviral vectors, adenoviral vectors, and plasmids.

[0026] 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 is (i) (a) the transmembrane domain of gp130, and (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, and (b) The Box1 and Box2 regions of G-CSFR, and (c) The C-terminal region of IL-2Rβ, or (iii) (a) The transmembrane domain of G-CSFR, and (b) The Box1 and Box2 regions of G-CSFR, and (c) The C-terminal region of IL-12Rβ2, or (iv) (a) The transmembrane domain of G-CSFR, and (b) The Box1 and Box2 regions of G-CSFR, and (c) The C-terminal region of IL-21R, or (v) (a) The transmembrane domain of IL-2Rβ+γc, and (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, and (b) The Box1 and Box2 regions of G-CSFR, and (c) The C-terminal region of IL-7Rα A nucleic acid encoding a chimeric receptor comprising. 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 part 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 part 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 part 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 part of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 43, or (h) A sequence encoding at least a part of the ICD of IL-2Rγc having the amino acid sequence of SEQ ID NO: 17.

[0027] In certain embodiments, described herein is an expression vector comprising the nucleic acid described herein. In certain embodiments, the vector is selected from the group consisting of retroviral vectors, lentiviral vectors, adenoviral vectors, and plasmids.

[0028] In certain embodiments, described herein is a cell comprising a nucleic acid encoding a chimeric receptor, wherein the chimeric receptor comprises: (a) an extracellular domain (ECD) of a granulocyte colony-stimulating factor receptor (G-CSFR) operably linked to a second domain; the second domain comprising (b) at least a portion of an intracellular domain (ICD) of a multi-subunit cytokine receptor selected from the group consisting of interleukin-2 receptor (IL-2R), interleukin-7 receptor (IL-7R), interleukin-12 receptor (IL-12R), and interleukin-21 receptor (IL-21R), 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 comprising at least one signal transduction molecule binding site of the intracellular domain of the cytokine receptor, optionally wherein the ICD comprises at least one signal transduction molecule binding site selected from the group consisting of the STAT3 binding site of G-CSFR; the STAT3 binding site of gp130; the SHP-2 binding site of gp130; the SHC binding site of IL-2Rβ; the STAT5 binding site of IL-2Rβ; the STAT3 binding site of IL-2Rβ; the STAT1 binding site of IL-2Rβ; the 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 wherein the ICD comprises the Box1 region and the Box2 region of a protein selected from the group consisting of G-CSFR and gp130; optionally 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, optionally wherein the transmembrane domain is a wild-type transmembrane domain; optionally,

[0029] 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, and optionally the cell is a stem cell, optionally the cell is a primary cell, and optionally the cell is a human cell, and is a cell comprising a nucleic acid encoding a chimeric receptor.

[0030] In certain embodiments, 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 is (i) (a) the transmembrane domain of gp130 and (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 and (b) the Box1 and Box2 regions of G-CSFR and (c) the C-terminal region of IL-2Rβ, or (iii) (a) the transmembrane domain of G-CSFR and (b) the Box1 and Box2 regions of G-CSFR and (c) the C-terminal region of IL-12Rβ2, or (iv) (a) the transmembrane domain of G-CSFR and (b) the Box1 and Box2 regions of G-CSFR and (c) the C-terminal region of IL-21R, or (v) (a) the transmembrane domain of IL-2Rβ+γc and (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 and (b) the Box1 and Box2 regions of G-CSFR and (c) The C-terminal region of IL-7Rα and 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, and optionally, the cell is a stem cell, optionally, the cell is a primary cell, and optionally, the cell is a human cell, a cell comprising a nucleic acid encoding a chimeric receptor.

[0031] 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-7R 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, and is constituted by a cell comprising the same.

[0032] In certain embodiments, described herein are cells comprising the expression vectors described herein, and optionally, the cells are immune cells, optionally T cells or NK cells. In certain embodiments, described herein are cells comprising the chimeric receptor of claim 1, and optionally, the cells are immune cells, optionally T cells, optionally NK cells, optionally NKT cells, optionally B cells, optionally plasma cells, optionally macrophages, optionally dendritic cells, and optionally, the cells are stem cells, and optionally, the cells are primary cells, and optionally, the cells are human cells.

[0033] In certain embodiments, described herein are cells comprising the chimeric receptor of claim 1, and optionally, the cells are immune cells, optionally T cells, optionally NK cells, optionally NKT cells, optionally B cells, optionally plasma cells, optionally macrophages, optionally dendritic cells, and optionally, the cells are stem cells, and optionally, the cells are primary cells, and optionally, the cells are human cells.

[0034] In certain embodiments, described herein is a method of selectively activating a chimeric receptor expressed on the surface of a cell, the method comprising contacting the chimeric receptor with G-CSF that selectively activates the chimeric receptor; the chimeric receptor comprising (a) an extracellular domain (ECD) of a G-CSFR (granulocyte colony-stimulating factor receptor) operably linked to a second domain; the second domain comprising (b) at least a portion of an intracellular domain (ICD) of a multi-subunit 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 comprising at least one signal transduction molecule binding site of the intracellular domain of the cytokine receptor, optionally wherein the at least one signal transduction molecule binding site is selected from the group consisting of the STAT3 binding site of G-CSFR; the STAT3 binding site of gp130; the SHP-2 binding site of gp130; the SHC binding site of IL-2Rβ; the STAT5 binding site of IL-2Rβ; the STAT3 binding site of IL-2Rβ; the STAT1 binding site of IL-2Rβ; the 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; and optionally wherein the ICD comprises the Box1 region and the 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 the 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, a method of selectively activating a chimeric receptor.;

[0035] In certain embodiments, described herein is a method of 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 comprises an ECD of G-CSFR operably linked to a second domain; the second domain is (i) (a) the transmembrane domain of gp130, and (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, and (b) the Box1 and Box2 regions of G-CSFR, and (c) the C-terminal region of IL-2Rβ, or (iii) (a) the transmembrane domain of G-CSFR, and (b) the Box1 and Box2 regions of G-CSFR, and (c) the C-terminal region of IL-12Rβ2, or (iv) (a) the transmembrane domain of G-CSFR, and (b) the Box1 and Box2 regions of G-CSFR, and (c) the C-terminal region of IL-21R, or (v) (a) the transmembrane domain of IL-2Rβ+γc, and (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, and (b) The Box1 and Box2 regions of G-CSFR, and (c) The C-terminal region of IL-7Rα is a method for selectively activating a chimeric receptor comprising the same.

[0036] In certain embodiments of the methods described herein, the activated chimeric receptor forms a homodimer, and optionally, 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; 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 the G-CSFR is a wild-type extracellular domain; the chimeric receptor is expressed in cells, optionally immune cells, optionally T cells, optionally NK cells, optionally NKT cells, optionally B cells, optionally plasma cells, optionally macrophages, optionally dendritic cells, optionally the cells are stem cells, optionally the cells are primary cells, and optionally the cells are human cells.

[0037] In certain embodiments of the methods described herein, the chimeric receptor 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 part of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 43, or (h) Comprising at least a part of the ICD of IL-2Rγc having the amino acid sequence of SEQ ID NO: 17; the transmembrane domain comprises the sequence described in (a) SEQ ID NO: 8, or (b) SEQ ID NO: 9, or (c) SEQ ID NO: 10, or (d) SEQ ID NO: 11.

[0038] In certain embodiments, described herein is a method of producing a chimeric receptor in a cell, comprising introducing into the cell the nucleic acid of any one of claims 13-16 or 19-22, or the 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, and optionally, the cell is a primary cell, and optionally, the cell is a human cell, a method of producing a chimeric receptor in a cell.

[0039] In certain embodiments, described herein is a method of treating a subject in need of treatment, comprising injecting into the subject cells that express a chimeric receptor and administering a cytokine that binds to 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) at least a portion of the intracellular domain (ICD) of a multi-subunit 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 signal transduction molecule binding site of the intracellular domain of the cytokine receptor; optionally, the ICD comprises a STAT3 binding site of G-CSFR; a STAT3 binding site of gp130; an SHP-2 binding site of gp130; an 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 region and a 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 the 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, a method of treating a subject in need of treatment.

[0040] In certain embodiments, described herein is a method of treating a subject in need of treatment, comprising injecting into the subject cells that express a chimeric receptor 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, and (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, and (b) the Box1 and Box2 regions of G-CSFR, and (c) the C-terminal region of IL-2Rβ, or (iii) (a) the transmembrane domain of G-CSFR, and (b) the Box1 and Box2 regions of G-CSFR, and (c) the C-terminal region of IL-12Rβ2, or (iv) (a) the transmembrane domain of G-CSFR, and (b) the Box1 and Box2 regions of G-CSFR, and (c) the C-terminal region of IL-21R, or (v) (a) the transmembrane domain of IL-2Rβ+γc, and (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, and (b) the Box1 and Box2 regions of G-CSFR, and (c) the C-terminal region of IL-7Rα A method for treating a subject in need of treatment, comprising

[0041] In certain embodiments of the method, the activating chimeric receptor forms a homodimer and, optionally, 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; 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, and 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 (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) comprising at least a portion of the ICD of IL-2Rγc 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.

[0042] In certain embodiments, the methods described herein are used to treat cancer. In certain embodiments, the method is used to treat autoimmune diseases. In certain embodiments, the method is used to treat inflammatory conditions. In certain embodiments, the method is used to prevent and treat graft rejection. In certain embodiments, the method is used to treat infectious diseases. In certain embodiments, the method further comprises administering at least one additional active agent, optionally, the additional active agent is an additional cytokine.

[0043] In certain embodiments, the methods described herein comprise: (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 of (ii) into a subject; and (iv) contacting the immune cells with a cytokine that binds to the chimeric receptor. In certain embodiments, the subject has received an immunosupressive treatment prior to administering or injecting the cells into the subject. In certain embodiments, the sample containing immune cells is isolated from the subject to whom the cells are to be administered or injected. In certain embodiments, the immune cells are contacted with a cytokine in vitro prior to the cells being administered or injected into the subject. In certain embodiments, the immune cells are contacted with a cytokine that binds to the chimeric receptor for a time sufficient to activate signal transduction from the chimeric receptor.

[0044] Described herein is a kit for treating a subject in need of treatment, comprising a cell that encodes the chimeric receptor described herein and is optionally an immune cell, and instructions for use, and optionally, the kit comprises a cytokine that binds to the chimeric receptor. Also described herein is a kit for generating a chimeric receptor expressed on a cell, comprising an expression vector encoding the chimeric receptor described herein and instructions for use, and optionally, the kit comprises a cytokine that binds to the chimeric receptor.

[0045] Described herein is a kit for generating a chimeric receptor expressed on a cell, comprising a cell that is optionally a bacterial cell and comprises an expression vector encoding the 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 receptor comprising a variant extracellular domain (ECD) of granulocyte colony-stimulating factor receptor (G-CSFR), wherein the variant ECD of G-CSFR comprises at least one mutation in the site II interface region, at least one mutation in the site III interface region, or a combination thereof, said receptor. [The present invention 1002] The receptor of the present invention 1001, wherein the at least one mutation in the site II interface region is located at an amino acid position of G-CSFR ECD selected from the group consisting of amino acid positions 141, 167, 168, 171, 172, 173, 174, 197, 199, 200, 202, and 288 of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1003] The receptor of the present invention 1002, wherein the at least one mutation in the site II interface region is selected from the group of G-CSFR ECD mutations consisting of R141E, R167D, K168D, K168E, L171E, L172E, Y173K, Q174E, D197K, D197R, M199D, D200K, D200R, V202D, R288D, and R288E. [The present invention 1004] The receptor of any one of the present invention, wherein the at least one mutation in the site III interface region is selected from the group of G-CSFR ECD mutations consisting of amino acid positions 30, 41, 73, 75, 79, 86, 87, 88, 89, 91, and 93 of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1005] The receptor of the present invention 1004, wherein the at least one mutation in the site III interface region is selected from the group of G-CSFR ECD mutations consisting of S30D, R41E, Q73W, F75KF, S79D, L86D, Q87D, I88E, L89A, Q91D, Q91K, and E93K. [The present invention 1006] The receptor of any one of the present invention, wherein the G-CSFR ECD comprises a combination of mutations of the design numbers in Table 6, said mutations corresponding to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1007] The receptor of any one of the present invention, wherein the G-CSFR ECD comprises the mutations R41E, R141E, and R167D. [The present invention 1008] The receptor of any one of the present invention, which is a chimeric receptor. [The present invention 1009] The receptor of any one of the present invention, which is expressed on the cell. [The present invention 1010] wherein 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, the receptor of the present invention 1009. [The present invention 1011] Any receptor of the present invention, wherein activation of the receptor by variant G-CSF elicits a cellular response selected from the group consisting of proliferation, survival, and enhanced activity of cells expressing the receptor. [The present invention 1012] A nucleic acid encoding any receptor of the present invention 1001-1011. [The present invention 1013] An expression vector comprising the nucleic acid of the present invention 1012. [The present invention 1014] A cell engineered to express any receptor of the present invention 1001-1011. [The present invention 1015] The cell of the present invention 1014, which is a T cell or an NK cell. [The present invention 1016] A variant granulocyte colony-stimulating factor (G-CSF) comprising at least one mutation in the site II interface region, at least one mutation in the site III interface region, or a combination thereof. [The present invention 1017] The variant G-CSF of the present invention 1016, wherein the at least one mutation in the site II interface region is located at an amino acid position selected from the group consisting of amino acid positions 12, 16, 19, 20, 104, 108, 109, 112, 115, 116, 118, 119, 122, and 123 of SEQ ID NO: 1. [The present invention 1018] The variant G-CSF of the present invention 1017, wherein the at least one mutation in the site II interface region is selected from the group of mutations consisting of S12E, S12K, S12R, K16D, L18F, E19K, Q20E, D104K, D104R, L108K, L108R, D109R, D112R, D112K, T115E, T115K, T116D, Q119E, Q119R, E122K, E122R, and E123R. [The present invention 1019] The at least one mutation in the Site III interface region is selected from the group of mutations consisting of amino acid positions 38, 39, 40, 41, 46, 47, 48, 49, and 147 of SEQ ID NO: 1, and the variant G-CSF of any one of the present inventions 1016 to 1018. [Present Invention 1020] The at least one mutation in the Site III interface region is selected from the group of mutations consisting of T38R, Y39E, K40D, K40F, L41D, L41E, L41K, E46R, L47D, V48K, V48R, L49K, and R147E, and the variant G-CSF of the present invention 1019. [Present Invention 1021] The variant G-CSF of any one of the present inventions 1016 to 1020, wherein the variant G-CSF comprises a combination of mutations of the design numbers in Table 6, and the mutations correspond to the amino acid positions of SEQ ID NO: 1. [Present Invention 1022] The variant G-CSF of the present invention 1021, comprising the mutations E46R, L108K, and D112R. [Present Invention 1023] The variant G-CSF of any one of the present inventions 1016 to 1022, which selectively binds to the receptor of any one of the present inventions 1001 to 1011. [Present Invention 1024] The variant G-CSF of the present invention 1023, wherein the receptor is expressed on the cell surface. [Present Invention 1025] The variant G-CSF of the present invention 1024, wherein the cell is an immune cell. [Present Invention 1026] The immune cell is 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, The variant G-CSF of the present invention 1025. [Present Invention 1027] The variant G-CSF of the present invention 1026, wherein the selective binding of the variant G-CSF to the receptor causes a cell response selected from the group consisting of proliferation, survival, and enhanced activity of the T cell or NK cell. [Present Invention 1028] A nucleic acid encoding the variant G-CSF of any one of the present inventions 1016 to 1027. [Present Invention 1029] An expression vector comprising the nucleic acid of the present invention 1028. [Present Invention 1030] A cell engineered to express the variant G-CSF of any one of the present inventions 1016 to 1027. [Present Invention 1031] The cell of the present invention 1030, which is an immune cell. [The present invention 1032] (a) Any receptor of the present inventions 1001 to 1011, and (b) Any variant G-CSF of the present inventions 1016 to 1027 A system for selectively activating a receptor expressed on the cell surface, comprising: The receptor includes at least one mutation in the site II interface region, the site III interface region, or a combination thereof, and the variant G-CSF includes at least one mutation in the amino acid sequence of G-CSF that binds to the site II interface region of the receptor, the site III interface region of the receptor, or a combination thereof, The variant G-CSF binds to the receptor preferentially over wild-type G-CSFR ECD, and the receptor binds to the variant G-CSF preferentially over wild-type G-CSF, said system. [The present invention 1033] The system of the present invention 1032, wherein the receptor and the variant G-CSF include a combination of mutations at the site II interface of the design numbers in Table 2, the mutation of the receptor corresponds to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2, and the mutation of the variant G-CSF corresponds to the amino acid positions of SEQ ID NO: 1. [The present invention 1034] The system of the present invention 1032, wherein the receptor and the variant G-CSF include a combination of mutations at the site III interface of the design numbers in Table 4, the mutation of the receptor corresponds to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2, and the mutation of the variant G-CSF corresponds to the amino acid positions of SEQ ID NO: 1. [The present invention 1035] The system of the present invention 1032, wherein the receptor and the variant G-CSF include a combination of mutations at the site II interface and the site III interface of the design numbers in Table 6, the mutation of the receptor corresponds to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2, and the mutation of the variant G-CSF corresponds to the amino acid positions of SEQ ID NO: 1. [The present invention 1036] The system of the present invention 1035, wherein the combination of mutations includes the mutation of design number 106, the variant G-CSF includes the E46R and D104K mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E and K168D mutations corresponding to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1037] The system of the present invention 1035, wherein the combination of the mutations includes the mutation of design number 117, the variant G-CSF includes the E46R, E122R, and E123R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E and R141E mutations corresponding to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1038] The system of the present invention 1035, wherein the combination of the mutations includes the mutation of design number 130, the variant G-CSF includes the E46R, L108K, and D112R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E and R167D mutations corresponding to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1039] The system of the present invention 1035, wherein the combination of the mutations includes the mutation of design number 134, the variant G-CSF includes the E46R, L108K, D112R, E122R, and E123R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, R141E, and R167D mutations corresponding to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1040] The system of the present invention 1035, wherein the combination of the mutations includes the mutation of design number 135, the variant G-CSF includes the E46R, T115K, E122R, and E123R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, R141E, L171E, and Q174E mutations corresponding to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1041] The system of the present invention 1035, wherein the combination of the mutations includes the mutation of design number 137, the variant G-CSF includes the E46R, L108K, and D112R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, R141E, and R167D mutations corresponding to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1042] The system of the present invention 1035, wherein the combination of the mutations includes the mutation of design number 300, the variant G-CSF includes the K40D, L41D, L108K, and D112R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the F75K, Q91K, and R167D mutations corresponding to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1043] The system of the present invention 1035, wherein the combination of the mutations includes the mutation of design number 301, the variant G-CSF includes the T38R, E46R, L108K, and D112R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, Q73E, and R167D mutations corresponding to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1044] The system of the present invention 1035, wherein the combination of the mutations includes the mutation of design number 302, the variant G-CSF includes the E46R, L108K, and D112R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, L86D, and R167D mutations corresponding to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1045] The system of the present invention 1035, wherein the combination of the mutations includes the mutation of design number 303, the variant G-CSF includes the L108K, D112R, and R147E mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the E93K and R167D mutations corresponding to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1046] The system of the present invention 1035, wherein the combination of the mutations includes the mutation of design number 304, the variant G-CSF includes the E46R, L108K, D112R, and R147E mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, E93K, and R167D mutations corresponding to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1047] The system of the present invention 1035, wherein the combination of the mutations includes the mutation of design number 305, the variant G-CSF includes the E19K, E46R, L108K, and D112R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, R167D, and R288E mutations corresponding to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1048] The system of the present invention 1035, wherein the combination of the mutations includes the mutation of design number 307, the variant G-CSF includes the S12E, K16D, E19K, and E46R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, D197K, D200K, and R288E mutations corresponding to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1049] The system of the present invention 1035, wherein the combination of the mutations includes the mutation of design number 308, the variant G-CSF includes the E19R, E46R, D112K mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, R167D, V202D, and R288E mutations corresponding to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1050] The system of the present invention 1035, wherein the combination of the mutations includes the mutation of design number 400, the variant G-CSF includes the E19K, E46R, D109R, and D112R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, R167D, M199D, and R288D mutations corresponding to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1051] The system of the present invention 1035, wherein the combination of the mutations includes the mutation of design number 401, the variant G-CSF includes the E19K, E46R, L108K, and D112R mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, R167D, and R288D mutations corresponding to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1052] The system of the present invention 1035, wherein the combination of the mutations includes the mutation of design number 402, the variant G-CSF includes the E19K, E46R, D112K, and T115K mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, R167E, Q174E, and R288E mutations corresponding to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1053] The system of the present invention 1035, wherein the combination of the mutations includes the mutation of design number 403, the variant G-CSF includes the E19R, E46R, and D112K mutations corresponding to the amino acid positions of SEQ ID NO: 1, and the receptor includes the R41E, R167D, and R288E mutations corresponding to the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2. [The present invention 1054] A method for selectively activating a receptor expressed on the surface of a cell, comprising contacting a receptor of any one of the present inventions 1001 to 1011 with a variant G-CSF of the present inventions 1016 to 1027. [The present invention 1055] The receptor is on an immune cell, optionally on a T cell, optionally on an NK cell, optionally on an NKT cell, optionally on a B cell, optionally on a plasma cell, Optionally on macrophages, Optionally on dendritic cells and expressed, Optionally the cells are stem cells, Optionally the cells are primary cells, Optionally the cells are human cells, The method of the present invention 1054. [The present invention 1056] The method of the present invention 1054, wherein the selective activation of the immune cells causes a cellular response selected from the group consisting of proliferation, survival, and enhanced activity of the immune cells. [The present invention 1057] A method for producing immune cells expressing any of the receptors of the present invention 1001-1011, the method comprising introducing the nucleic acid of the present invention 1012 or the expression vector of the present invention 1013 into the cells. [The present invention 1058] A method for treating a subject in need of treatment, the method comprising injecting the cells of the present invention 1014 into the subject. [The present invention 1059] The method of the present invention 1058, further comprising administering to the subject any of the variant G-CSFs of the present invention 1016-1027. [The present invention 1060] The method of the present invention 1058 or 1059, which is used for treating cancer. [The present invention 1061] The method of the present invention 1058 or 1059, which is used for treating inflammatory conditions. [The present invention 1062] The method of the present invention 1058 or 1059, which is used for treating graft rejection. [The present invention 1063] The method of the present invention 1058 or 1059, which is used for treating infectious diseases. [The present invention 1064] The method of the present invention 1058 or 1059, wherein the method further comprises administering at least one additional activator, and optionally the additional activator is an additional cytokine. [The present invention 1065] A method for treating a subject in need of treatment, comprising: (i) isolating a sample containing immune cells; (ii) transducing or transfecting the immune cells with a nucleic acid sequence encoding a variant cytokine receptor of the present invention 1001-1011; (iii) administering or injecting the immune cells of (ii) into the subject; (iv) contacting the immune cells with a variant G-CSF of the present invention 1016-1027 that binds to the variant receptor The method as described above. [The present invention 1066] The method of the present invention 1065, wherein the subject has received immune ablation therapy before the cells are administered or injected into the subject. [The present invention 1067] The method of the present invention 1065, wherein the sample containing the immune cells is isolated from the subject to whom the cells are administered or injected. [The present invention 1068] The method of the present invention 1065, wherein the immune cells are contacted with the cytokine in vitro before the cells are administered or injected into the subject. [The present invention 1069] The method of the present invention 1065, wherein the immune cells are contacted with the cytokine that binds to the chimeric receptor for a time sufficient to activate signal transduction from the chimeric receptor. [The present invention 1070] A kit for treating a subject in need of treatment, comprising cells encoding any of the variant receptors of the present invention 1001-1011 and instructions for use, and optionally, the kit comprising a variant G-CSF of the present invention 1016-1027 that binds to the variant receptor, and optionally, the cells being immune cells. [The present invention 1071] A kit for creating a system for selectively activating a receptor expressed on the cell surface, (a) a nucleic acid of the present invention 1012 or an expression vector of the present invention 1013, and (b) any of the variant G-CSFs of the present invention 1016-1027, a nucleic acid of the present invention 1028, or an expression vector of the present invention 1029, and (c) instructions for use The kit comprising. [The present invention 1072] A kit for creating a chimeric receptor expressed on a cell, comprising cells containing an expression vector encoding any of the variant receptors of the present invention 1001-1011 and instructions for use, and optionally, the cells being bacterial cells, and optionally, the kit comprising a variant G-CSF that binds to the variant receptor.

Brief Description of the Drawings

[0046] These and other features, aspects, and advantages of the present invention will be better understood with reference to the following description and the accompanying drawings.

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Modes for Carrying Out the Invention

[0100] Detailed Description of the Invention Briefly stated, as described in more detail below, this specification describes methods and compositions for the selective activation of cells using variant cytokine receptors and cytokine pairs, wherein the cytokine receptor comprises a variant extracellular domain (ECD) of the granulocyte colony-stimulating factor receptor (G-CSFR). In certain embodiments, the methods and compositions described herein are useful for the exclusive activation of cells for adoptive cell transfer therapy. Accordingly, included herein are methods for generating cells that express a variant receptor that is selectively activated by a cytokine that does not bind to its native receptor. Also disclosed herein is a method of treating a subject in need of treatment, the method comprising administering to the subject cells that express a receptor comprising a variant ECD of G-CSFR and co-administering a variant of G-CSF that binds to the variant ECD of G-CSFR. In certain aspects, the compositions and methods described herein address unmet needs for the selective activation of cells for adoptive cell transfer and may reduce or eliminate the need for immune depletion of the subject prior to adoptive cell transfer or the need to administer broad-acting stimulatory cytokines such as IL-2.

[0101] Definitions The terms used in the claims and specification are defined as described below unless otherwise specified.

[0102] The term "treatment" means any therapeutically beneficial result in the treatment of a disease state, such as a cancer disease state, and includes its prevention, reduction in severity or progression, remission, or cure.

[0103] The term "in vivo" refers to processes that occur within a living organism.

[0104] The term "mammal" as used herein includes both human and non-human mammals and includes, but is not limited to, humans, non-human primates, dogs, cats, mice, cows, horses, and pigs.

[0105] The term "sufficient amount" means an amount sufficient to produce the desired effect, e.g., an amount sufficient to selectively activate a receptor expressed on a cell.

[0106] The term "therapeutically effective amount" is an amount effective to ameliorate the symptoms of a disease. Since prevention can be considered a form of treatment, a therapeutically effective amount can be a "prophylactically effective amount".

[0107] The term "operably linked" refers to a nucleic acid or amino acid sequence that is placed in a functional relationship with another nucleic acid or amino acid sequence. Generally, "operably linked" means that the linked nucleic acid or amino acid sequences are contiguous and, in the case of a secretory leader, contiguous and in the reading phase.

[0108] As used herein, the term "extracellular domain" (ECD) refers to the domain external to the plasma membrane of a receptor (e.g., G-CSFR) 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 SEQ ID NO: 7.

[0109] 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.

[0110] 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 of the cell surface receptor when the receptor is expressed on the cell surface.

[0111] The term "cytokine" refers to a small protein (about 5-20 kDa) that binds to a cytokine receptor and, when bound and activated to a cytokine receptor expressed on a cell, can induce cell signaling. Examples of cytokines include, but are not limited to, interleukins, lymphokines, colony-stimulating factors, and chemokines.

[0112] The term "cytokine receptor" refers to a receptor that binds to cytokines, including type 1 and type 2 cytokine receptors. Examples of cytokine receptors include, but are not limited to, G-CSFR, IL-2R (interleukin-2 receptor), IL-7R (interleukin-7 receptor), IL-12R (interleukin-12 receptor), and IL-21R (interleukin-21 receptor).

[0113] The term "chimeric receptor" as used herein refers to a transmembrane receptor engineered to have at least a portion of at least one domain (e.g., ECD, ICD, TMD, or C-terminal region) derived from the sequences of one or more different transmembrane proteins or receptors.

[0114] As used herein, the terms "site II interface", "site II region", "site II interface region", or "site II" refer to the larger of the two G-CSF:G-CSFR 2:2 heterodimer binding interfaces between G-CSF and G-CSFR, and is located at the interface between the cytokine receptor homolog (CRH) domains of G-CSF and G-CSFR.

[0115] As used herein, the terms "site III interface", "site III region", "site III interface region", or "site III" refer to the smaller of the two G-CSF:G-CSFR 2:2 heterodimer binding interfaces between G-CSF and G-CSFR, and is located at the interface between the N-terminal Ig-like domains of G-CSF and G-CSFR.

[0116] As used herein, the terms "at least a portion" or "a portion" in certain embodiments refer to more than 75%, more than 80%, more than 90%, more than 95%, more than 99% of the length of consecutive nucleic bases or amino acids of the SEQ ID NOs described herein. In certain embodiments, at least a portion of a domain or binding site (e.g., ECD, ICD, transmembrane, C-terminal region, or signaling molecule binding site) described herein can be more than 75%, more than 80%, more than 90%, more than 95%, more than 99% identical to the SEQ ID NOs described herein.

[0117] The term "wild-type" refers to the native 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 of that species of protein or gene.

[0118] The terms "variant cytokine-receptor pair", "variant cytokine and receptor pair", "variant cytokine and receptor design(s)", "variant cytokine-receptor switch", or "orthogonal cytokine-receptor pair" refer to a pair of genetically engineered proteins that have been modified by changing amino acids such that they (a) do not bind to a native cytokine or cognate receptor and (b) specifically bind to a corresponding engineered (variant) ligand or receptor.

[0119] As used herein, the term "variant receptor" or "orthogonal receptor" refers to a genetically engineered receptor of a variant cytokine-receptor pair and includes chimeric receptors.

[0120] As used herein, the term "variant ECD" refers to the genetically engineered extracellular domain of a receptor of a variant cytokine-receptor pair (e.g., G-CSFR).

[0121] As used herein, the term "variant cytokine", "variant G-CSF", or "orthogonal cytokine" refers to a genetically engineered cytokine of a variant cytokine-receptor pair.

[0122] As used herein, "does not bind" or "is unable to bind" refers to the absence of detectable binding or only weak binding, i.e., having a binding affinity much lower than that of the native ligand.

[0123] As used herein, the terms “selectively activate” or “selective activation,” when referring to cytokines and variant receptors, mean that the cytokine preferentially binds to the variant receptor and that the binding of the cytokine to the variant receptor activates the receptor. In certain embodiments, the cytokine selectively activates a chimeric receptor that has co-evolved to specifically bind the cytokine. In certain embodiments, the cytokine is a wild-type cytokine that selectively activates a chimeric receptor expressed on a cell, but the native wild-type receptor for the cytokine is not expressed intracellularly.

[0124] 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, the activity being the activity observed for the native receptor upon stimulation with the native cytokine.

[0125] The term “immune cell” refers to any cell known to have a function that supports the immune system of an organism (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 include all subpopulations of cells, whether rare or abundant in vivo. In certain embodiments, immune cells are so identified by carrying known markers (e.g., cell surface markers) of immune cell types and subpopulations.

[0126] The term “T cell” refers to mammalian immune effector cells that can be characterized by the expression of CD3 and / or the T cell antigen receptor, and these cells can be engineered to express orthogonal cytokine receptors. In some embodiments, the T cell is a naive CD8 + T cell, cytotoxic CD8 + T cell, 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., T R 1, natural T Reg , induced T Reg ; memory T cells, e.g., selected from central memory T cells, effector memory T cells, NKT cells, and γδ T cells.

[0127] 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.

[0128] The term "G-CSF" refers to granulocyte colony-stimulating factor. G-CSF may also be referred to as colony-stimulating factor 3 and CSF3. Human G-CSF is encoded by a gene having Ensembl identification number: ENSG00000108342. Human G-CSF is encoded by a cDNA sequence corresponding to GeneBank accession number KP271008.1.

[0129] "JAK" can 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 a gene with the Ensembl identification number: ENSG00000162434. Human JAK1 is encoded by a cDNA sequence corresponding to the GeneBank accession number NM_002227. Human JAK2 is encoded by a gene with the Ensembl identification number: ENSG00000096968. Human JAK2 is encoded by a cDNA sequence corresponding to the GeneBank accession number NM_001322194. Human JAK3 is encoded by a gene with the Ensembl identification number: ENSG00000105639. Human JAK3 is encoded by a cDNA sequence corresponding to the GeneBank accession number NM_000215. Human TYK2 is encoded by a gene with the Ensembl identification number: ENSG00000105397. Human TYK2 is encoded by a cDNA sequence corresponding to the GeneBank accession number NM_001385197.

[0130] STAT can also be called a signal transducer and activator of transcription. STAT is a family of seven STAT proteins: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. Human STAT1 is encoded by a gene with the Ensembl identification number: ENSG00000115415. Human STAT1 is encoded by a cDNA sequence corresponding to the GeneBank accession number: NM_007315. Human STAT2 is encoded by a gene with the Ensembl identification number: ENSG00000170581. Human STAT2 is encoded by a cDNA sequence corresponding to the GeneBank accession number NM_005419. Human STAT3 is encoded by a gene with the Ensembl identification number: ENSG00000168610. Human STAT3 is encoded by a cDNA sequence corresponding to the GeneBank accession number: NM_139276. Human STAT4 is encoded by a gene with the Ensembl identification number: ENSG00000138378. Human STAT4 is encoded by a cDNA sequence corresponding to the GeneBank accession number: NM_003151. Human STAT5A is encoded by a gene with the Ensembl identification number: ENSG00000126561. Human STAT5A is encoded by a cDNA sequence corresponding to the GeneBank accession number: NM_003152. Human STAT5B is encoded by a gene with the Ensembl identification number: ENSG00000173757. Human STAT5B is encoded by a cDNA sequence corresponding to the GeneBank accession number: NM_012448. Human STAT6 is encoded by a gene with the Ensembl identification number: ENSG00000166888. Human STAT6 is encoded by a cDNA sequence corresponding to the GeneBank accession number: NM_003153.

[0131] SHC can also be called 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 a gene with the Ensembl identification number: ENSG00000160691. Human SHC1 is encoded by a cDNA sequence corresponding to the GeneBank accession number: NM_183001. Human SHC2 is encoded by a gene with the Ensembl identification number: ENSG00000129946. Human SHC2 is encoded by a cDNA sequence corresponding to the GeneBank accession number: NM_012435. Human SHC3 is encoded by a gene with the Ensembl identification number: ENSG00000148082. Human SHC3 is encoded by a cDNA sequence corresponding to the GeneBank accession number: NM_016848.

[0132] SHP-2 can also be called non-receptor protein tyrosine phosphatase 11 (PTPN11) and protein tyrosine phosphatase 1D (PTP-1D). Human SHP-2 is encoded by a gene with the Ensembl identification number: ENSG00000179295. Human SHP-2 is encoded by a cDNA sequence corresponding to the GeneBank accession number: NM_001330437.

[0133] PI3K can also be called phosphatidylinositol-4,5-bisphosphate 3-kinase. The catalytic subunit of PI3K can be called PIK3CA. Human PIK3CA is encoded by a gene with the Ensembl identification number: ENSG00000121879. Human PIK3CA is encoded by a cDNA sequence corresponding to the GeneBank accession number: NM_006218.

[0134] Abbreviations used in this application include: ECD (extracellular domain), ICD (intracellular 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). IL-2Rγ may also be referred to herein as IL-2RG, IL-2Rgc, γc, or IL-2Rγc. For selected chimeric cytokine receptor designs: "G-CSFRwt-ICDIL-2Rb" is also referred to herein as "G / IL-2Rb"; "G-CSFRwt-ICDgc" is also referred to herein as "G / gc"; "G-CSFR137-ICDgp130-IL-2Rb" is also referred to herein as "G2R-2 with 137ECD"; "G-CSFR137-ICDIL-2Rb + GCSFR137-ICDgc" is also referred to herein as "G2R-1 with 137ECD". IL-2Rγ (i.e., IL-2RG, IL-2Rgc, γc, or IL-2Rγc).

[0135] It should 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.

[0136] It is understood that when a range of values is provided, each intervening value between the upper and lower limits of that range, down to one tenth of the unit of the lower limit, is specifically disclosed, unless the context clearly dictates otherwise. Each smaller range between any of the recited values, or between any other recited value and an intervening value within the recited range, is included within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded within the range, and any range that includes either, neither, or both of these limits within these smaller ranges is included within the invention, subject to any specific exclusionary limitations recited within the stated range. Ranges that exclude any one or both of the recited limits, if the recited range includes one or both of the limits, are also included within the invention.

[0137] Variant Cytokines and Receptor Design Described herein are variant cytokine and receptor pairs for selectively activating a variant receptor. The variant receptors of the disclosure include the extracellular domain of G-CSFR; the variant cytokines include G-CSF (granulocyte colony-stimulating factor) that binds to and activates the variant receptor. In certain embodiments, the variant receptor is a chimeric receptor that includes the ECD of G-CSFR and at least a portion of the ICD of a receptor different from G-CSFR.

[0138] Variant G-CSF and variant G-CSFR ECD pair In certain aspects, the variant G-CSF and receptor designs described herein include at least one site II interface region mutation, at least one site III interface region mutation, and combinations thereof. In certain aspects, the variant G-CSF and receptor designs described herein include at least one site II or site III interface region mutation described in Table 2, 4, or 6.

[0139] In certain embodiments, at least one mutation on the variant receptor at the Site II interface region is located at an amino acid position of the G-CSFR extracellular domain selected from the group consisting of amino acid positions 141, 167, 168, 171, 172, 173, 174, 197, 199, 200, 202, and 288 of the G-CSFR extracellular domain (SEQ ID NO: 2).

[0140] In certain embodiments, at least one mutation on the variant G-CSF at the Site II interface region is located at an amino acid position of G-CSF selected from the group consisting of amino acid positions 12, 16, 19, 20, 104, 108, 109, 112, 115, 116, 118, 119, 122, and 123 of G-CSF (SEQ ID NO: 1).

[0141] In certain embodiments, at least one mutation on the variant receptor at the Site II interface region is selected from the group of G-CSFR extracellular domain mutations consisting of R141E, R167D, K168D, K168E, L171E, L172E, Y173K, Q174E, D197K, D197R, M199D, D200K, D200R, V202D, R288D, and R288E.

[0142] In certain embodiments, at least one mutation on the variant G-CSF at the Site II interface region is selected from the group of G-CSF mutations consisting of K16D, R, S12E, S12K, S12R, K16D, L18F, E19K, E19R, Q20E, D104K, D104R, L108K, L108R, D109R, D112R, D112K, T115E, T115K, T116D, Q119E, Q119R, E122K, E122R, and E123R.

[0143] In certain embodiments, at least one mutation on the variant at the Site III interface region is selected from the group of G-CSFR extracellular domain mutations consisting of amino acid positions 30, 41, 73, 75, 79, 86, 87, 88, 89, 91, and 93 of SEQ ID NO: 2.

[0144] In certain embodiments, at least one mutation on the Site III interface region is selected from the group of G-CSF mutations consisting of amino acid positions 38, 39, 40, 41, 46, 47, 48, 49, and 147 of SEQ ID NO: 1.

[0145] In certain embodiments, at least one mutation on the Site III interface region is selected from the group of G-CSFR extracellular domain mutations consisting of S30D, R41E, Q73W, F75K, S79D, L86D, Q87D, I88E, L89A, Q91D, Q91K, and E93K.

[0146] In certain embodiments, at least one mutation on the variant G-CSF Site III interface region is selected from the group of G-CSF mutations consisting of T38R, Y39E, K40D, K40F, L41D, L41E, L41K, E46R, L47D, V48K, V48R, L49K, and R147E.

[0147] The variant cytokine and receptor pairs described herein may contain mutations in the Site II region only, the Site III region only, or both the Site II and Site III regions.

[0148] The variant cytokine and receptor pairs described herein can have any number of the Site II and / or Site III mutations described herein. In certain embodiments, the variant G-CSF and receptor have the mutations described in Table 6. In certain embodiments, the variant receptor and / or variant G-CSF can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations described herein.

[0149] In certain embodiments, the variant receptors described herein comprise a G-CSFR ECD domain that 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% amino acid identity with the G-CSFR ECD of the SEQ ID NOs described herein. In certain embodiments, the chimeric receptor comprises an ECD of G-CSFR having the amino acid sequence of SEQ ID NO: 2, 3, 6, or 8.

[0150] In certain embodiments, the variant G-CSFs described herein comprise an amino acid sequence that 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% amino acid identity with the G-CSFR ECD of SEQ ID NO: 1.

[0151] In certain embodiments, the ECD of G-CSFR comprises at least one amino acid substitution selected from the group consisting of R41E, R141E, and R167D.

[0152] Variant cytokines and / or receptors can be produced recombinantly not only directly but also as fusion polypeptides with heterologous polypeptides, which heterologous polypeptides are, for example, signal sequences or other polypeptides having specific cleavage sites at the N-terminus of the mature protein or polypeptide. Generally, the signal sequence may be a component of the vector or part of the coding sequence inserted into the vector. The selected heterologous signal sequence is preferably one that is recognized and processed by the host cell (i.e., cleaved by signal peptidase). In mammalian cell expression, a native signal sequence may be used, or other mammalian signal sequences, such as signal sequences from secreted polypeptides of the same or related species, and viral secretion leaders may be suitable. In certain embodiments, the signal sequence is the signal sequence of G-CSFR or GM-CSFR. In certain embodiments, the signal sequence is SEQ ID NO: 11 or SEQ ID NO: 12.

[0153] In certain embodiments, variant receptors and / or variant G-CSF are modified either naturally or synthetically (e.g., glycosylation, PEG) to enhance stability. For example, in certain embodiments, the variant cytokine is fused to the Fc domain of IgG, albumin, or other molecules to extend the half-life, e.g., by PEGylation, glycosylation, etc., as known in the art. Fc fusion can also promote alternative Fc receptor-mediated properties in vivo. The "Fc region" can be a naturally occurring or synthetic polypeptide homologous to the IgG C-terminal domain produced by digesting IgG with papain. The IgG Fc has a molecular weight of about 50 kDa. The variant cytokine may contain the entire Fc region or a smaller portion that retains the ability to extend the circulatory half-life of a chimeric polypeptide that is a part thereof. Further, full-length or fragmented Fc regions can be variants of the wild-type molecule.

[0154] When a variant cytokine binds to a variant receptor, the variant receptor activates signal transduction that is transmitted through the native intracellular components to provide a biological activity that mimics its native response, which is specific to cells engineered to express the variant receptor. In certain embodiments, the variant receptor and G-CSF pair do not bind to their native wild-type G-CSF or native wild-type G-CSFR. Thus, in certain embodiments, the variant receptor does not bind to endogenous cognate cytokines, including the native counterpart of the variant cytokine, while the variant cytokine does not bind to any endogenous receptor, including the native counterpart of the variant receptor. In certain embodiments, the variant cytokine binds to the native receptor with a significantly reduced affinity compared to the binding of the native cytokine to the native cytokine receptor. In certain embodiments, the affinity of the variant 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 the affinity of the native cytokine for the native cytokine receptor. In certain embodiments, the variant cytokine binds to the native receptor with a K -4 greater than 1X10 -5 M, greater than 1X10 -6 M, greater than 1X10 -7 M, greater than 1X10 -8 M, greater than 1X10 -9 M, or greater than 1X10 D . In certain embodiments, the variant cytokine receptor binds to the native cytokine with a significantly reduced affinity compared to the binding of the native cytokine receptor to the native cytokine. In certain embodiments, the variant cytokine receptor binds to the native cytokine with less than 10-fold, less than 100-fold, less than 1,000-fold, or less than 10,000-fold the affinity of the native cytokine for the native cytokine receptor. In certain embodiments, the variant cytokine receptor binds to the native cytokine with a K -4 greater than 1X10 -5 M, greater than 1X10 -6 M, greater than 1X10 -7Greater than M, 1×10 -8 Greater than M, or 1×10 -9 K greater than M D binds to the native cytokine. In some embodiments, the affinity of the variant cytokine for the variant receptor is comparable to the affinity of the native cytokine for the native receptor, e.g., having an affinity of 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 may be higher than that, 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. The affinity can be determined by any number of assays well known to those skilled in the art. For example, the affinity can be determined by a competitive binding experiment that measures receptor binding using a single concentration of labeled ligand in the presence of various concentrations of unlabeled ligand. Typically, the concentration of the unlabeled ligand varies over at least six orders of magnitude. From the competitive binding experiment, the IC 50 can be determined. As used herein, "IC 50 " refers to the concentration of unlabeled ligand required to inhibit 50% of the association between the receptor and the labeled ligand. The IC 50 is an indicator of ligand-receptor binding affinity. A low IC 50 represents high affinity, while a high IC 50 represents low affinity.

[0155] Binding of a variant cytokine to a variant cytokine receptor expressed on the surface of a cell may or may not affect (compared to native cytokine receptor activity) the function of the variant cytokine receptor; native activity is not required or desired in all cases. In certain embodiments, binding of a variant cytokine to a variant cytokine receptor will induce one or more features of native cytokine signaling. In certain embodiments, binding of a variant cytokine to a variant cytokine receptor expressed on the surface of a cell causes a cell response selected from the group consisting of proliferation, survival, and enhanced activity.

[0156] (Table 1) Sequences of human WT G-CSF and human WT G-CSFRIg-CRH domain TIFF0007708745000001.tif181153

[0157] (Table 2) G-CSF E and G-CSFR E Site II designs having mutations in TIFF0007708745000002.tif240161

[0158] (Table 4) Site III designs TIFF0007708745000003.tif127128

[0159] (Table 6) Examples of designs obtained from combinations of Site II and III designs TIFF0007708745000004.tif219153TIFF0007708745000005.tif59153

[0160] Chimeric receptor In certain embodiments, the variant receptors described herein are chimeric receptors. The chimeric receptors can include any of the variant G-CSFR ECD domains described herein. In certain embodiments, the chimeric receptors further include at least a portion of the intracellular domain (ICD) of a different cytokine receptor. The intracellular domain of the different cytokine receptor can be selected from the group consisting of gp130 (glycoprotein 130), IL-2Rβ or IL-2Rb (interleukin-2 receptor β), IL-2Rγ or γc or IL-2RG (interleukin-2 receptor γ), IL-7Rα (interleukin-7 receptor α), IL-12Rβ2 (interleukin-12 receptor β2), and IL-21R (interleukin-21 receptor). In certain embodiments, at least a portion of the intracellular domain comprises an amino acid sequence that shares 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 with the amino acid sequence of the cytokine receptor ICD described herein. In certain embodiments, at least a portion of the cytokine receptor ICD shares 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 sequence identity with SEQ ID NO: 4, 7, or 9.

[0161] In certain embodiments, described herein is a chimeric cytokine receptor comprising an extracellular domain (ECD) of a G-CSFR (granulocyte colony-stimulating factor receptor) operably linked to a second domain; the second domain comprises at least a portion of an intracellular domain (ICD) of a multi-subunit cytokine receptor, e.g., IL-2R. In certain aspects, the chimeric cytokine receptor comprises a portion of the ICDs of Tables 15A and 15B. In certain aspects, the chimeric cytokine receptor comprises a transmembrane domain selected from Tables 15A and 15B. In certain aspects, the chimeric cytokine receptor ICD comprises the Box1 and Box2 regions of Tables 15A, 15B, and 16. In certain aspects, the chimeric cytokine receptor comprises at least one signaling molecule binding site of Tables 15A, 15B, and 16.

[0162] In certain aspects, the chimeric receptor described herein comprises an amino acid sequence in the N-terminal to C-terminal order of the sequences disclosed in each of Tables 17-20. In certain aspects, the chimeric receptor described herein comprises a nucleic acid sequence in the 5’ to 3’ order of the sequences disclosed in each of Tables 17-20. In certain aspects, 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% amino acid identity with the amino acid sequence in the N-terminal to C-terminal order of the sequences disclosed in each of Tables 17-20. In certain aspects, 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 with the nucleic acid sequence in the 5’-3’ order of the sequences disclosed in each of Tables 17-20.

[0163] 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 with the ICD sequence numbers 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: 26, 29, 31, 39, 41, 43, 45, 47, or 49. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of IL-2Rβ, i.e., IL-2Rb, having the nucleic acid sequence of SEQ ID NO: 54, 57, 59, 67, 69, 71, 73, 75, or 77. 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: 51 or the nucleic acid sequence of SEQ ID NO: 79. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of IL-7R having the amino acid sequence of SEQ ID NO: 53 or the nucleic acid sequence of SEQ ID NO: 81. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO: 45 or 55. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of IL-21R having the nucleic acid sequence of SEQ ID NO: 35 or 37. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of IL-12Rβ2 having the amino acid sequence of SEQ ID NO: 33, 42, or 46. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of IL-12Rβ2 having the nucleic acid sequence of SEQ ID NO: 61, 70, or 74. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO: 30, 32, 34, 36, 38, 40, 44, 50, or 52. 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: 58, 60, 62, 64, 66, 68, 72, 78, or 80. 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: 28 or 48. 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: 56 or 76.In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of IL-2Rγ having the amino acid sequence of SEQ ID NO: 27 (i.e., IL-2RG, IL-2Rgc, γc, or IL-2Rγc). In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of IL-2Rγ having the nucleic acid sequence of SEQ ID NO: 55 (i.e., IL-2RG, IL-2Rgc, γc, or IL-2Rγc).

[0164] In certain embodiments, at least a portion of the ICDs described herein comprises at least one signal transduction molecule binding site. In certain embodiments, the at least one signal transduction molecule binding site is the STAT3 binding site of G-CSFR; the STAT3 binding site of gp130; the SHP-2 binding site of gp130; the Shc binding site of IL-2Rβ; the STAT5 binding site of IL-2Rβ; the STAT3 binding site of IL-2Rβ; the STAT1 binding site of IL-2Rβ; the STAT5 binding site of IL-7Rα; the phosphatidylinositol 3-kinase (PI3K) binding site of IL-7Rα; the STAT5 binding site of IL-12Rβ2; the STAT4 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. In certain embodiments, the at least one signal transduction molecule binding site comprises a sequence further comprising the amino acids described in Table 16.

[0165] In certain embodiments, at least a portion of the ICDs described herein comprises the Box1 and Box regions of gp130 or G-CSFR. In certain embodiments, the Box1 region comprises the amino acid sequence described in Table 2. In certain embodiments, the Box1 region comprises an amino acid sequence having more than 50% identity to the Box1 sequence described in Table 16.

[0166] In certain embodiments, the intracellular domains of the different cytokine receptors are wild-type intracellular domains.

[0167] In certain embodiments, the chimeric variant receptors described herein further comprise at least a portion of the transmembrane domain (TMD) of different cytokine receptors. The TMDs of different cytokine receptors can be selected from the group consisting of gp130 (glycoprotein 130), IL-2Rβ (interleukin-2 receptor β), IL-2Rγ or γc (IL-2 receptor γ), IL-7Rα (interleukin-7 receptor α), IL-12Rβ2 (interleukin-12 receptor β2), and IL-21R (interleukin-21 receptor). In certain embodiments, at least a portion of the TMD comprises an amino acid sequence that shares 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 with the amino acid sequence of the cytokine receptor TMD described herein. In certain embodiments, at least a portion of the cytokine receptor TMD shares 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 sequence identity with SEQ ID NO: 4, 5, 7, or 9.

[0168] In certain embodiments, the chimeric receptor described herein comprises at least a portion of the G-CSFR ECD domain, transmembrane domain (TMD), and ICD, arranged in order from the N-terminus to the C-terminus, as shown in the chimeric receptor designs of FIGS. 20, 23, and 24.

[0169] In certain embodiments, the chimeric receptor comprises the G-CSFR ECD of SEQ ID NO: 3, a portion of the gp130 TMD and ICD of SEQ ID NO: 4, and a portion of the IL-2Rβ ICD of SEQ ID NO: 5. In certain embodiments, the chimeric receptor comprises the G-CSFR ECD of SEQ ID NO: 6, and a portion of the IL-2Rβ ICD of SEQ ID NO: 7. In certain embodiments, the chimeric receptor comprises the G-CSFR ECD of SEQ ID NO: 8, and a portion of the IL-2Rγ ICD of SEQ ID NO: 9.

[0170] Binding of a variant or wild-type cytokine to a chimeric cytokine receptor expressed on the surface of a cell may or may not affect (as compared to native cytokine receptor activity) the function of the variant cytokine receptor; native activity is not required or desired in all cases. In certain embodiments, binding of a variant cytokine to a chimeric cytokine receptor will induce one or more features of native cytokine signaling. In certain embodiments, binding of a variant 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.

[0171] Nucleic acids encoding variant cytokines and receptors Included in the present disclosure are nucleic acids encoding any one of the receptors and variant G-CSF described herein.

[0172] Variant receptors or variant G-CSF can be produced not only directly by recombination, but also as fusion polypeptides with heterologous polypeptides, such as signal sequences, or other polypeptides having specific cleavage sites at the N-terminus of the mature protein or polypeptide. Generally, the signal sequence can be a component of the vector or part of the coding sequence inserted into the vector. The selected heterologous signal sequence is preferably one that is recognized and processed (i.e., cleaved by signal peptidase) by the host cell. In mammalian cell expression, native signal sequences can be used, or other mammalian signal sequences, such as signal sequences from secreted polypeptides of the same or related species, and viral secretion leaders may be suitable. In certain embodiments, the signal sequence can be an amino acid sequence comprising a signal sequence in the N-terminal region of SEQ ID NO: 2, 3, 6, or 8. In certain embodiments, the signal sequence can be the amino acid sequence of MARLGNCSLTWAALIILLLPGSLE (SEQ ID NO: 11).

[0173] Expression vectors encoding variant cytokines or receptors Also described herein are expression vectors and kits of expression vectors comprising one or more nucleic acid sequences (s) encoding one or more of the variant receptors or variant G-CSFs described herein.

[0174] In certain embodiments, the nucleic acid encoding the variant receptor or variant G-CSF is inserted into a replicable vector for expression. Such vectors can be used to introduce the nucleic acid sequence(s) into a host cell so as to express the variant receptor or cytokine described herein. Many such vectors are available. The components of the vector generally include, but are not limited to, one or more of an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence. Vectors include viral vectors, plasmid vectors, integrating vectors, and the like. The vector can be, for example, a plasmid or viral vector such as a retroviral vector, an adenoviral vector, a lentiviral vector, or a transposon-based vector or synthetic mRNA. The vector can be transfected or transduced into a cell (e.g., a T cell, an NK cell, or other cell).

[0175] Expression vectors usually contain a selectable gene, also commonly referred to as a selectable marker. The selectable marker gene encodes a protein necessary for the survival or growth of transformed host cells that grow in a selective culture medium. Host cells not transformed with a vector containing a selectable gene do not survive in the culture medium. Typical selectable genes encode a protein that (a) confers resistance to an antibiotic or other toxin, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complements an auxotrophic deficiency, or (c) supplies an essential nutrient not available from complex media.

[0176] In certain embodiments, the expression vector contains a promoter that is recognized by the host organism and operably linked to the variant protein coding sequence. A promoter is an untranslated sequence (generally within about 100 to 1000 bp) located upstream (5') of the start codon of a structural gene that controls the transcription and translation of the specific nucleic acid sequence to which it is operably linked. Such promoters are typically divided into two classes, inducible promoters and constitutive promoters. An inducible promoter is a promoter that initiates increased levels of transcription from DNA under its control in response to some change in culture conditions, such as the presence or absence of nutrients or a change in temperature. A number of promoters recognized by various potential host cells are well known.

[0177] Transcription from vectors in mammalian host cells can be controlled by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses (e.g., mouse stem cell virus), hepatitis B virus, and most preferably, simian virus 40 (SV40), such as heterologous mammalian promoters, e.g., actin promoter, PGK (phosphoglycerate kinase), or immunoglobulin promoter, or a promoter obtained from a heat shock promoter, provided that such a promoter is compatible with the host cell line. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 origin of replication.

[0178] Transcription by higher eukaryotes is often increased by inserting enhancer sequences into the vector. Enhancers are cis - acting elements of DNA, usually about 10 - 300 bp in length, which act on the promoter to facilitate its transcription. Enhancers are found within the 5' and 3' sides of the transcription unit, within introns, and within the coding sequence itself, independent of their relative orientation and position. Many enhancer sequences derived from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin) are known. However, typically, enhancers derived from eukaryotic viruses are used. Examples include the SV40 enhancer on the late side of the origin of replication, the cytomegalovirus immediate - early promoter enhancer, the polyoma enhancer on the late side of the origin of replication, and the adenovirus enhancer. The enhancer may be spliced into the expression vector at the 5' or 3' position of the coding sequence, but is preferably located at the 5' site derived from the promoter.

[0179] Expression vectors used in eukaryotic host cells also contain sequences necessary for transcription termination and mRNA stabilization. Such sequences can generally be obtained from the 5' - and optionally 3' - untranslated regions of eukaryotic or viral DNA or cDNA. Construction of suitable vectors containing one or more of the above components uses standard techniques.

[0180] In certain embodiments, disclosed herein is a lentiviral vector encoding a chimeric receptor disclosed herein. In certain embodiments, the lentiviral vector comprises the HIV - 1 5'LT and 3'LTR. In certain embodiments, the lentiviral vector comprises the EF1a promoter. In certain embodiments, the lentiviral vector comprises the SV40 poly - terminator sequence. In certain embodiments, the vector is psPAX2, Addgene® 12260, pCMV - VSV - G, or Addgene® 8454.

[0181] In some embodiments, nucleic acid and polypeptide sequences having a high sequence identity to the sequences described herein, e.g., 95, 96, 97, 98, 99% or greater sequence identity, are also described. The term percent sequence “identity” in the context of two or more nucleic acid or polypeptide sequences refers to the percentage of particular nucleotides or amino acid residues that are identical when the sequences or subsequences are compared and aligned for maximum correspondence 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, percent “identity” can exist over regions of the sequences being compared, e.g., over functional domains, or over the entire length of the two sequences being compared.

[0182] For sequence comparison, typically one sequence acts as a reference sequence, to which the test sequence is compared. When sequence comparison algorithms are used, the test and reference sequences are input 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.

[0183] Optimal alignment of arrays for comparison can be carried out, 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 (generally, see Ausubel et al.).

[0184] One example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, 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 / ).

[0185] Cells expressing variant receptors and variant cytokines Also described herein are cells expressing variant receptors. The above expression vectors for variant cytokine or receptor expression can be transfected or transduced into host cells, including engineered immune cells.

[0186] In certain embodiments, the present disclosure provides a cell comprising one or more of the variant receptors or variant cytokines described herein. The cell may comprise a nucleic acid or vector encoding the variant receptor or variant cytokine described herein. The present disclosure also provides a method of making a cell that expresses a variant receptor. In certain aspects, the cell is made by introducing the nucleic acid or expression vector described herein into the cell. The nucleic acid or expression vector can be introduced into the cell by any process including, but not limited to, transfection, transduction with a viral vector, transfer, or gene editing. Any gene editing technique known in the art can be used, including, but not limited to, techniques consisting of clustered regularly interspaced short palindromic repeats (CRISPR-Cas) systems, zinc finger nucleases, transcription activator-like effector-based nucleases, and meganucleases.

[0187] 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, naive CD8 + T cell, cytotoxic CD8 + T cell, naive CD4 + T cell, helper T cell, e.g., T H 1, T H 2, T H 9, T H 11, T H 22, T FH ; regulatory T cell, e.g., T R 1, natural T Reg -, inducible T Reg; Memory T cells, such as, but not limited to, central memory T cells, effector memory T cells, NKT cells, γδ T cells, etc. In certain embodiments, the cells 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, plasma cells, etc. In certain embodiments, the cells are natural lymphoid cells, including, but not limited to, NK cells, etc. In certain embodiments, the cells are myeloid cells, including, but not limited to, macrophages, dendritic cells, myeloid-derived suppressor cells, etc.

[0188] In certain embodiments, the cells are stem cells, including, but not limited to, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, etc.

[0189] In some embodiments, the cells are genetically modified by ex vivo procedures prior to transplantation into a subject. The cells can be provided in unit doses for treatment and can be allogeneic, autologous, etc. with respect to the intended recipient.

[0190] 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 surfaces. As summarized below, there are various types of T cells.

[0191] Helper T helper cells (Th cells) assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and memory B cells, as well as the activation of cytotoxic T cells and macrophages. Th cells express CD4 on their surface. Th cells become activated when peptide antigens are presented to them 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, Th3, Th17, Th9, or Tfh, which secrete different cytokines to promote different types of immune responses.

[0192] Cytotoxic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells and are also involved in transplant rejection. Most CTLs express CD8 on their surface. These cells recognize targets by binding to antigens associated with MHC class I, which is present on the surface of all nucleated cells.

[0193] Memory T cells are a subset of antigen-specific T cells that persist long-term even after an infection has resolved. Memory T cells expand rapidly into large numbers of effector T cells upon re-exposure to their homologous antigen, providing the immune system with a "memory" of past infections. Memory T cells include 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.

[0194] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are essential for maintaining immune tolerance. Their main role is to shut down T cell-mediated immunity towards the end of an immune response and to suppress autoreactive T cells that have escaped the process of negative selection in the thymus. Two major classifications of CD4+ Treg cells are described: natural Treg cells and adaptive Treg cells.

[0195] Innate Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) are generated in the thymus and are associated with the interaction 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.

[0196] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can arise during a normal immune response. The cells can be natural killer cells (or 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.

[0197] In certain embodiments, the cells expressing the variant receptors or variant cytokines described herein are tumor-infiltrating lymphocytes (TILs) or tumor-associated lymphocytes (TALs). In certain embodiments, TILs or TALs include CD4+ T cells, CD8+ T cells, natural killer (NK) cells, and combinations thereof.

[0198] 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 embodiments, the CAR-T cells are derived from T cells in the patient's own blood (i.e., autologous). In certain embodiments, the CAR-T is derived from T cells of another healthy donor (i.e., allogeneic).

[0199] In certain embodiments, the T cells described herein are engineered T cell receptors (eTCR-T cells) genetically engineered to produce a specific T cell receptor for use in immunotherapy. In certain embodiments, the eTCR-T cells are derived from T cells in the patient's own blood (i.e., autologous). In certain embodiments, the eTCR-T cells are derived from donor T cells (i.e., allogeneic).

[0200] NK cells (which belong to the group of natural lymphocytes) 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 and then enter the circulatory system from there.

[0201] In certain embodiments, the cells expressing the chimeric cytokine receptor described herein are B cells. B cells include, but are 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.

[0202] In certain embodiments, the cells expressing the chimeric cytokine receptor described herein are myeloid cells including, but not limited to, macrophages, dendritic cells, and myeloid-derived suppressor cells.

[0203] The cells expressing the variant receptor or variant cytokine described herein can be of any cell type. In certain embodiments, the cells expressing the variant receptor or variant cytokine described herein are hematopoietic cells. The immune cells according to the present invention (e.g., T cells or NK cells) can be generated ex vivo from the patient's own peripheral blood (first party), in the setting of hematopoietic stem cell transplantation from a donor peripheral blood (second party), or from a peripheral blood from an unrelated donor (third party). Alternatively, the immune cells described herein can be derived from the ex vivo differentiation of induced or embryonic progenitor cells into immune cells. Alternatively, immortalized immune cell lines that retain effector function and can act as therapeutic agents can be used (e.g., T cell or NK cell lines that retain lysis function, plasma cell lines that retain antibody production function, or dendritic cell lines or macrophages that retain phagocytosis and antigen presentation functions, etc.). In all of these embodiments, the variant receptor-expressing cells are generated by introducing DNA or RNA encoding each variant receptor(s) by one of many means, including transduction by viral vectors or transfection by DNA or RNA.

[0204] The cells described herein can be immune cells derived from a subject that have been manipulated ex vivo to express a variant receptor and / or variant cytokine. The immune cells can be derived from a peripheral blood mononuclear cell (PBMC) sample or a tumor sample. The immune cells can be activated and / or expanded, for example, by treatment with an anti-CD3 monoclonal antibody and / or IL-2, prior to being transfected with a nucleic acid encoding a molecule that provides a variant receptor or variant cytokine according to the first aspect of the present invention. The immune cells of the present invention can be prepared by (i) isolating a sample containing cells from a subject or one of the other sources described above, and (ii) transducing or transfecting the immune cells with one or more nucleic acid sequences encoding a variant receptor or variant cytokine.

[0205] The cells can be cultured in conventional nutrient media that are modified as necessary for the induction of promoters, the selection of transformants, or the amplification of genes encoding the desired sequences. 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 Medium ((DMEM), Sigma) are suitable for culturing host cells. Any of these media can be supplemented, as necessary, 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 can also be included at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc. are those that have been used previously for the host cells selected for expression and will be apparent to those skilled in the art.

[0206] Next, the immune cells can be purified, for example, based on the expression of the antigen-binding domain of the antigen-binding polypeptide. In certain embodiments, the cells are selected by the expression of a selectable marker (such as a protein, a fluorescent marker, or an epitope tag) or by any method known in the art for the selection, isolation, and / or purification of cells.

[0207] Kit The present disclosure also describes a kit for generating cells that express at least one of the variant receptors or variant G-CSFs described herein. In certain embodiments, the kit includes at least one expression vector encoding at least one variant receptor and instructions for use. In certain aspects, the kit further includes an expression vector encoding a variant cytokine in a pharmaceutical formulation or a variant G-CSF that binds to at least one of the variant receptors described herein. In certain embodiments, the kit includes cells containing an expression vector encoding a variant receptor described herein.

[0208] In certain embodiments, the kit includes cells containing an expression vector encoding a chimeric antigen receptor (CAR) / engineered T cell receptor (eTCR) such as a CAR / TCR (e.g., an engineered non-natural TCR receptor). In certain embodiments, the kit includes an expression vector encoding a chimeric antigen receptor (CAR) / engineered T cell receptor (eTCR) such as a CAR / eTCR. In certain embodiments, the kit includes an expression vector encoding a variant receptor described herein and a chimeric antigen receptor (CAR) / engineered T cell receptor (eTCR) such as a CAR / eTCR.

[0209] In certain aspects, the kit described herein further includes a variant cytokine. In certain embodiments, the kit further includes at least one additional variant cytokine. In certain aspects, the kit further includes at least one variant cytokine in a pharmaceutical formulation. In certain embodiments, the components are provided in a dosage form, in liquid or solid form, in any convenient packaging.

[0210] Additional reagents may be provided for the growth, selection, and preparation of the cells provided or generated as described herein. For example, the kit can include components for cell culture, growth factors, differentiating agents, reagents for transfection or transduction, and the like.

[0211] 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 package, package insert, etc. The instructions can also be provided as a computer-readable medium on which the information is recorded. Further, the instructions can be provided with the address of a website that can be used to access the information.

[0212] Method for selective activation of variant receptors The present disclosure provides a method for selectively activating a variant receptor expressed on the surface of a cell, the method comprising contacting a variant receptor described herein with a cytokine that selectively activates a chimeric receptor. In certain embodiments, the cytokine that selectively activates the chimeric receptor is variant G-CSF. The G-CSF can be wild-type G-CSF or G-CSF that includes one or more mutations that result in preferential binding and activation of G-CSF to the variant receptor as compared to the native (wild-type) cytokine receptor.

[0213] In certain embodiments, the selective activation of the variant receptor by binding of the cytokine to the variant receptor results in homodimerization, heterodimerization, or a combination thereof.

[0214] In certain embodiments, activation of the variant receptor results in activation of downstream signaling molecules. In certain embodiments, the variant receptor activates a signaling molecule or pathway that is transmitted through a native intracellular signaling molecule to provide a biological activity that mimics its native response, which is specific for cells engineered to express the variant receptor. In certain embodiments, activation of downstream signaling molecules includes activation of cell cycle progression, proliferation, survival, and / or activation of cellular signaling pathways that stimulate enhanced activity. In certain embodiments, the signaling pathway or molecule that is activated is, but is not limited to, Jak1, Jak2, Jak3, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6, Shc, ERK1 / 2, and Akt. In certain embodiments, activation of the variant receptor results in an increase in cell proliferation following administration of a cytokine that binds to the receptor. In certain embodiments, the degree of proliferation is 0.1 to 10 times the proliferation observed when cells are stimulated with IL-2.

[0215] Method for adoptive cell transfer The present invention provides a method for treating and / or preventing a disease, comprising the step of administering to a subject a cell (e.g., a pharmaceutical composition described below) that expresses a variant receptor and / or variant cytokine described herein.

[0216] Methods for treating a disease relate to the therapeutic use of the cells described herein, such as T cells, NK cells, or any other immune or non-immune cells expressing a variant receptor. The cells can be administered to a subject having an existing disease or condition to reduce, mitigate, or improve at least one symptom associated with the disease and / or to decelerate, reduce, or arrest the progression of the disease. Methods for preventing a disease relate to the prophylactic use of the cells of the present disclosure. Such cells can be administered to a subject who has not yet been affected by the disease and / or does not exhibit symptoms of the disease to prevent or impair the cause of the disease and / or to reduce or prevent the occurrence of at least one symptom associated with the disease. The subject can be considered to have a predisposition to the disease or to be at risk of developing it.

[0217] In some embodiments, the compositions, methods, and kits of the subject matter are used to enhance an immune response. In some embodiments, the immune response is effected against a condition 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 of cytokines (e.g., intramuscularly, intraperitoneally, intravenously, etc.).

[0218] The method can include: (i) isolating a sample containing immune cells; (ii) transforming or transfecting such cells with a nucleic acid sequence or vector expressing, for example, a variant receptor; (iii) administering (i.e., injecting) the cells of (ii) to a subject; and (iv) administering a variant cytokine that stimulates the injected cells. In certain embodiments, the subject may have received an immunosuppressive treatment prior to administering the cells to the subject. In certain embodiments, the subject has not received an immunosuppressive treatment prior to administering the cells to the subject. In certain embodiments, the subject may have received an immunosuppressive treatment with reduced severity, dosage, and / or duration that would otherwise be required without using the variant receptor described herein prior to administering the cells to the subject.

[0219] A sample containing immune cells can be isolated from a subject or other source, 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 induced by in vitro methods, such as inducing differentiation from stem cells or other forms of progenitor cells.

[0220] In some embodiments, the immune cells are contacted with the variant cytokine in vivo, i.e., the immune cells are transferred to a recipient and an effective amount of the variant cytokine is administered to the recipient to contact the immune cells at their original location, such as in lymph nodes. In some embodiments, the contact is performed in vitro. When the cells are contacted with the variant cytokine in vitro, the cytokine is added to the cells in a dosage and for a period sufficient to activate signal transduction from the receptor, which can utilize features of the natural intracellular machinery, such as accessory proteins, co-receptors, etc. The activated cells can be used for any purpose, including but not limited to experimental purposes related to determination of antigen specificity, cytokine profiling, and delivery in vivo.

[0221] In certain embodiments, a therapeutically effective number of cells are administered to the subject. In certain embodiments, the subject is administered or infused with cells expressing the variant receptor on multiple separate occasions. In certain embodiments, at least 1x10 6 cells / kg, at least 1x10 7 cells / kg, at least 1x10 8 cells / kg, at least 1x10 9 cells / kg, at least 1x10 10Cells / kg or more are administered, which may be limited by the number of cells obtained during collection, for example, transfected T cells. The transfected cells can be injected into the subject in any physiologically acceptable medium, usually intravascularly, but may be introduced into any other convenient site where the cells can find a site suitable for growth.

[0222] In certain embodiments, a therapeutically effective amount of a variant cytokine is administered to the subject. In certain embodiments, the subject is administered the variant cytokine on a plurality of separate occasions. In certain embodiments, the amount of variant cytokine administered is an amount sufficient to achieve a therapeutically desirable result (e.g., reduce the symptoms of the subject's disease). In certain embodiments, the amount of variant cytokine administered is an amount sufficient to stimulate the progression, proliferation, survival, and / or functional activity of the cell cycle of cells expressing the variant cytokine receptor described herein. In certain embodiments, the variant cytokine is administered in the dosage and / or period required to achieve a therapeutically desirable result. In certain embodiments, the variant cytokine is administered in a dosage and / or period sufficient to stimulate the progression, proliferation, survival, and / or functional activity of the cell cycle of cells expressing the variant cytokine receptor described herein. The dosage and frequency may vary depending on the drug, the method of administration, the nature of the cytokine, etc. It will be understood by those skilled in the art that such guidelines are adjusted according to individual circumstances. The dosage can also be varied for local administration (e.g., intranasal administration, inhalation, etc.) versus systemic administration (e.g., intramuscular, intraperitoneal, intravascular, etc.).

[0223] Indications for adoptive cell transfer The present disclosure provides cells expressing the variant receptor described herein for use in the treatment and / or prevention of a disease. The present invention also relates to the use of cells expressing the variant receptor described herein in the manufacture of a medicament for the treatment and / or prevention of a disease.

[0224] Diseases that can be treated and / or prevented by the method of the present invention can be, but are not limited to, cancerous diseases such as cholangiocarcinoma, 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 lymphoma, pancreatic cancer, prostate cancer, sarcoma, and thyroid cancer.

[0225] The diseases to be treated and / or prevented may be autoimmune diseases. Autoimmune diseases are characterized by T and B lymphocytes that abnormally target self-proteins, polypeptides, peptides, and / or other self-molecules, causing damage and / or dysfunction of organs, tissues, or cell types (e.g., pancreas, brain, thyroid, or gastrointestinal tract) in the body, thereby causing the clinical symptoms of the disease. Autoimmune diseases include diseases that affect specific tissues and diseases that can affect multiple tissues, which can be partially determined by whether the response is directed against antigens limited to specific tissues or antigens widely distributed in the body. Autoimmune diseases include, but are not limited to, type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, autoimmune thyroid diseases, and Graves' disease.

[0226] The diseases to be treated and / or prevented may be inflammatory diseases such as cardiac fibrosis. Generally, inflammatory conditions or disorders typically involve the immune system attacking the body's own cells or tissues, which can cause abnormal inflammation, potentially leading to chronic pain, redness, swelling, stiffness, and damage to normal tissues. Inflammatory diseases are those 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, atherosclerosis, arthritis, myositis, scleroderma, gout, Sjogren's syndrome, ankylosing spondylitis, antiphospholipid antibody syndrome, and psoriasis.

[0227] In certain embodiments, the method is used to treat infectious diseases.

[0228] In certain embodiments, the condition to be treated is to prevent and treat graft rejection. In certain embodiments, the condition to be treated and / or prevented is allograft rejection. In certain aspects, the allograft rejection is acute allograft rejection.

[0229] The disease to be treated and / or prevented can include transplantation of cells, tissues, organs, or other anatomical structures into an affected individual. The cells, tissues, organs, or other anatomical structures can be derived from the same individual (autologous or "self" transplantation) or a different individual (allogeneic or "allograft" transplantation). The cells, tissues, organs, or other anatomical structures can also be produced using in vitro methods, including cell cloning, induction of cell differentiation, or production using synthetic biomaterials.

[0230] The present invention provides a method for treating and / or preventing a disease, comprising one or more steps of administering to a subject the variant cytokine and / or cells (e.g., in the pharmaceutical composition described above).

[0231] Methods for treating and / or preventing a disease relate to the therapeutic use of the cells of the present disclosure. As used herein, the cells can be administered to a subject having an existing disease or condition to reduce, mitigate, or improve at least one symptom associated with the disease and / or to decelerate, reduce, or arrest the progression of the disease. Methods for preventing a disease relate to the prophylactic use of the cells of the present disclosure. Such cells can be administered to a subject who has not yet developed 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 can be considered to have a predisposition to the disease or to be at risk of developing it. The method can include (i) isolating a sample containing immune cells, (ii) transforming or transfecting such cells with a nucleic acid sequence or vector provided by the invention, (iii) administering the cells of (ii) to the subject, and (iv) administering a variant cytokine that stimulates the infused cells. A sample containing immune cells can be isolated from the subject or another source, for example, as described above. The immune cells can be isolated from the subject's own peripheral blood (first party), in the setting of a hematopoietic stem cell transplant from a donor peripheral blood (second party), or from a peripheral blood from an unrelated donor (third party).

[0232] The treatment can be combined with other active agents, such as, for example, 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 CTLA-4), but is not limited thereto. Additional cytokines may also be included (e.g., interferon γ, tumor necrosis factor α, interleukin 12, etc.).

[0233] Method of using stem cells expressing variant cytokine receptors The present invention provides a method for treating and / or preventing a condition or disease, comprising the step of administering stem cells that express a variant receptor and / or a variant cytokine as described herein. In certain embodiments, the stem cells that express a variant cytokine receptor and / or a variant cytokine as described herein are used in regenerative medicine, cell / tissue / organ transplantation, tissue reconstruction, or tissue repair.

[0234] Pharmaceutical composition of the present invention The present disclosure also relates to a pharmaceutical composition containing a plurality of cells that express a variant receptor as described herein and / or a cytokine as described herein. The present disclosure also relates to a pharmaceutical composition containing a variant cytokine as described herein. The cells of the present invention can be formulated into a pharmaceutical composition. These compositions can include, in addition to one or more cells that express a variant receptor as described herein, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials must be non-toxic and must not interfere with the effectiveness of the active ingredient. The pharmaceutical composition may optionally include one or more additional pharmaceutically active polypeptides and / or compounds. Such formulations can be, for example, in a form suitable for intravenous infusion.

[0235] In the case of cells expressing the variant receptors and variant cytokines described herein according to the present disclosure to be administered to a subject, the administration is preferably at a "therapeutically effective amount" sufficient to show benefit to the subject. A "prophylactically effective amount" can also be administered if sufficient to show benefit to the subject. The actual amount of cytokine or number of cells administered, as well as the rate and duration of administration, depend on the nature and severity of the disease being treated. Determination of the treatment regimen, such as dosage, is within the responsibility of the general practitioner and other physicians and usually takes 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 the practitioner. Examples of the above techniques and protocols can be found in Remington’s Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0236] The pharmaceutical composition can be administered alone or in combination with other treatments, either concomitantly or sequentially, depending on the condition to be treated.

Examples

[0237] The following are examples of specific embodiments for carrying out the present invention. These examples are provided for illustrative purposes only and are in no way intended to limit the scope of the present invention. Although efforts are made to ensure the accuracy of the numerical values used (e.g., amounts, temperatures, etc.), some experimental errors and deviations should of course be tolerated.

[0238] The practice of the present invention will employ conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, cell culture, adoptive cell transfer, and pharmacology within the skill of the art, unless otherwise indicated. Such techniques are fully explained in the literature. See, for example, 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).

[0239] Example 1: Rational design of the exclusive site II interface of G-CSF:G-CSFR(CRH) Wild-type (WT) G-CSF WT : G-CSFR WT The complex is a 2:2 heterodimer. G-CSF has two binding interfaces with the extracellular domain (ECD) of G-CSFR. The larger interface between G-CSF and the extracellular cytokine receptor homology (CRH) domain of G-CSFR is called Site II. The smaller interface between G-CSF and the N-terminal Ig-like (Ig) extracellular domain of G-CSFR is called Site III (see Figure 1).

[0240] Co-evolved, engineered (E) G-CSF E : G-CSFR ECytokine: To design receptor pairs, a 2:2 complex of WT G-CSF and the Ig-CRH extracellular domain of G-CSFR (Protein data Bank ID 2D9Q, Tamada et al. PNAS 2006) was separated into two different partial complexes consisting of G-CSF:G-CSFR(CRH) and G-CSF:G-CSFR(Ig), respectively, which include the site II and site III interfaces (see Figure 1). See Table 1 for the sequences.

[0241] Method Co-evolved exclusive G-CSF E :G-CSFR E To create mutant pairs (designs), a computational design workflow was adopted (see Figure 2). First, an exclusive design at site II was created.

[0242] G-CSF WT :G-CSFR(CRH) WT In silico structural analysis of the site II interface interaction of G-CSF:G-CSFR(CRH) showed that most molecular interactions, which account for a total attractive AMBER energy of 109.64 kcal / mol at site II, are carried out by charged residues (see Figure 3). Detailed examination revealed that most are electrostatic and hydrogen bond interactions, for example, between R167 of G-CSFR(CRH) and D112 / D109 of G-CSF, which account for 28% of the total attractive AMBER energy at site II. Another important electrostatic interaction exists between R141 of G-CSFR(CRH) and E122 / E123 of G-CSF (see Figure 4), which accounts for 21.4% of the total attractive AMBER energy at site II (see Figure 3). Similarly, the salt bridge between E19 of the cytokine and R288 of the receptor CRH domain accounts for 17.3%, and arginine is further stabilized by electrostatic and hydrogen bond interactions to D200 of the receptor CRH domain.

[0243] Each design at site II is between G-CSF E and G-CSFR(CRH) Econsists of mutant pairs. First, while creating the positive design at Site II, for example, through charge reversal by mutating the basic residue on the binding partner side to an acidic residue or vice versa, packing and hydrophobic interactions were maintained by additional mutations as necessary. Mutant pair G-CSF E :G-CSFR(CRH) E was packed using the mean field packing workflow of ZymeCAD™. The in-silico model of the designed Site II was visually inspected for structural integrity and evaluated using the ZymeCAD™ metric. Specifically, the G-CSF E mutant was designed to have an in-silico dAMBER binding affinity of less than 10 kcal / mol for the corresponding G-CSFR(CRH) E mutant (pair interaction). This metric compares the AMBER affinity, which is the sum of the Lennard Jones affinity and the electrostatic affinity, with the AMBER affinity of the WT:WT cytokine-receptor pair. Designs with a dAMBER_folding greater than 80 kcal / mol were excluded. The dAMBER_folding metric scores the total change in Lennard Jones binding folding and electrostatic folding upon mutation. Also, designs that obtained a score higher than 400 kcal / mol in dDDRW apostability were excluded. This metric represents the change in knowledge-based potential stability when the protein mutates from its apo form.

[0244] Next, the G-CSF E mutants of each design were packed by ZymeCAD™ into complexes with WT G-CSFR (and vice versa for G-CSFR E with G-CSF WT ), resulting in the following two complexes G-CSF WT :G-CSFR(CRH) E and G-CSF E :G-CSFR(CRH) WTUnder mispairing conditions when forming, the metrics of each positive design were evaluated. Using ZymeCAD (trademark), for mispaired orientations, the in-silico ddAMBER metric was calculated (ddAMBER_affinity_Awt_Bmut is the difference obtained by subtracting the AMBER affinity of the mispaired complex G-CSF WT :G-CSFR(CRH) E from the AMBER affinity of the paired and engineered complex, and ddAMBER_affinity_Amut_Bwt is the difference obtained by subtracting the AMBER affinity of the mispaired complex G-CSF E :G-CSFR(CRH) WT from the AMBER affinity of the paired and engineered complex. A design that minimizes the mispaired ddAMBER affinity metric was considered to be selective for its paired binding partner rather than for binding to the wild-type cytokine or receptor binding.

[0245] All site II designs were clustered, and the packing metrics of G-CSF E :G-CSFR(CRH) E 、G-CSF WT :G-CSFR(CRH) E 、G-CSF E :G-CSFR(CRH) WT were examined to evaluate the pairing strength (positive design) and selectivity for mispairing with WT G-CSF and G-CSFR(CRH) (negative design), and the designs were ranked.

[0246] Results The designs described in Table 2 are, in silico, G-CSF E :G-CSFR(CRH) EIt has the packing metric in ZymeCAD™ that is advantageous for pairing (see Table 3), and shows satisfactory interactions in silico, such as the presence of salt bridges, hydrogen bonds, and the absence of violent collisions (see Figure 5). These designs also showed high selectivity against mispairing with WT G-CSF or G-CSFR(CRH) (see Table 3).

[0247] Therefore, the Site II mutations of the same variant G-CSF and receptor designs shown in Table 2 are predicted to show preferential binding over wild-type G-CSFR and G-CSF, respectively.

[0248] (Table 3) Site II designs with the AMBER metric shown in kcal / mol from triplicate in silico mean-field packing using ZymeCAD™ TIFF0007708745000006.tif238155

[0249] Example 2: In vitro screening of site II design The selected Site II designs were screened in a pull-down experiment in the form of G-CSF E :G-CSFR(CRH) E for their ability to form Site II complexes when co-expressed in a baculovirus-based insect cell system. Also, by co-expressing each design of the G-CSF E mutant with G-CSFR(CRH) WT and vice versa by co-expressing each design of the G-CSFR(CRH) E mutant with GCSF WT it was evaluated whether the design could form a mispairing complex with the WT receptor or cytokine. Expression of G-CSF E alone was confirmed by single infection with the cytokine mutant.

[0250] Method Briefly, the pair of G-CSFR (CRH) variants corresponding to the Site II G-CSF design were cloned separately into an insect cell transfection vector. G-CSF WT (residues 1-173, Table 1) and the variants were cloned into a modified pAcGP67b transfer vector (Pharmingen) with an N-terminal secretion signal and a C-terminal TEV-cleavable Twin Strep tag with sequence TIFF0007708745000007.tif4136 in-frame. Among the receptor extracellular domains, only the CRH domain (residues 98-308, Table 1) with the Site II design variant was cloned into a modified pAcGP67b transfer vector with an N-terminal secretion signal and a TEV-cleavable hexahistidine (SEQ ID NO: 89) tag with sequence TIFF0007708745000008.tif4128 in-frame. All constructs were synthesized and codon-optimized for expression in insect cells (Genscript). The transfer vector DNA was prepared by Midi-prep (ThermoScientific, cat. K0481), endotoxin-free, with an A 260 / 280 absorbance ratio of 1.8 - 2.0. Recombinant virus production was achieved by co-transfecting recombinant, linearized baculovirus DNA and vector DNA into Spodoptera frugiperda 9 (Sf9) cells using the attachment method according to the manufacturer's (Expression Systems, California) instructions. Approximately 1 hour before transfection, 0.46×10 6 cells / ml -1Then, 2 mL of healthy logarithmic-phase Sf9 cells were seeded into each well of a 6-well tissue culture plate (Greiner, cat.657-160). The transfection mixture was prepared as follows: 100 μl of transfection medium (Expression Systems, California, cat.95-020-100) was placed into each of two sterile 1.5 ml Microfuge tubes A and B. To tube A, 0.4 μg of recombinant BestBac 2.0Δ v-cath / chiA linearized DNA (Expression Systems, California, cat.91-002) and 2 μg of vector DNA were added. To tube B, 1.2 μl of 5X Express 2 TR transfection reagent (Expres2ION, cat.S2-55A-001) was added. Solutions A and B were incubated at approximately 24 °C for 5 minutes and then combined and incubated for 30 minutes. After incubation, 800 μl of transfection medium was added to each transfection reaction to increase the volume to 1 ml. The old ESF921 medium was removed from the wells and replaced with 1 mL of the transfection mixture by dropping it gently without disturbing the cell monolayer. The plate(s) was / were gently rocked back and forth and side to side to evenly disperse the transfection mixture and incubated at 27 °C for 4 hours. After 4 hours, the transfection mixture was removed from the co-transfection plate(s), and 2 mL of fresh ESF921 insect cell culture medium (Expression Systems, California, cat.96-001-01) containing gentamicin at 10 μg / ml (cat.15750-060) was added by dropping it. To prevent evaporation, the plate was wrapped with Saran wrap, placed in a sterile plastic box, and incubated at 27 °C for 4 to 5 days. On the 4th or 5th day after transfection, the P1 supernatant was collected, clarified by centrifugation at 5000 rpm for 5 minutes, transferred to a new sterile tube, protected from light, and stored at 4 °C.

[0251] The recombinant P1 stock generated as described above was further amplified to obtain a high-titer, low-passage P2 stock for protein expression studies. In the following workflow, a P1 seed stock of the virus collected from co-transfection was used as an inoculum to generate 50 - 100 mL of the virus. 1.5x10 6 cells / ml -1 of logarithmic-phase Sf9 cells in 50 mL were seeded into a 250 mL shake flask (Fisher Scientific, cat. PBV 250), and 0.5 mL of the P1 virus stock was added. The cells were incubated at 27 °C with shaking at 135 rpm and observed for infection. On days 5 - 7 after infection, the P2 virus supernatant was collected and clarified by centrifugation at 4000 rpm for 10 minutes. To minimize titer reduction, 10% heat-inactivated FBS (VWR, cat. 97068-085) was added, and the P2 virus was stored at 4 °C in the dark.

[0252] The P2 virus stock was tested for protein expression on a small scale. Using the P2 virus, the G-CSF E mutant and the corresponding G-CSFR E mutant were co-infected into Trichoplusia ni (Tni) cells in a 12-well plate. The P2 stocks of G-CSF WT and GCSFR (CRH) WT were used to perform separate co-infections for each design mutant and for the G-CSF E mutant alone. For each reaction, 20 μl of the P2 virus was inoculated into 2 mL of healthy logarithmic-phase Tni at 2x10 6 cells / ml -1 . The plates were incubated at 27 °C with shaking at 135 rpm for approximately 70 hours. The supernatant was clarified by centrifugation at 5000 rpm for 3 minutes, and the secreted protein was analyzed for the G-CSF E mutant and G-CSF WTPull-down was performed in batch mode with streptactin-XT superflow (IBA Lifesciences, cat. 2-4010-010) via the upper Twin-Strep Tag (TST). Briefly, to each 1.8 mL of reaction supernatant, 0.2 mL of 10X HEPES buffered saline (HBS: 20 mM HEPES pH 8, 150 mM NaCl) was added with 20 μl of bed volume (b.v) of purified beads at 1X, and the reaction was incubated for 30 minutes with inversion mixing at 24 °C. Another 20 μl of b.v. of purified beads was added and the incubation was continued for another 30 minutes. The beads were pelleted by centrifugation at 2200 rpm for 3 minutes, the supernatant was removed and washed with 1X HBS buffer. The protein was eluted with 30 μl of BXT elution buffer (100 mM Tris-CL pH 8, 150 mM NaCl, 1 mM EDTA, 50 mM biotin (IBA Lifesciences, cat. 2-1042-025)), boiled in SDS-PAGE sample buffer and analyzed on a 12% Bolt Bis-Tris plus, 12-well gel (Thermo Fisher Scientific, cat. NW00122BOX) at 200 V for 30 minutes under reducing conditions.

[0253] Results Site II designs #6, 7, 8, 9, 15, 17, 30, 34, 35, 36 showed expression of G-CSF E alone, and G-CSF E sufficiently stable paired G-CSF pulled down via the upper Twin Strep tag E :G-CSFR E formed a complex (see Figure 6). For example, Site II design #6 after pull-down of the engineered complex showed, on SDS-PAGE, a band of approximately 22 kDa for its G-CSF E variant and a band of approximately 33 kDa for the co-expressed G-CSFR(CRH) E variant (see Figure 6).

[0254] Site II designs #8, 9, 15, and 34 are selective against mispairing with WT G-CSF and WT G-CSFR in co-expression assays (see Figure 7), because they were not pulled down by WT G-CSF (see lower panel of Figure 7) and the WT receptor was not pulled down by the G-CSF E variant (see upper panel of Figure 7). Site II G-CSFR E Designs 30 and 35 were selective against mispairing with WT G-CSF in co-expression assays because they were not pulled down by WT G-CSF (see lower panel of Figure 7), but the reciprocal G-CSF E design was not selective against mispairing with WT G-CSFR in co-expression assays because the WT G-CSFR was pulled down (see upper panel of Figure 7). Site II designs 6, 7, 17, and 36 pulled down the WT G-CSFR and were pulled down by WT G-CSF, and thus were not selective against mispairing with WT G-CSF or WT G-CSFR in co-expression assays (see Figure 7).

[0255] Site II receptor variants having a mutation at residue R288 did not express in the form of the G-CSFR (CRH) receptor chain lacking the Ig domain. Designs containing R288 were combined with site III designs on the Ig domain and evaluated for paired complex formation by SPR (see Example 6). Thus, several site II designs were identified that formed a sufficiently stable, paired G-CSF E :G-CSFR E complex that was also selective against mispairing with WT G-CSF and WT G-CSFR.

[0256] Thus, the various site II mutations of the same variant G-CSF and receptor designs shown in Table 2 show preferential binding to the wild-type G-CSFR and G-CSF, respectively.

[0257] Example 3: Rational design of the exclusive site III interface of G-CSF:G-CSFR(Ig) The binding activity effect of the site III receptor Ig domain that binds to G-CSF contributes to the formation of the G-CSF:G-CSFR(Ig-CRH) complex with a 2:2 heterodimer stoichiometry (see Figure 1). The selective design that exists only at site II with the WT site III interface is completely exclusive 2:2 G-CSF E :G-CSFR(Ig-CRH) E appears to be insufficient to create the complex. To prioritize the binding of paired co-evolutionary designs that are more selective than mispairing binding to the WT cytokine or receptor, the in silico design workflow described in Example 1 was applied to the site III interface to create a selective site III design (see Figure 2).

[0258] Method First, a structural analysis of the site III interface interaction was performed. The site III interface contributes 55.64 kcal / mol of AMBER energy to the G-CSF:G-CSFR complex and has a generally smaller interface area of 571.5 Å 2 compared to site II in the 692.6 Å 2 interface region. A detailed examination of the site III interface reveals fewer electrostatic and hydrogen bond interactions compared to the site II interface (see Figure 8). Important interactions at site III are, for example, salt bridges between E46 of G-CSF and R41 of the receptor Ig domain, and between R147 of G-CSF and E93 of the receptor Ig domain (see Figure 9). Both interactions account for 15.9% and 15.4% respectively of the total attractive AMBER energy of site III. Further interactions are made, for example, via Q87 of the receptor Ig domain, forming a hydrogen bond interaction with the side chain amide to the backbone of G-CSF site III, and having Lennard Jones interactions with side chains around the cytokine such as E46 and L49. This interaction accounts for 12.3% of the total attractive AMBER energy of site III.

[0259] Next, G-CSF EThe variant's co-evolved G-CSFR(Ig) E Positive designs were created in silico at Site III with favorable AMBER binding affinities for the variant (pair interaction). This was done, for example, by charge inversion or change of shape complementarity while maintaining Lennard Jones and hydrogen bond interactions. The variant pair G-CSF E :G-CSFR(Ig) E was packed with the mean field packing workflow of ZymeCAD™. The in silico models of the designed Site III were visually inspected for structural integrity and evaluated with the ZymeCAD™ metrics described in Example 1. Next, the G-CSF E variants of each design were packed with ZymeCAD™ with WT G-CSFR(Ig) (and its reverse G-CSFR(Ig) E with G-CSF WT ), and the metrics were evaluated under conditions of mispairing with the WT cytokine and receptor. As previously described for Site II in Example 1, for the Site III designs using ZymeCAD™, the ddAMBER affinity metric (G-CSF WT :G-CSFR(Ig) E G-CSF E :G-CSFR(Ig) WT ) was calculated (see Table 5).

[0260] The Site III designs were clustered and the packing metrics of all three in silico complexes G-CSF E :G-CSFR(Ig) E G-CSF WT :G-CSFR(Ig) E G-CSF E :G-CSFR(Ig) WT were considered in conjunction with visual inspection, and the strength of pairing and selectivity against mispairing with the WT were evaluated for ranking the designs.

[0261] Results The designs described in Table 4 are G-CSFE : G-CSFR(Ig) E has a ZymeCAD™ in-silico packing metric that is favorable for the pairing of E and has high selectivity against mispairing with WT G-CSF or G-CSFR(Ig). Thus, the various Site III mutations of the same variant G-CSF and receptor designs shown in Table 4 are predicted to exhibit preferential binding over wild-type G-CSFR and G-CSF, respectively.

[0262] (Table 5) Site III designs in AMBER metric in kcal / mol from triplicate in-silico mean field packing in ZymeCAD™ TIFF0007708745000009.tif142157

[0263] Example 4: Combinations of sites II and III for creating variant, co-evolved cytokine receptor switches Enable binding and signal transduction through engineered complexes of 2:2 heterodimers and have highly selective or completely abolished cross-reactivity with wild-type cytokines or receptors. A fully selective design pair of G-CSF E : G-CSF(Ig-CRH) E To develop E , the selected Site II and III designs of Examples 1 and 3 were combined (see Table 6) and tested in vitro.

[0264] Similar to combining the designs in Table 6, any other combination of the Site II design of Example 1 (Table 2) and the Site III design of Example 3 (Table 4) enables variant signal transduction for a fully selective G-CSF E : G-CSFR(Ig-CRH) E Combinations of designs were obtained. Combinatorial designs 401 and 402 were tested for their ability to form engineered G:CSF E : G-CSFR(Ig-CRH) E complexes and their ability to bind WT cytokines or receptors in the co-expression assay described in Example 2.

[0265] Method Co-expression assays with pull-down via the cytokine TST tag were performed as described in Example 2 above, except that the receptor construct included Ig and CRH domains (residues 2 - 308, Table 1) called G-CSFR(Ig-CRH).

[0266] Results Combinatorial designs 401 and 402 were completely selective in the co-expression assay in that the engineered cytokines pulled down their co-evolved, engineered receptors but not the WT receptor, and conversely, WT G-CSF did not pull down the engineered receptor (see Figure 10).

[0267] These results indicate that receptors containing variant G-CSF, as well as variant G-CSFR ECD designs combining selected site II and site III mutants, can specifically bind to their engineered cytokine-receptor pairs and do not bind to the wild-type receptor or cytokine.

[0268] Example 5: Generation of G-CSF and G-CSFR wild-type and mutants Recombinant proteins were expressed and purified from insect cells to generate wild-type and engineered cytokine and receptor variants and compare their biophysical properties.

[0269] Methods G-CSF E and G-CSF WT were cloned as described above. The preparative-scale production of recombinant proteins was performed as follows in 2 - 4 L of healthy logarithmic-phase Tni cells. 2×10 6 cells / ml -1To 800 mL of Tni, 20 μL of cytokine invariant P2 virus per 2 mL of cells was inoculated, and incubated at 27 °C for 70 hours while shaking at 135 rpm. After incubation, the cells were pelleted by centrifugation at 5500 rpm for 15 minutes, and the supernatant was filtered twice, first through a 1 μm A / E type glass fiber filter (PALL, cat.61631), and then through a 0.45 μm PVDF membrane filter (Sigma Aldrich, cat.HVLP04700). Protease inhibitor cocktail III (Sigma Aldrich, cat.539134) was added, the supernatant buffer was exchanged with HBS (20 mM HEPES pH8, 150 mM NaCl), and concentrated to 300 mL by tangential flow. The protein was purified in batch mode by 3 x 3 mL of b.v Streptactin-XT superflow, and incubated with stirring at 4 °C for 2 x 1 hour and 1 x overnight. Before elution, the resin was washed with 10 CV of HBS buffer. The protein was eluted with 4 x 5 mL of BXT elution buffer. The eluate was analyzed by nanodrop A280 and reduced SDS-PAGE, concentrated to about 2 mL, and the TST purification tag was cleaved by mixing and incubating with TEV at a TEV:protein ratio of 1:80 at 18 °C overnight. The cleaved protein was confirmed by SDS-PAGE (see Figure 11) before loading onto an SX75 16 / 600 or SX200 16 / 600 size exclusion column (GE Healthcare, cat.28-9893-33 or 28-9893-35) equilibrated with 20 mM BisTris pH6.5, 150 mM NaCl. Protein-containing fractions were analyzed by reduced SDS-PAGE, pooled, and the concentration was measured by nanodrop A280 measurement.

[0270] For protein purification of the receptor variant, wild type and G-CSFR(Ig-CRH) EThe variants were cloned as described above, and the receptor constructs for purification contained Ig domains in addition to the CRH domain (residues 3 - 308 of Uniprot ID Q99062, Table 1). Virus stocks were prepared and used for infection at a scale of 2 - 4 L as described above. The clarified supernatant was buffer-exchanged into Ni-NTA binding buffer (20 mM HEPES pH8, 1 M NaCl, 30 mM imidazole) and concentrated to 300 mM as described above. The protein was purified in batch binding mode by 3 x 3 mL of b.v Ni-NTA superflow and incubated with stirring at 4°C for 2 x 1 hour and 1 x overnight. The resin was washed with 10 CV of binding buffer before elution. The protein was eluted with 4 x 5 mL of Ni-NTA elution buffer (20 mM HEPES pH8, 1 M NaCl, 250 mM imidazole). The eluate was analyzed, buffer-exchanged into 20 mM Bis-Tris pH6.5, 150 mM NaCl, concentrated, and cleaved overnight as described above. The cleaved protein was subsequently loaded onto an SX75 16 / 600 or SX200 16 / 600 size exclusion column (GE Healthcare) equilibrated with 20 mM BisTris pH6.5, 150 mM NaCl (see Figure 12). Protein-containing fractions were analyzed by reducing SDS-PAGE, pooled, and the concentration was measured by nanodrop A280 measurement.

[0271] Results Wild type, G-CSF E , and G-CSFR E The purity of the variants after SEC was over 90% as judged by reducing SDS-PAGE (see Figures 11 and 12). The yields after SEC per liter of culture of G-CSF of designs 401 and 402 E were 2.7 mg and 1.6 mg, respectively. The yields after SEC per liter of production of G-CSFR of designs 401 and 402 E were 1.7 mg and 1.5 mg, respectively. WT G-CSF was purified with a yield after SEC of 2.1 mg per liter of culture, and WT G-CSFR was purified with a yield after SEC of 3.1 mg per liter of culture.

[0272] These results confirm that the methods used to purify the variant G-CSF and receptor were efficient in obtaining purified proteins for use in the analysis of biophysical properties in vitro.

[0273] Example 6: Determination of the affinity of the design for paired and mispaired binding partners by SPR To determine the affinity of the designed cytokine for the co-evolved receptor variant, the affinity of G-CSF WT for G-CSFR E was measured. Also, the affinity of G-CSF E for G-CSFR WT and the affinity of G-CSF WT for G-CSFR E were measured by SPR for a subset of the designs (mismatched).

[0274] Method The SPR binding assay was performed at 25 °C using a Biacore T200 instrument (GE Healthcare, Mississauga, ON, Canada) with PBS-T (PBS + 0.05% (v / v) Tween 20) running buffer. The CM5 Series S sensor chip, Biacore amine coupling kit (NHS, EDC, and 1 M ethanolamine), and 10 mM sodium acetate buffer were all purchased from GE Healthcare. The PBS running buffer containing 0.05% Tween 20 (PBS-T) was purchased from Teknova Inc. (Hollister, CA). The designs were evaluated in three different immobilization orientations.

[0275] G-CSF E To determine the binding affinity of G-CSF for G-CSFR E the G-CSFR E variant was captured by standard amine coupling as described by the manufacturer (GE LifeSciences). Briefly, immediately after EDC / NHS activation, G-CSFR in 10 mM NaOAc, pH 5.0 EA 5 μg / mL solution was injected at a flow rate of 5 μL / min until a receptor density of approximately 700 - 900 RU was reached. The remaining active groups were quenched by injecting 1 M ethanolamine hydrochloride - NaOH pH 8.5 at 10 μL / min for 420 seconds. Using single - cycle kinetics, corresponding G - CSF E mutants in a two - fold dilution series starting from 200 nM and a blank buffer control were sequentially injected at 25 μL / min for 300 seconds, with a dissociation phase of 1800 seconds, to obtain a series of sensorgrams referenced to the buffer blank. The same sample titrations were also performed in a reference cell where the variant was not captured. The chip was regenerated by 1 pulse of 10 mM glycine / HCl, pH 2.0 (30 μL / min for 30 seconds) in preparation for the next injection cycle.

[0276] G - CSF WT to G - CSFR E To evaluate the binding affinity to G - CSF E was captured on the chip at a density of approximately 700 - 900 RU as described above. Using single - cycle kinetics, each G - CSFR WT in a two - fold dilution series starting from 200 nM and a blank buffer control were sequentially injected at 25 μL / min for 300 seconds, with a total dissociation time of 1800 seconds, to obtain a series of sensorgrams referenced to the buffer blank. The same sample titrations were also performed in a reference cell where the variant was not captured and the chip was regenerated as described above.

[0277] G - CSF E to G - CSFR WT To evaluate the binding affinity to G - CSFR, recombinant G - CSFR WT purified as described in Example 5 was captured as described in this example. Using single - cycle kinetics, each G - CSF E in a two - fold dilution series starting from 200 nM and a blank buffer control were sequentially injected as described above. The same sample titrations were also performed in a reference cell where the variant was not captured. The G - CSFR WT surface was regenerated as described above.

[0278] As a control, the binding of WTG-CSF to WT G-CSFR(Ig-CRH) was evaluated in each experiment and used to calculate the fold change in K within each independent measurement. D It was used to calculate the fold change.

[0279] Using Biacore™ T200 evaluation software v3.0, double-reference sensorgrams from two or three repeated injections were analyzed and fitted to a 1:1 Langmuir binding model.

[0280] Results By fitting the association and dissociation phases of the curves, the kinetic-derived affinity constant (K D ) was obtained. The K of WT G-CSF for WT G-CSFR(Ig-CRH) D was in the range of 1.8 - 2.5E-9. When the kinetic parameters could not be fitted, an attempt was made to derive the steady-state affinity constant. In these cases, the KD fold change was calculated from the K D derived from the steady state of the WT G-CSF:WT G-CSFR(Ig-CRH) pair and is shown in Table 7.

[0281] Designs 9, 130, 134, 137, 307, 401, and 402 showed an affinity for co-evolved binding partners with a difference within two-fold of the WT:WT KD (see Table 7 and Figure 13).

[0282] The G-CSFR(Ig-CRH) E mutants of Designs 9, 30, and 34 showed more than 700-fold weaker affinity for WT G-CSF compared to WT G-CSFR(Ig-CRH). The G-CSFR(Ig-CRH) of Design #35 E had low selectivity for mispairing with the WT cytokine and the affinity for WT G-CSF decreased to approximately 1 / 19 of the WT:WT affinity. The G-CSFR(Ig-CRH) of Designs 130, 134, 401, 402, 300, 3003, 304, and 307 EThe variant was most selective against mispairing with WT G-CSF and showed no significant binding to WT G-CSF at titrated concentrations (see Table 8, Figure 13).

[0283] G-CSFs of Designs 124, 130, 401, 402, 300, 303, 304, and 307 E The variant showed no significant binding to WT G-CSFR (Ig-CRH) at titrated concentrations. G-CSFs of Designs 9, 30, and 34 E The variant showed at least about 20-fold weaker K D to WT G-CSFR (Ig-CRH) compared to WT:WT KD. G-CSF of Design #134 E showed an affinity about 500-fold weaker to WT G-CSFR (Ig-CRH) (see Table 9, Figure 13). G-CSFs of Designs 35 and 117 E The variant shows binding to WT G-CSFR (Ig-CRH) similar to WT:WT KD.

[0284] These results indicate that the selected variant G-CSF designs do not bind to the wild-type G-CSFR ECD or bind with a significantly reduced affinity to the wild-type G-CSFR ECD (at least about 20-fold weaker KD).

[0285] (Table 7) Corresponding G-CSFR (Ig-CRH) compared to the WT:WT binding affinity measured by SPR E G-CSF for the variant E Change in binding affinity (KD) of the variant TIFF0007708745000010.tif120128 ss represents the affinity constant derived from the steady state.

[0286] (Table 8) Change in binding affinity of WT G-CSF to Design G-CSFR (Ig-CRH) compared to the WT:WT binding affinity measured by SPR E to the corresponding G-CSFR (Ig-CRH) TIFF0007708745000011.tif113128 ss shows the affinity constant derived from the steady state.

[0287] (Table 9) G-CSF for wild-type G-CSFR (Ig-CRH) compared to WT:WT binding heteroaffinity measured by SPR E Changes in binding affinity TIFF0007708745000012.tif127128

[0288] Example 7: Designed G-CSF E Measurement of the thermal stability of mutants G-CSF compared to WT cytokine and receptor E and G-CSFR E To measure the thermal stability of the G-CSF and G-CSFR mutants, differential scanning calorimetry (DSC) was performed.

[0289] Method The thermal stability of the variants was evaluated as follows by differential scanning calorimetry (DSC): 950 μL of a purified sample at a concentration of 1–2 mg / mL was used for DSC analysis using Nano DSC (TA instruments, New Castle, DE). At the start of each analysis, a buffer blank was injected for baseline stabilization. Each sample was scanned from 25 to 95 °C at a rate of 60 °C / h under a nitrogen pressure of 60 psi. The resulting thermograms were referenced and analyzed using NanoAnalyze software to measure the melting temperature (Tm), an indicator of thermal stability.

[0290] Results The thermal stability of the engineered variants is reported as the difference in the most prominent transition (highest enthalpy) between the engineered molecule and the wild-type molecule of equivalent size, measured under the same conditions and experimental setup. The measured Tm of WT GCSF varied between 52.2 and 55.4 °C among independent experiments, and the Tm of WT G-CSFR was 50.5 °C. G-CSF tested except for Designs #15 and 34 EThe variants showed a Tm that differed by less than 5 °C from that of WT G-CSF (see Table 10 and Figure 14). All receptor variants tested showed the same thermal stability as the WT receptor (see Table 10 and Figure 14).

[0291] These results indicate that variant G-CSF and receptors with variant G-CSFR ECD designs have similar thermal stability to wild-type G-CSF and G-CSFR, and that site II and / or site III mutations do not impair the thermal stability of either G-CSF or G-CSFR ECD.

[0292] (Table 10) Changes in melting temperature (Tm) of designed cytokines and receptor variants compared to wild type measured by DSC TIFF0007708745000013.tif31128TIFF0007708745000014.tif210103 * Measured in 150 mM NaCl, 20 mM BisTris pH 6.5

[0293] Example 8: G-CSF by UPLC-SEC E Measurement of the monodispersity of mutants G-CSF compared to WT G-CSF E To measure the monodispersity of the variants, the variants were analyzed by UPLC-SEC.

[0294] Method For the SEC-purified protein sample, an Acquity BEH125 SEC column (4.6x150mm, stainless steel, 1.7μm particles) (Waters LTD, Mississauga, ON) was set at 30 °C and attached to an Agilent Technologies 1260 infinity II system equipped with a PDA detector to perform UPLC-SEC. The analysis time was composed of a 7-minute runtime at a flow rate of 0.4 mL / min using a running buffer of 150 mM NaCl, 20 mM HEPES pH 8.0 or 150 mM NaCl, 20 mM BisTris pH 6.5. Elution was monitored by UV absorbance in the range of 210 - 500 nm, and the chromatogram was extracted at 280 nm. Peak integration was performed using OpenLAB™ CDS ChemStation™ software.

[0295] Results WT G-CSF and G-CSF of Designs 34, 35, and 130 E The variants were 100% monodisperse (see Table 11). Variants 8, 9, 15, 117, 135 showed lower monodispersity of 65.3 - 79.5%. The Design #134 cytokine showed 57.3% monodispersity at pH 8.0 but improved to 86.6% monodispersity at pH 6.5. The improvement in monodispersity at a lower pH of the mobile phase may be due to a shift in pI (e.g., from the calculated pI 5.41 in WT G-CSF to the calculated pI 8.35 in Design #134 G-CSF). E to the calculated pI 8.35 in Design #134 G-CSF).

[0296] These results show that some of the variant G-CSF designs have the same 100% monodispersity as wild-type G-CSF, suggesting that a subset of Site II and / or Site III variants do not impair the monodispersity of G-CSF, while other variant G-CSF designs show reduced monodispersity, which was shown to increase at low pH.

[0297] (Table 11) G-CSF measured by UPLC-SEC EMonodispersity of design variants TIFF0007708745000015.tif85128*150 mM of NaCl, in BisTris pH 6.5

[0298] Example 9: Construction of a chimeric G-CSF receptor with an intracellular IL-2 receptor signaling domain Design G-CSF E Whether cytokine variants can signal through the design G-CSFR (Ig-CRH) E receptor variant and cause immune cell proliferation, a single-chain chimeric G-CSF receptor (G-CSFR WT -ICD gp130-IL-2Rβ ) was constructed using the G-CSFR ECD fused to the gp130 transmembrane (TM) domain and intracellular signaling domain (ICD), as well as the IL-2Rβ intracellular signaling domain. Also, a chimeric G-CSFR consisting of two subunits designed to co-express as a heterodimeric receptor was utilized: 1) G-CSFR WT -ICD IL-2Rβ subunit consists of the G-CSFR ECD fused to the IL-2Rβ TM and ICD, and 2) G-CSFR WT -ICD γc subunit consists of the G-CSFR ECD fused to the common γ chain (γc, IL-2Rγ2Rγ) TM and ICD.

[0299] Method A single-chain chimeric receptor construct was designed to contain the G-CSFR signal peptide and ECD, then the gp130 TM and partial ICD, and the IL-2Rβ partial ICD (Table 12). The heterodimeric chimeric receptor constructs were designed to contain: 1) the G-CSFR signal peptide and ECD, then the IL-2Rβ TM and ICD (Table 13); and 2) the G-CSFR signal peptide and ECD, then the γc TM and ICD (Table 14). The chimeric receptor constructs were cloned into a lentiviral transfer plasmid, and the construct sequences were confirmed by Sanger sequencing. The transfer plasmid and the lentiviral packaging plasmids (psPAX2, pVSVG) were co-transfected into the lentiviral packaging cell line HEK293T / 17 cells (ATCC) as follows: The cells were plated overnight in DMEM containing 10% fetal bovine serum and penicillin / streptomycin and the medium was changed 2 - 4 hours prior to transfection. Plasmid DNA and water were mixed in a polypropylene tube and CaCl2 (0.25 M) was added dropwise. After incubating for 2 - 5 minutes, 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 onto the cells and incubated overnight at 37°C and 5% CO2. The next day, the HEK293T / 17 medium was changed and the cells were incubated for an additional 24 hours. Early the next morning, the cell supernatant was collected from the plate, gently centrifuged to remove debris, and filtered through a 0.45 μm filter. The supernatant was spun at 25,000 rpm for 90 minutes using the SW-32Ti rotor of a Beckman Optima L-XP ultracentrifuge. The supernatant was removed and the pellet was resuspended in an appropriate volume of Opti-MEM medium. The viral titer was measured by adding serial dilutions of the virus to BAF3 cells (RPMI for growth containing 10% fetal bovine serum, penicillin, streptomycin, and 100 IU / ml of hIL-2).Forty-eight to seventy-two hours after transduction, cells were incubated with anti-human G-CSFR APC-conjugated antibody (1:50 dilution) and eBioscience™ Fixable Viability Dye eFluor™ 450 (1:1000 dilution) for 15 minutes at 4°C, washed, and analyzed on a Cytek Aurora or BD FACS Calibur flow cytometer. Using the push force titer measured by this method, the 32D-IL-2Rβ cell line (grown in RPMI containing 10% fetal bovine serum, penicillin, streptomycin, and 300 IU / ml of hIL-2) was transduced with lentiviral supernatant encoding the chimeric receptor construct at an MOI of 0.5. Transduction was performed by adding an appropriate amount of viral supernatant to the cells, incubating for 24 hours, and replacing the cell medium. Three to four days after transduction, the expression of human G-CSFR was confirmed by flow cytometry as described above. Before performing the BrdU assay, cells were treated with G-CSF. WT and grown therein for approximately 14 to 28 days.

[0300] As described above, G-CSF WT The 32D-IL-2Rβ cells grown therein were washed three times with PBS, and the relevant assay site cytokines (cytokine-free, hIL-2 (300 IU / ml), G-CSF WT (30 ng / ml) or G-CSF E (30 ng / ml) and replated in fresh medium for 48 hours. The BrdU assay procedure was performed according to the instructions of the BD Pharmingen™ APC BrdU Flow Kit (557892), with the following additional steps: Cells were co-incubated with BrdU and eBioscience™ Fixable Viability Dye eFluor™ 450 (1:5000) for 30 minutes. Analysis flow cytometry was performed using a Cytek Aurora instrument.

[0301] Results In the BrdU assay, the single-chain chimeric receptor construct G-CSFR WT -ICD gp130-IL-2Rβor the heterodimeric receptor construct G-CSFR WT -ICD IL-2Rβ +G-CSFR WT -ICD γc Cells transfected with WT (30 ng / ml) showed equivalent or superior proliferation compared to hIL-2 (300 IU / ml) in response to G-CSF. The cells did not proliferate in the absence of cytokine (see Figure 15).

[0302] These results indicate that upon stimulation with G-CSF, single-chain and heterodimeric chimeric receptor constructs can be activated to induce cell proliferation.

[0303] Example 10: G-CSFR of design 137 investigated by BrdU E -ICD IL-2 Transduced with wild-type or designed G-CSF 137 Proliferation of 32D-IL-2Rβ cells treated with The combination design of Sites II / III that was sufficiently selective as determined by SPR or co-expression assays was tested in vitro for its ability to induce the proliferation of 32D-IL-2Rβ cells.

[0304] Method The point mutations of Design 137 were introduced into the constructs described in Tables 12-14. Cloning and expression of the G-CSFR 137 -ICD gp130-IL-2Rβ (homodimer) or G-CSFR 137 -ICD IL-2Rβ +G-CSFR 137 -ICD γc (heterodimer) constructs followed the same procedure as above. Before performing the BrdU assay, the cells were grown in G-CSF 137 for approximately 14-28 days.

[0305] G-CSF 137 32D-IL-2Rβ cells grown in G-CSF were assayed for BrdU using the above procedure in the presence of G-CSF 137 (30 ng / ml), G-CSF WTAssays were performed for proliferation in the presence of G-CSF (30 ng / ml), hIL-2 (300 IU / ml), or no cytokine.

[0306] Results In the BrdU assay, cells transduced with the single-chain chimeric receptor construct G-CSFR 137 -ICD gp130-IL-2Rβ or the heterodimeric receptor construct G-CSFR 137 -ICD IL-2Rβ +G-CSFR 137 -ICD γc showed equal or superior proliferation compared to hIL-2 (300 IU / ml) in the presence of G-CSF 137 (30 ng / ml). Cells did not proliferate in the absence of cytokine and showed inferior proliferation in the presence of G-CSF WT (30 ng / ml) (see Figure 16).

[0307] These results indicate that variant G-CSF specifically activates engineered receptors; conversely, engineered receptors are activated by variant G-CSF but are significantly less activated than wild-type G-CSF. Thus, variant G-CSF can specifically activate chimeric receptors by the variant G-CSFR ECD and specifically induce the proliferation of cells expressing the chimeric receptors.

[0308] Example 11: Wild-type G-CSFR-ICD investigated by BrdU IL-2 Transduced with wild-type or designed G-CSF E Proliferation of 32D-IL-2Rβ cells treated with Subsequently, the ability of the Site II / III combinatorial designs that were able to restore paired signaling in 32D-IL-2R2Rβ cells to induce the proliferation of 32D-IL-2R2Rβ cells transduced with the single-chain chimeric receptor construct G-CSFR WT -ICD gp130-IL-2Rβ or the heterodimeric receptor construct G-CSFR WT -ICD IL-2Rβ +G-CSFR WT -ICD γc was tested.

[0309] Method G-CSFR WT -ICD gp130-IL-2Rβ (homodimer) or G-CSFR WT -ICD IL-2Rβ +G-CSFR WT -ICD γc (heterodimer) constructs were cloned and expressed according to the same procedure as above. Before performing the BrdU assay, the cells were grown in G-CSF WT for approximately 14 to 28 days.

[0310] G-CSF WT 32D-IL-2Rβ cells grown in G-CSF were assayed for growth in G-CSF 137 (30 ng / ml), G-CSF WT (30 ng / ml), hIL-2 (300 IU / ml), or in the absence of cytokines.

[0311] Results In the BrdU assay, cells transduced with the single-chain chimeric receptor construct G-CSFR WT -ICD gp130-IL-2Rβ or the heterodimeric receptor construct G-CSFR WT -ICD IL-2Rβ and G-CSFR WT -ICD γc showed inferior growth in G-CSF 137 (30 ng / ml) compared to G-CSF WT (30 ng / ml). The cells did not grow in the absence of cytokines (see Figure 17).

[0312] These results indicate that the variant G-CSF does not efficiently bind to the wild-type G-CSFR, that the variant G-CSF specifically activates the engineered receptor but not the wild-type G-CSFR, and that it induces cell proliferation.

[0313] Example 12: WT or G-CSFR of design 137 analyzed by Western blot E -ICDIL-2 Transduced with wild-type or designed G-CSF 137 Signal transduction of 32D-IL-2Rβ cells treated with Proliferation signaling can be restored through the engineered cytokine receptor complex of 32D-IL-2Rβ cells, and G-CSF WT The combination design of Sites II / III, which did not significantly signal through the binding of WT-GCSFR-ICD-IL2, was also evaluated by Western blot for its ability to activate downstream signaling molecules in response to G-CSF WT or G-CSF 137 The ability to activate downstream signaling molecules in response to G-CSF was evaluated by Western blot.

[0314] Method G-CSFR WT -ICD gp130-IL-2Rβ (homodimer) or G-CSFR WT -ICD IL-2Rβ +G-CSFR WT -ICD γc (heterodimer) constructs were cloned and expressed according to the same procedure as above. Before performing the Western blot assay, the cells were grown in G-CSF 137 or G-CSF WT for approximately 14 - 28 days. Non-transduced cells were maintained in IL-2.

[0315] To perform Western blot, the cells were washed three times with PBS and incubated in cytokine-free medium for 16 - 20 hours. The cells were incubated without cytokine, with IL-2 (300 IU / ml), G-CSF 137 (30 ng / ml), or G-CSF WT(30 ng / ml), and stimulated at 37 °C for 20 minutes. The cells were washed once with a washing 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 mini tablets (A32961). The cells were lysed on ice for 10 minutes in the above washing buffer supplemented with 0.2% Igepal CA630 (Sigma). After centrifugation at 13,000 rpm for 10 minutes at 4 °C, the supernatant (cytoplasmic fraction) was collected. The pellet was dissolved and resuspended in the above washing buffer supplemented with 0.42 M NaCl and 20% glycerol. The cells were lysed on ice for 30 minutes with frequent vortexing and then centrifuged at 13,000 rpm for 20 minutes at 4 °C, and the nuclear fraction (supernatant) was collected. The cytoplasmic and nuclear fractions were reduced for 10 minutes (70 °C) and electrophoresed on a NuPAGE™ 4–12% Bis-Tris protein gel. The gel was transferred to a nitrocellulose membrane (20 V for 60 minutes in a Trans-Blot® SD semi-dry transfer cell), dried, and blocked with Odyssey® blocking buffer (927-50000) in TBS for 1 hour. The blot was incubated overnight at 4 °C with primary antibodies (1:1,000) in Odyssey® blocking buffer in TBS containing 0.1% Tween 20. The primary antibodies used were obtained from Cell Signaling Technologies: 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-Stat3 (Tyr705) (D3A7) XP® rabbit mAb #9145, Phospho-Stat5 (Tyr694) (C11C5) rabbit mAb #9359, and histone H3 (96C10) mouse mAb #3638.The blot was washed three times with TBS containing 0.1% Tween 20 and incubated with the secondary antibody (1:10,000) in TBS buffer containing 0.1% Tween 20 for 30 - 60 minutes at room temperature. The secondary antibodies were obtained from Cell Signaling Technologies: anti-mouse IgG (H+L) (DyLight™ 800 4X PEG conjugate) #5257 and anti-rabbit IgG (H+L) (DyLight™ 800 4X PEG conjugate) #5151. The blot was washed and exposed using a LI-COR Odyssey imager.

[0316] Results In non-transformed 32D-IL-2Rβ cells, activated IL-2R-related signaling molecules in response to stimulation with IL-2 alone were detected. G-CSFR WT -ICD gp130-IL-2Rβ Or G-CSFR WT -ICD IL-2Rβ +G-CSFR WT -ICD γc In 32D-IL-2Rβ cells expressing any of WT these, a similar pattern of activated signaling molecules in response to IL-2 or G-CSF was observed. G-CSFR WT -ICD gp130-IL-2Rβ Expressing or G-CSFR WT -ICD IL-2Rβ +G-CSFR WT -ICD γc Activation of IL-2R-related signaling molecules in response to G-CSF stimulation was not observed in 32D-IL-2Rβ cells expressing 137 G-CSFR 137 -ICD gp130-IL-2Rβ Or G-CSFR 137 -ICD IL-2Rβ +G-CSFR 137 -ICD γc In 32D-IL-2Rβ cells expressing any of 137 these, a similar pattern of activated signaling molecules in response to IL-2 or G-CSF was observed. G-CSFR 137 -ICD gp130-IL-2Rβexpressing or G-CSFR 137 -ICD IL-2Rβ +G-CSFR 137 -ICD γc G-CSF of 32D-IL-2Rβ cells expressing WT Activation of IL-2R2R2R-related signaling molecules in response to G-CSF stimulation was not observed.

[0317] These results demonstrate that variant G-CSF can activate a chimeric receptor expressing the variant G-CSFR ECD and induce the characteristics of native cytokine signaling in cells expressing the chimeric receptor.

[0318] Methods of Examples 13 to 30 Primary cells and cell lines: The lentiviral packaging cell line HEK293T / 17 (ATCC) was cultured in DMEM containing 10% fetal bovine serum and penicillin / streptomycin. BAF3-IL-2Rβ cells were pre-generated by stable transfection of the human IL-2Rβ subunit into the BAF3 cell line 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 pre-generated by stable transfection of the human IL-2Rβ subunit into the 32D cell line and grown in RPMI-1640 containing 10% fetal bovine serum, penicillin, streptomycin, and 300 IU / ml of hIL-2, or other indicated cytokines. Human PBMC-derived T cells (Hemacare) were grown in TexMACS™ medium (Milenyi Biotec, 130-097-196) containing 3% human AB serum (Sigma-Aldrich, H4522) and 300 IU / ml of hIL-2, or other indicated cytokines. Human tumor-associated lymphocytes (TAL) were generated by culturing primary ascites samples for 14 days in T cell medium (the following 50:50 mixture): 1) RPMI-1640 containing 10% fetal bovine serum, 50 μM β-mercaptoethanol, 10 mM HEPES, 2 mM L-glutamine, penicillin, 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, TAL were cultured in T cell medium containing 300 IU / ml of hIL-2 or other indicated cytokines. 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).

[0319] 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 the lentiviral packaging plasmid were co-transfected into HEK293T / 17 cells using the calcium phosphate transfection method as follows: The cells were plated overnight and the medium was changed 2 - 4 hours before transfection. Plasmid DNA and water were mixed in a polypropylene tube, and CaCl2 (0.25 M) was added dropwise. After incubating for 2 - 5 minutes, the DNA was precipitated by mixing with an equal volume of 2x HEPES-buffered saline (0.28 M NaCl, 1.5 mM Na2HPO4, 0.1 M HEPES). The precipitated DNA mixture was added onto the cells and incubated overnight at 37 °C with 5% CO2. The next day, the HEK293T / 17 medium was changed and the cells were incubated for an additional 24 hours. The following morning, the cell supernatant was collected from the plate, gently centrifuged to remove debris, and the supernatant was filtered through a 0.45 micron filter. The supernatant was spun at 25,000 rpm for 90 minutes using the SW-32Ti rotor of a Beckman Optima L-XP ultracentrifuge. The supernatant was removed and the pellet was resuspended in an appropriate amount of Opti-MEM medium. The virus titer was determined by adding serial dilutions of the virus to BAF3-IL-2Rβ cells. 48 - 72 hours after transduction, the 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 measured by this method, the 32D-IL-2Rβ cell line was transduced with the lentiviral supernatant encoding the chimeric receptor construct at a multiplicity of infection (MOI) of 0.5. Transduction was performed by adding an appropriate amount of virus supernatant to the cells, incubating for 24 hours, and then changing the medium.Three to four days after transfection, the expression of human G-CSFR was measured by flow cytometry as described above.

[0320] Lentiviral transfection of human primary T cells: For transfection of PBMC-derived T cells and TAL, cells were thawed and plated according to the manufacturer's guidelines in the presence of Human T Cell TransAct™ (Miltenyi Biotec, 130-111-160). Twenty-four hours after activation, lentiviral supernatant was added at an MOI of 0.125 - 0.5. Forty-eight hours after activation, the cells were split into fresh medium to remove residual virus and activation reagent. Two to four days after transfection, the transfection efficiency was measured by flow cytometry as described above. In experiments measuring the transfection efficiency of CD4+ and CD8+ fractions separately, 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) were used together with Fixable Viability Dye eFluor™ 450 (1:1000).

[0321] Human T Cell and 32D-IL-2Rβ Proliferation Assay: Human primary T cells or 32-IL-2Rβ cells expressing the indicated chimeric receptor constructs generated above were washed three times in PBS and replated in fresh medium or, as indicated, their medium was gradually exchanged. Complete medium was exchanged to contain either wild-type human G-CSF (self-generated or NEUPOGEN®, Amgen Canada), mutant G-CSF (self-generated), hIL-2, or no cytokine. Every 3 - 5 days, cell viability and density were measured by trypan blue exclusion and the fold expansion relative to the starting cell number was calculated. Expression of G-CSFR was evaluated by flow cytometry as described above.

[0322] CD4+ and CD8+ Human TAL Proliferation Assay:: To examine the proliferation of the CD4+ and CD8+ fractions of TAL, ex vivo ascites samples were thawed and the CD4+ and CD8+ fractions were enriched using the Human CD4+ T Cell Isolation Kit (Miltenyi Biotec, 130 - 096 - 533) and the Human CD8+ T Cell Isolation Kit (Miltenyi Biotec, 130 - 096 - 495), respectively. After proliferation in cytokine-containing medium, the immunophenotype of the cells was evaluated 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).

[0323] Primary human T cell immunophenotype analysis assay: After proliferation in cytokine-containing medium, the immunophenotype of T cells was evaluated by flow cytometry 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 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), CD95 (1:33, PE-cyanine 7 conjugate, eBioscience™, 25-0959-42) together with Fixable Viability Dye eFluor™ 450 or 5106 (1:1000).

[0324] Retroviral transduction: The pMIG transfer plasmid (plasmid #9044, Addgene) was modified by restriction endonuclease cloning to remove IRES-GFP (from BglII to PacI site) and an annealing primer encoding a custom multiple cloning site was introduced. 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. Twenty-four hours after transfection, the medium was replaced with 5 ml of fresh complete medium. Forty-eight hours after transfection, the cell supernatant was collected 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.

[0325] Forty-eight hours before harvesting the retrovirus supernatant, 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 harvesting the retrovirus supernatant, C57Bl / 6J mice (self-generated) were euthanized under an approved animal experiment protocol administered by the University of Victoria Animal Experiment Committee. Spleens were harvested and mouse T cells were isolated as follows: Spleens were manually removed and filtered through a 100-micron filter. Red blood cells were lysed by incubating in ACK lysis buffer (Gibco, A1049201) for 5 minutes at room temperature and then washed once with serum-containing medium. CD8a-positive cells or Pan-T cells were isolated using specific bead-based separation kits (Miltenyi Biotec, 130-104-075 or 130-095-130, respectively). Cells were added to plates coated with anti-CD3-antibody and anti-CD28-antibody or 300 IU / mL of human IL-2 (Proleukin) in mouse T cell growth medium (RPMI-1640 containing 10% FBS, penicillin / streptomycin, 0.05 mM β-mercaptoethanol, and 2 ng / ml of mouse IL-2 (Peprotech, 212-12)) and incubated at 37°C, 5% CO2 for 24 hours. On the day of transduction, approximately half of the medium was replaced with the retrovirus supernatant generated above. Using this retrovirus supernatant, the cells were spinfected at 1000 g for 90 minutes at 30°C. The plates were returned to the incubator for 0 - 4 hours, and then approximately half of the medium was replaced with fresh T cell growth medium. As described above, retroviral transduction was repeated 24 hours later for a total of two transductions. Twenty-four hours after the last transduction, the T cells were split into 6-well plates and removed from antibody stimulation.

[0326] Forty-eight to seventy-two hours after transduction, transduction efficiency was evaluated by flow cytometry to detect 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.

[0327] 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 cytokines: cytokine-free, hIL-2 (300 IU / ml), wild-type or engineered G-CSF (concentrations indicated in individual experiments). The BrdU assay procedure was performed according to the instructions of the BD Pharmingen™ APC BrdU Flow Kit (BD Biosciences, 557892), with the following additional steps: 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 evaluate the proliferation of mouse T cells expressing the chimeric receptor, additional staining with human G-CSFR (1:20 dilution), CD4 (1:50 dilution), and CD8 (1:50 dilution) was performed on ice for 15 minutes before fixation.

[0328] 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 left standing for 16 - 20 hours in cytokine-free medium. Cells were treated with cytokine-free, IL-2 (300 IU / ml), wild-type G-CSF (concentrations indicated in individual experiments), or G-CSF 137(30 ng / ml) was used to stimulate at 37 °C for 20 minutes. 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 mini tablets (A32961). The cells were lysed in the above washing buffer, and 0.2% NP-40 (Sigma) was added on ice for 10 minutes. 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 washing buffer supplemented with 0.42 M NaCl and 20% glycerol. The nuclei were incubated for 30 minutes on ice with frequent vortexing, centrifuged at 13,000 rpm at 4 °C for 20 minutes, and then the supernatant (nuclear fraction) was collected. The cytoplasmic fraction and nuclear fraction were reduced for 10 minutes (70 °C) and electrophoresed on a NuPAGE™ 4–12% Bis-Tris protein gel. The gel was transferred to a nitrocellulose membrane (20 V for 60 minutes in a Trans-Blot® SD semi-dry transfer cell), dried, and blocked with Odyssey® blocking buffer (927-50000) in TBS for 1 hour. The blot was incubated overnight at 4 °C with a primary antibody (1:1,000) in Odyssey® blocking buffer in TBS containing 0.1% Tween 20.The 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. The 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 minutes at room temperature. The 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. The blots were washed and exposed with a LI-COR Odyssey imager.

[0329] Flow cytometry for detecting phosphorylated proteins: The human primary T cells, 32D-IL-2Rβ cells, or mouse primary T cells generated as described above were washed three times in PBS and allowed to stand for 16 - 20 hours in cytokine-free medium. The cells were stimulated at 37°C for 20 minutes without cytokines, with IL-2 (300 IU / ml), or with wild-type G-CSF (100 ng / ml) 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. The cells were pelleted and fixed with BD Phosflow™ Fixation Buffer I (BD Biosciences, 557870) for 15 minutes at room temperature. After washing the cells, they were permeabilized with BD Phosflow™ Perm Buffer III (BD Biosciences, 558050) for 15 minutes on ice. The cells were washed twice and resuspended in a buffer containing 20 μl of BD Phosflow™ PE Mouse anti-Stat3 (pY705) (BD Biosciences, 612569) or PE Mouse IgG2a, κ Isotype Control (BD Biosciences, 558595). The cells were washed and flow cytometry was performed using a Cytek Aurora instrument.

[0330] Example 13: Proliferation of human T cells expressing G2R-1, G-CSFR / IL-2RΒ subunit only, MYC-tagged G-CSFR / γ-C subunit only, or full-length G-CSFR PBMC-derived T cells or tumor-associated lymphocytes (TAL) were transduced with a lentivirus encoding the chimeric receptor construct shown in Figure 1. After washing the cells, they were replated with the indicated cytokines. The 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 assisted in detection by flow cytometry and did 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 PMBC-derived T cells and TAL expressing the G2R-1 chimeric cytokine receptor (Figure 22). Note that the transduction efficiency of the lentivirus was less than 100% such that less than 100% of the T cells expressed the indicated chimeric cytokine receptor. This is thought to be the reason for the lower proliferation rate mediated by G2R-1 compared to IL-2. Similarly, an increase in proliferation was observed in 32D-IL-2Rβ cells (which stably express the human IL-2Rβ subunit) expressing the G-CSFR chimeric receptor subunits G2R-1 and G2R-2 and stimulated with G-CSF (Figure 21). 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 seen in 32D-IL-2Rβ cells expressing only the G / γc chimeric receptor subunit (Figure 2).

[0331] These results showed that G-CSF can stimulate the proliferation and survival of PMBC-derived T cells and TAL 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.

[0332] Example 14: G-CSFR ECD is expressed on the surface of cells transduced with G / IL-2RΒ, G2R-1, and G2R-2 Flow cytometry was performed on 32D-IL-2Rβ cell lines, PBMC-derived human T cells, and human tumor-associated lymphocytes after transduction with a lentiviral vector encoding the G2R-2 chimeric cytokine receptor (shown schematically in Figures 23 and 25) to measure whether cells expressed G-CSFR ECD on the cell surface. G-CSFR-positive cells were detected in all transduced cell types (Figure 26). In another experiment, 32D-IL-2Rβ cells expressing G / IL-2Rβ, G2R-1, and G2R-2 chimeric receptors were positive for G-CSFR ECD by flow cytometry (lower panels of Figures 21B - D).

[0333] These results indicate that the G / IL-2Rβ, G2R-1, and G2R-2 chimeric receptors are expressed on the cell surface.

[0334] Example 15: Proliferation of cells expressing G2R-2 compared to non-transduced cells T cells derived from human PBMCs and human tumor-associated lymphocytes were transduced with the G2R-2 receptor construct (Figures 4 and 6) using lentivirus, washed, and replated with the indicated cytokines. In some experiments, T cells were also periodically reactivated by stimulation with TransAct. Viable cells were counted every 3 - 4 days. Proliferation of PMBC-derived T cells (Figure 27A) and tumor-associated lymphocytes (two independent experiments in Figures 27B, C) was observed after stimulation with G-CSF (100 ng / ml) in cells expressing the G2R-2 chimeric receptor, but not in non-transduced cells.

[0335] These results indicate that activation of the G-CSF-inducible G2R-2 chimeric receptor is sufficient to induce the proliferation and survival of immune cells.

[0336] Example 16: Proliferation and immune phenotype of CD4 or CD8 selected human tumor - associated lymphocytes expressing G2R - 2 compared to non - transfected cells CD4-selected and CD8-selected human T cells were either transduced with a lentiviral vector encoding G2R-2 (Figure 25) or left untransduced as indicated. Cells were washed and replated with the indicated cytokines and counted every 3 - 4 days. Proliferation of CD4 or CD8-selected TALs 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 28 and 29). In Figure 28, each line represents the results from one of five patient samples.

[0337] Immunophenotypic analysis by flow cytometry revealed that T cells cultured with G-CSF or IL-2 retained their CD4+ or CD8+ characteristics (Figure 30A) and lacked the NK cell phenotype (CD3-CD56+) (Figure 30A) under these culture conditions and exhibited the CD45RA-CCR7-T effector memory (T EM ) phenotype (Figure 30B).

[0338] To confirm that stimulation with G-CSF promotes the progression of the cell cycle of T cells expressing G2R-2, a BrdU assay was performed (Figure 31). T cells were selected by culture with IL-2 or G-CSF as indicated prior to the assay. Both tumor-infiltrating lymphocytes (Figure 31A) and PBMC-derived T cells (Figure 31B) were evaluated.

[0339] These results indicate that G-CSF can selectively activate the progression of the cell cycle and long-term proliferation of primary human TALs through the activation of the chimeric cytokine receptor G2R-2. These results also indicate that activation of the G2R-2 chimeric receptor by homodimer formation is sufficient to activate cytokine-like signaling and proliferation in TALs. Furthermore, TALs expressing G2R-2 maintain cytokine dependence such that they undergo cell death upon withdrawal of G-CSF, similar to the response to withdrawal of IL-2. TALs cultured with G-CSF maintain an immunophenotype similar to that of TALs cultured with IL-2.

[0340] Example 17: Proliferation of primary mouse T cells expressing G2R - 2 in response to G - CSF. To evaluate the proliferation of primary mouse T cells expressing G2R-2 or single-chain G / IL-2Rβ (a component of G2R-1) and mock-transduced cells upon stimulation with G-CSF, a BrdU incorporation assay was performed. All cells were grown with IL-2 for 3 days prior to the assay. Cell surface expression of G2R-2 or G / IL-2Rβ was confirmed by flow cytometry (Figure 32A). As shown, the cells were then plated with IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or without cytokine. Promotion of cell cycle progression after stimulation with G-CSF was observed in cells expressing G2R-2 rather than in non-transduced cells or cells expressing single-chain G / IL-2Rβ (Figure 32B, C). Panels B and C show the results for all live cells or G-CSFR+ cells, respectively.

[0341] These results indicate that the G2R-2 chimeric receptor is more efficient than the single-chain G / IL-2Rβ receptor and activates cytokine-like signaling and proliferation in mouse T cells in response to G-CSF-induced homodimerization.

[0342] Example 18: Activation of cytokine - related intracellular signaling events in human primary T cells expressing G2R - 2 in response to G - CSF or IL - 2 To confirm that the chimeric cytokine receptor can actually activate cytokine signaling similar to IL-2, the ability of the cytokine receptor to activate various signaling molecules was evaluated. Tumor-associated lymphocytes expressing G2R-2 and PBMC-derived T cells were pre-expanded in G-CSF, and non-transduced 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 without cytokine, and Western blotting was performed on cell lysates using antibodies against the indicated signaling molecules (Figure 33). Panels A and B show the results for TAL, and panel C shows the results for PBMC-derived T cells. T cells expressing G2R-2, when stimulated with G-CSF, activated IL-2-related signaling molecules to a similar extent as seen after IL-2 stimulation of non-transduced or transduced cells (except for the expected exception that G-CSF induced Jak2 phosphorylation while IL-2 induced Jak3 phosphorylation).

[0343] These results confirm that the G2R-2 chimeric receptor can activate IL-2 receptor-like cytokine receptor signaling when stimulated with G-CSF.

[0344] Example 19: In mouse primary T cells expressing G2R - 2, cytokine signaling is activated in response to G - CSF. To evaluate 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 evaluated by Western blot of cell lysates from primary mouse T cells expressing G2R-2 or G / IL-2Rβ and mock-transduced cells. All cells were grown in IL-2 for 3 days prior to the assay. Cells were then washed and stimulated with IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. T cells expressing G2R-2, when stimulated with G-CSF, activated IL-2-related signaling molecules to a similar extent as seen after IL-2 stimulation of non-transduced or transduced cells (except for the expected exception that G-CSF induced Jak2 phosphorylation whereas IL-2 induced Jak3 phosphorylation) (Figure 34). In contrast, G / IL-2Rβ did not activate cytokine signaling upon exposure to G-CSF.

[0345] These results confirm in primary mouse T cells that the G2R-2 chimeric receptor can activate IL-2 receptor-like cytokine receptor signaling by homodimerization upon G-CSF stimulation, whereas the single-chain G / IL-2Rβ alone cannot activate cytokine signaling by homodimerization in response to G-CSF.

[0346] Example 20: Expression of the chimeric receptor results in the proliferation of 32D - IL - 2Rβ cells and primary mouse T cells after stimulation with orthogonal G - CSF. To determine whether cells expressing chimeric cytokine receptors can be selectively activated in response to orthogonal G-CSF, 32D-IL-2Rβ cells or primary mouse T cells were transduced with chimeric receptors G2R-1 and G2R-2 (G2R-1 WT ECD, G2R-2 WT ECD) containing wild-type G-CSFR ECD, and chimeric receptors G2R-1 and G2R-2 (G2R-1 134 ECD, G2R-2 134 ECD) containing G-CSFR ECD with amino acid substitutions R41E, R141E, and R167D. Cells were stimulated with either IL-2, wild-type G-CSF, or orthogonal G-CSF (130 G-CSF) that can bind to G2R-1 134 ECD and G2R-2 134 ECD but has significantly reduced binding to wild-type G-CSFR. A BrdU incorporation assay was performed to evaluate the ability of cytokine stimulation to promote cell cycle progression (Figure 3). 32D-IL-2Rβ cells expressing G2R-2 134 ECD showed cell cycle progression when stimulated with 130 G-CSF (bearing amino acid substitutions E46R, L108K, and D112R; 30 ng / ml), but no cell cycle progression occurred when stimulated with wild-type G-CSF (30 ng / ml). The orthogonality of the engineered cytokine:receptor ECD pairs was further demonstrated by stimulating primary mouse T cells in a "cross-cross" proliferation assay, in which cells expressing G2R-3 (Figure 23) with WT, 130, 134, 304, or 307 ECD were stimulated with WT, 130, 304, or 307 cytokine (100 ng / ml) (Figure 36). 130 ECD has amino acid substitutions: R41E and R167D. 304 ECD has amino acid substitutions: R41E, E93K, and R167D, and 304 cytokine has amino acid substitutions: E46R, L108K, D112R, and R147E. 307 ECD has amino acid substitutions: R41E, D197K, D200K, and R288E, and 307 cytokine has amino acid substitutions: S12E, K16D, E19K, and E46R. Panels A and B of Figure 36 represent replicate experiments.

[0347] These results indicate that cells expressing the orthogonal chimeric cytokine receptor can be selectively activated and undergo cell cycle progression when stimulated with the orthogonal G-CSF CSF.

[0348] Example 21: Intracellular signaling is activated in 32D - IL2Rβ cells and primary human T cells expressing an orthogonal chimeric cytokine receptor and stimulated with orthogonal G - CSF. To determine whether cells expressing the chimeric cytokine receptor can 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 (G2R-1 WT ECD and G2R-2 WT ECD) containing the wild-type G-CSFR ECD and chimeric receptors G2R-1 and G2R-2 (G2R-1 134 ECD, G2R-2 134 ECD) containing the G-CSFR ECD with amino acid substitutions R41E, R141E, and R167D. The cells were stimulated with either IL-2 (300 IU / ml), wild-type G-CSF (30 ng / ml), or orthogonal G-CSF (130 G-CSF-E46R_L108K_D112R; 30 ng / ml) that can bind to G2R-1 134 ECD and G2R-2 134 ECD but has significantly reduced binding to the wild-type G-CSFR. To evaluate the ability of the cells to activate cytokine signaling upon exposure to the cytokine, Western blot was performed on cell lysates (Figure 37). Cells expressing G2R-2 134 ECD showed evidence of cytokine signaling upon stimulation with 130G-CSF but not wild-type G-CSF. Furthermore, cells expressing G2R-2 WT ECD were unable to activate cytokine signaling when stimulated with 130G-CSF.

[0349] Operated cytokines: Orthogonality of the receptor pairs was further demonstrated by subjecting primary mouse T cells to Western blot analysis, in which 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 signal transduction events were measured (Figure 38A). IL-2 (300 IU / ml) and IL-12 (10 ng / ml) functioned as control cytokines. Cells expressing G2R-3 WT ECD showed evidence of cytokine signal transduction upon stimulation with IL-2, IL-12, or WT G-CSF. Cells expressing G2R-3 134 ECD showed evidence of cytokine signal transduction upon stimulation with IL-2, IL-12, or 130 G-CSF. Cells expressing G2R-3 304 ECD showed evidence of cytokine signal transduction upon stimulation with IL-2, IL-12, or 304 G-CSF.

[0350] Cell surface expression of the three ECD variants of G2R-3 was confirmed by flow cytometry (Figure 38B).

[0351] These results indicate that cells expressing orthogonal chimeric cytokine receptors can selectively activate intracellular cytokine signal transduction events when stimulated with orthogonal G-CSF.

[0352] Example 22: Expression of G2R - 3 results in proliferation, cell cycle progression, and cytokine - related intracellular signaling and immune phenotype in primary human T cells To determine whether the G2R-3 chimeric receptor can promote cytokine signaling-related events when stimulated with G-CSF in primary human T cells, TALs were transduced with a lentiviral vector encoding G2R-3. A T cell proliferation assay was performed to test 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 non-transduced counterparts, primary TALs expressing G2R-3 proliferated in culture in response to G-CSF (Figure 40A). To determine whether cytokine signaling events were activated upon stimulation with G-CSF, cell lysates were subjected to Western blot to evaluate intracellular signaling. Cells were harvested from the proliferation 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 exception of the expected discrepancy that G-CSF induced Jak2 phosphorylation while IL-2 induced Jak3 phosphorylation (Figure 40B).

[0353] To evaluate the progression of the cell cycle upon stimulation with G-CSF, a BrdU incorporation assay was performed. Cells were harvested from the proliferation assay, washed, and then re-plated in the presence of IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. Primary TALs expressing G2R-3 demonstrated progression of the cell cycle in response to G-CSF (Figure 40C).

[0354] G-CSF-induced proliferation of cells expressing G2R-3 was also demonstrated using human T cells derived from primary PBMCs (Figure 41). Cells expressing G2R-3 WT ECD proliferated in response to WT G-CSF but not in medium alone (Figure 41A). To demonstrate that the cells were continuously dependent on exogenous cytokines, on day 21 of culture, cells under G-CSF proliferation conditions were washed and replated in WT G-CSF (100 ng / mL), IL-7 (20 ng / mL) + IL-15 (20 ng / mL), or medium alone. Only cells replated in the presence of G-CSF or IL-7 + IL-15 maintained viability over time.

[0355] Expression of G-CSFR ECD, as evaluated by flow cytometry, remained stable between days 21 and 42 of proliferation in both CD4+ and CD8+ T cells (Figure 41B).

[0356] Western blot showed that primary PBMC-derived T cells expressing G2R-3 exhibited IL-2-related signaling events in response to G-CSF (Figure 42A). Flow cytometry-based immunophenotypic analysis was performed on primary PBMC-derived T cells that had proliferated for 42 days in WT G-CSF or IL-7 + IL-15. Cells expressing G2R-3 WT ECD and cultured in G-CSF retained a phenotype similar to that of non-transduced cells cultured in IL-7 + IL-15, mainly showing the CD62L+, CD45RO+ phenotype indicative of the stem cell-like memory T cell phenotype (T SCM ). (Figure 42B, C). Similarly, the fractions of central memory (T CM ), effector memory (T EM ), and terminally differentiated (T TE ) T cells were also similar.

[0357] These results confirm that the G2R-3 chimeric cytokine receptor can activate cytokine signaling events and promote cell cycle progression and proliferation in primary cells. The immunophenotype of T cells expressing G2R-3 and proliferating long-term in G-CSF is similar to that of non-transduced cells proliferating in IL-7 + IL-15.

[0358] Example 23: Orthogonal G - CSF induces proliferation and expansion in primary human T cells expressing G2R - 3 with an orthogonal ECD. It was evaluated whether the chimeric cytokine receptor G2R-3 with 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 G-CSF. Primary PBMC-derived human T cells were transduced with a lentiviral vector encoding GR-3 304 ECD or G2R-3 307 (R41E_D197K_D200K_R288E) ECD. A T cell proliferation assay was performed to evaluate the fold increase in cell growth when cultured in IL-2 (300 IU / ml), 304 G-CSF (100 ng / ml), 307 G-CSF (100 ng / ml), or without cytokines. Live cells were counted every 3 - 4 days. T cells expressing G2R-3 304 ECD were grown in culture medium in response to IL-2 or 304 G-CSF (Figure 43A). T cells expressing G2R-3 307 ECD were grown in culture medium in response to IL-2 or 307 G-CSF (Figure 43B). Only non-transduced T cells were grown in response to IL-2 (Figure 43C).

[0359] The BrdU incorporation assay was performed in a cross-over design to evaluate cell cycle progression upon stimulation with 130, 304, and 307 G-CSF. Cells were harvested from the proliferation assay, washed, and replated in the presence of 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 G2R-3 304 ECD showed cell cycle progression in response to 130 or 304 G-CSF, but not to 307 G-CSF (Figure 44). T cells expressing G2R-3 307 ECD showed cell cycle progression in response to 307 G-CSF, but not to 130 or 304 G-CSF. All T cells showed cell cycle progression in response to IL-2.

[0360] These results indicate that the chimeric receptors G2R-3 304 ECD and G2R-3 307 ECD can induce selective cell cycle progression and proliferation of primary human CD4+ and CD8+ T cells when stimulated with the orthogonal 304 or 307 G-CSF, respectively. Furthermore, 130 G-CSF can stimulate the proliferation of cells expressing G2R-3 304 ECD, but not cells expressing G2R-3 307 ECD.

[0361] Example 24: G - CSFR ECD is expressed on the surface of primary human tumor - associated lymphocytes (TAL) transfected with G21R - 1, G21R - 2, G12R - 1, and G2R - 3 chimeric receptor constructs. To evaluate whether the chimeric cytokine receptor constructs can be expressed on the surface of primary human tumor-associated lymphocytes (TAL), TAL were transduced with lentiviral vectors encoding the G21R-1, G21R-2, G12R-1, and G2R-3 chimeric receptors and the cells were tested for G-CSFR ECD expression on the cell surface by flow cytometry (Figure 39). G-CSFR ECD-positive cells were detected for all four chimeric cytokine receptor designs.

[0362] These results indicate 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 indicate that the design of the G-CSFR ECD chimeric receptor is expressed on the surface of primary cells.

[0363] Example 25: G - CSFR ECD is expressed on the surface of primary mouse T cells transfected 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 mouse T cells were transduced with retroviral vectors encoding the G12R-1 and G21R-1 chimeric receptors and analyzed by flow cytometry (Figure 45).

[0364] This result indicates that the G-CSFR ECD is expressed on the surface of primary mouse CD4+ and CD8+ T cells transduced with retroviral vectors encoding G12R-1 and G21R-1.

[0365] Example 26: 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 can 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 receptors. Cells were intracellularly stained with a phospho-STAT3 (p-STAT3)-specific antibody and evaluated by flow cytometry to measure the degree of STAT3 phosphorylation, which is a measure of STAT3 activation (Figure 46). Stimulation with G-CSF (100 ng / ml) increased the number of cells expressing phosphorylated STAT3 in a 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 show an increase upon stimulation with IL-21.

[0366] These results indicate that the G21R-1 and G21R-2 chimeric receptors can activate IL-21-related cytokine signaling events when stimulating G-CSF in primary human T cells.

[0367] Example 27: G - CSF induces intracellular signaling events in primary mouse T cells expressing G21R - 1 or G - 12R - 1. To determine whether the chimeric cytokine receptor G21R-1 can activate cytokine signaling events, primary mouse T cells were transduced with a retroviral vector encoding G21R-1 and evaluated by flow cytometry to detect phosphorylated STAT3 upon stimulation with G-CSF. Live cells were gated with CD8 or CD4, and the percentage of phospho-STAT3-positive stained cells was measured for the 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 (not non-transduced cells) showed an increase in the amount of phosphorylated STAT3 (Figures 47A and 47B). Western blot was performed to evaluate the intracellular cytokine signaling of cells expressing G21R-1 or G12R-1 upon stimulation with G-CSF. As expected, cells expressing G21R-1 and stimulated with G-CSF showed an increase in the phosphorylation of STAT3 and a slight increase in phospho-STAT4 and phospho-STAT5 (Figure 47C). Also as expected, in cells expressing G12R-1, strong phosphorylation of STAT4 was seen in response to G-CSF. G-CSF did not induce signaling events in mock-transduced cells. (Note that in the G12R-1 group, it seems that the positive control (hIL-12 10 ng / ml) did not induce signaling events; this may be due to insufficient binding of human IL-12 to the mouse IL-12R.)

[0368] The results indicate that G21R-1 and G12R-1 can induce cytokine signaling events in primary mouse T cells upon stimulation with G-CSF.

[0369] Example 28: G - CSF induces proliferation and intracellular signaling events in primary mouse 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 evaluate cytokine signaling events and cell proliferation mediated by chimeric cytokine receptors, primary mouse 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. To evaluate the progression of the cell cycle upon stimulation with G-CSF, a BrdU incorporation assay was performed. Cells were harvested, washed, and replated in the presence of IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or without cytokine. G-CSF-induced cell cycle progression was seen in primary mouse T cells expressing G2R-2, G2R-3, G7R-1, G21 / 7R-1, or G27 / 2R-1 (Figs. 48A, 48B), or G21 / 2R-1, G12 / 2R-1, or G21 / 12 / 2R-1 (Figs. 49A, 49B). Expression of the G-CSFR ECD was also detectable by flow cytometry (Figs. 48C, 49C).

[0370] By Western blot, multiple cytokine signaling events were observed in cells expressing the indicated chimeric cytokine receptors in response to G-CSF (100 ng / ml), but not in mock-transduced cells (Figures 48D and 49D). In general, the observed cytokine signaling events were as expected based on the signaling domains incorporated into the various ICD designs (Figures 23 and 24). As an example, the G7R-1 chimeric receptor induced phosphorylation of STAT5 (Figure 48D), which was expected to result from the incorporation of the STAT5 binding site from IL-7Rα (Figure 23). As a second example, the G21 / 2R-1 chimeric receptor induced phosphorylation of STAT3 (Figure 49D), which was expected to result from the incorporation of the STAT3 binding site from G-CSFR (Figure 24). As a third example, the G12 / 2R-1 chimeric receptor induced phosphorylation of STAT4, which was expected to result from the incorporation of the STAT4 binding site from IL-12Rβ2 (Figure 24). Other chimeric cytokine receptors showed other distinct different patterns of intracellular signaling events.

[0371] 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 are able to induce cytokine signaling events and proliferation in primary mouse T cells when stimulated with G-CSF. Furthermore, by incorporating different signaling domains into the ICD of the chimeric receptor, different patterns of intracellular signaling events can be generated.

[0372] Example 29: Orthogonal G - CSF induces expansion, proliferation, cytokine - related intracellular signaling, and immune phenotype in primary human T cells expressing G12 / 2R - 1 with an orthogonal ECD. To determine whether the chimeric cytokine receptor G12 / 2R-1 with 134 ECD can induce proliferation and expansion in response to stimulation with the orthogonal ligand 130 G-CSF, primary PBMC-derived human T cells were transduced using a lentiviral vector encoding G12 / 2R-1 134 ECD. A T cell proliferation assay was performed to evaluate the fold increase in cell proliferation when cultured with IL-2 (300 IU / ml), 130 G-CSF (100 ng / ml), or no cytokine. Viable cells were counted every 4 - 5 days. Primary human T cells expressing G12 / 2R-1 134 ECD proliferated in culture in response to IL-2 or 130 G-CSF (Figure 50A), but showed limited transient proliferation in medium alone.

[0373] On day 19 of this experiment, T cells expanded with 130 G-CSF or IL-2 were washed three times and re-plated with IL-2, 130 G-CSF, or medium alone. In medium alone, T cells showed a decrease in viability and number (Figure 50B). In contrast, T cells re-plated with IL-2 or 130 G-CSF showed continued viability and stable numbers.

[0374] 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 50C). A BrdU incorporation assay was performed to evaluate cell cycle progression upon stimulation with 130 G-CSF.

[0375] To evaluate cell cycle progression by BrdU assay, cells were harvested from the proliferation assay, washed, and replated in the presence of IL-2 (300 IU / ml), IL-2, and IL-12 (10 ng / ml), 130 G-CSF (300 ng / ml), or no cytokine. Primary human T cells expressing G12 / 2R-1 134 ECD showed cell cycle progression in response to 130 G-CSF, IL-2, or IL-2 + IL-12, while non-transduced cells responded only to IL-2 or IL-2 + IL-12 (Figure 51A).

[0376] After a 16-day culture period, immunophenotypic analysis was performed by flow cytometry using antibodies against CD62L and CD45RO to compare T cells expressing G12 / 2R-1 134 ECD that had proliferated in 130 G-CSF with non-transduced cells that had proliferated in IL-2. The two T cell populations showed similar proportions of stem cell-like memory (T SCM ), central memory (T CM ), effector memory (T EM ), and terminally differentiated (T TE ) phenotypes (Figures 51B, C).

[0377] A similar experiment was performed using the chimeric cytokine receptor G12 / 2R-1 with 304 ECD (instead of 134 ECD). Primary PBMC-derived human T cells were transduced with a lentiviral vector encoding G12 / 2R-1 304 ECD. A T cell proliferation assay was performed to evaluate the fold increase in cell growth when cultured in IL-2 (300 IU / ml), 130 G-CSF (100 ng / ml), 304 G-CSF (100 ng / ml), or medium alone. Live cells were counted every 4 - 5 days. T cells expressing G12 / 2R-1 with 304 ECD could proliferate in the presence of IL-2, 130 G-CSF, or 304 G-CSF, but not in medium alone, while non-transduced cells could proliferate in response to IL-2 only (Figure 52A).

[0378] To evaluate cell cycle progression by BrdU assay, T cells expressing G12 / 2R-1 304 ECD pre-proliferated with either 130 G-CSF or 304 G-CSF were recovered from the proliferation assay, washed, and then re-plated 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 medium only. T cells expressing G12 / 2R-1 304 ECD showed cell cycle progression in response to 130 or 304 G-CSF, but not in response to 307 G-CSF or medium only (Figure 52B).

[0379] This result indicates that G12 / 2R-1 134 ECD can induce cell cycle progression and proliferation of primary human CD4+ and CD8+ T cells when stimulated with orthogonal 130 G-CSF. The T cell memory phenotype of cells expressing G12 / 2R-1 134 ECD and proliferated with 130 G-CSF is similar to that of non-transduced cells proliferated with IL-2. Furthermore, G12 / 2R-1 304 ECD can induce selective cell cycle progression and proliferation of T cells when stimulated with 130 or 304 G-CSF, but not in response to 307 G-CSF.

[0380] Example 30: Orthogonal G-CSF induces distinct intracellular signaling events in primary human T cells expressing G2R-3 or G12 / 2R-1 with an orthogonal ECD To evaluate intracellular signaling events, primary PBMC-derived human T cells were transduced with a lentiviral vector encoding G2R-3 304 ECD or G12 / 2R-1 304 ECD. Western blot was performed to evaluate intracellular cytokine signaling in cells expressing G2R-3 304 ECD or G12 / 2R-1 304 ECD, or non-transduced cells, at the time of stimulation with 304 G-CSF (100 ng / mL), IL-2 (300 IU / mL), IL-2, and IL-12 (10 ng / mL), or medium alone. Strong phosphorylation of STAT5 was detected in response to stimulation with either IL-2+IL-12 or IL-2 alone in both transduced and non-transduced T cells (Figure 53). Strong phosphorylation of STAT4 was detected in response to stimulation with both IL-2+IL-12, but only weak phosphorylation of STAT4 was detected in response to stimulation with IL-2 alone. In cells expressing G2R-3 304 ECD, weak phosphorylation of STAT4 and strong phosphorylation of STAT5 were detected in response to stimulation with 304 G-CSF, similar to the pattern seen in response to IL-2 alone. In cells expressing G12 / 2R-1 304 ECD, strong phosphorylation of STAT4 and STAT5 were detected in response to stimulation with 304 G-CSF, similar to the pattern seen in response to IL-2+IL-12. Non-transduced T cells did not show a response to 304 G-CSF.

[0381] These results indicate that G12 / 2R-1 with 304 ECD can induce cytokine signaling events, including strong phosphorylation of STAT4 and STAT5, in response to stimulation with 304 G-CSF. Different patterns of signaling events were seen in cells expressing G2R-3 304 ECD after stimulation with 304 G-CSF, including strong phosphorylation of STAT5 but not strong phosphorylation of STAT4.

[0382] The present invention has been particularly shown and described with reference to preferred embodiments and various alternative embodiments, but it will be understood by those skilled in the relevant art that various changes in form and detail may be made herein without departing from the spirit and scope of the invention.

[0383] All references, issued patents, and patent applications cited in the text of this specification are hereby incorporated by reference in their entirety for all purposes.

[0384] (Table 12) G-CSFR WT -ICD gp130-IL-2Rβ sequence TIFF0007708745000016.tif228153

[0385] (Table 13) G-CSFR WT -ICD IL-2Rβ sequence TIFF0007708745000017.tif222152

[0386] (Table 14) G-CSFR WT -ICD γC sequence TIFF0007708745000018.tif171153

[0387] (Table 15A) Chimeric cytokine receptor TIFF0007708745000019.tif65139

[0388] (Table 15B) Chimeric cytokine receptor TIFF0007708745000020.tif105140

[0389] (Table 16) TIFF0007708745000021.tif97144

[0390] (Table 17) Signal peptide. The signal peptide is composed of any one of the following: TIFF0007708745000022.tif72149

[0391] (Table 18) Wild-type G-CSFR extracellular domain (ECD): The G-CSFR ECD is composed of any one of the following: TIFF0007708745000023.tif85149TIFF0007708745000024.tif225149TIFF0007708745000025.tif153149

[0392] (Table 19) Transmembrane domain (TM). The TM is composed of any one of the following: TIFF0007708745000026.tif103149

[0393] (Table 20) Intracellular domain (ICD). The ICD is composed of any one of the following: TIFF0007708745000027.tif161149TIFF0007708745000028.tif222149TIFF0007708745000029.tif226149TIFF0007708745000030.tif221149TIFF0007708745000031.tif226149TIFF0007708745000032.tif226149TIFF0007708745000033.tif172149

Claims

**Claim 1** (a) A receptor comprising a variant extracellular domain (ECD) of the granulocyte colony-stimulating factor receptor (G-CSFR), wherein the variant ECD of the G-CSFR comprises at least one mutation in the site II interface region and at least one mutation in the site III interface region, said receptor, and (b) A variant G-CSF comprising at least one mutation in the site II interface region and at least one mutation in the site III interface region A system for selectively activating a receptor expressed on the cell surface, comprising i) A variant G-CSF comprising the E46R, L108K, and D112R mutations at the amino acid positions of SEQ ID NO: 1, and a receptor comprising the R41E and R167D mutations at the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2, or ii) A variant G-CSF comprising the E46R, L108K, D112R, and R147E mutations at the amino acid positions of SEQ ID NO: 1, and a receptor comprising the R41E, E93K, and R167D mutations at the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2, or iii) A variant G-CSF comprising the S12E, K16D, E19K, and E46R mutations at the amino acid positions of SEQ ID NO: 1, and a receptor comprising the R41E, D197K, D200K, and R288E mutations at the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2, or iv) A variant G-CSF comprising the E46R, L108K, D112R, E122R, and E123R mutations at the amino acid positions of SEQ ID NO: 1, and a receptor comprising the R41E, R141E, and R167D mutations at the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2, or v) A variant G-CSF comprising the E46R, L108K, and D112R mutations at the amino acid positions of SEQ ID NO: 1, and a receptor comprising the R41E, R141E, and R167D mutations at the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2, or vi) A variant G-CSF comprising the K40D, L41D, L108K, and D112R mutations at the amino acid positions of SEQ ID NO: 1, and a receptor comprising the F75K, Q91K, and R167D mutations at the amino acid positions of amino acids 2 to 308 of SEQ ID NO: 2, or vii) Variant G-CSF comprising the T38R, E46R, L108K, and D112R mutations at the amino acid positions of SEQ ID NO: 1, and a receptor comprising the R41E, Q73E, and R167D mutations at the amino acid positions of amino acids 2-308 of SEQ ID NO: 2, or viii) Variant G-CSF comprising the L108K, D112R, and R147E mutations at the amino acid positions of SEQ ID NO: 1, and a receptor comprising the E93K and R167D mutations at the amino acid positions of amino acids 2-308 of SEQ ID NO: 2, or ix) Variant G-CSF comprising the E19K, E46R, L108K, and D112R mutations at the amino acid positions of SEQ ID NO: 1, and a receptor comprising the R41E, R167D, and R288D mutations at the amino acid positions of amino acids 2-308 of SEQ ID NO: 2, or x) Variant G-CSF comprising the E19K, E46R, D112K, and T115K mutations at the amino acid positions of SEQ ID NO: 1, and a receptor comprising the R41E, R167E, Q174E, and R288E mutations at the amino acid positions of amino acids 2-308 of SEQ ID NO: 2 comprising the said system. **Claim 2**: A nucleic acid encoding the system according to claim 1. **Claim 3**: An expression vector comprising the system according to claim 1. **Claim 4**: A method for selectively activating a receptor expressed on the surface of a cell, comprising contacting the receptor of the system according to claim 1 with variant G-CSF. **Claim 5**: The method according to claim 4, wherein the cell is i) an immune cell selected from the group consisting of T cells, NK cells, NKT cells, B cells, plasma cells, macrophages, and dendritic cells, ii) a stem cell, iii) a primary cell, or iv) a human cell. **Claim 6**: A method for producing immune cells expressing the system according to claim 1, the method comprising introducing the nucleic acid according to claim 2 or the expression vector according to claim 3 into the cells. **Claim 7**: A pharmaceutical composition for treating a subject in need of treatment, comprising cells and the system according to claim 1. **Claim 8**: The pharmaceutical composition according to claim 7, for use in treating cancer, an inflammatory condition, graft rejection, or an infectious disease. **Claim 9**: A pharmaceutical composition for treating a subject in need of treatment, comprising immune cells transfected or transduced with a nucleic acid sequence encoding the system according to claim 1, wherein the immune cells are (i) isolating a sample containing immune cells; (ii) transfecting or transducing the immune cells with a nucleic acid sequence encoding the system according to claim 1; prepared by a process comprising the pharmaceutical composition.

10. A kit for treating a subject in need of treatment, comprising a cell comprising the system according to claim 1 and instructions for use.

11. A kit for generating a system for selectively activating a receptor expressed on the cell surface, (a) the nucleic acid according to claim 2 or the expression vector according to claim 3; (b) instructions for use comprising the kit.

12. A kit for generating a receptor expressed on a cell, comprising a cell comprising an expression vector encoding the system according to claim 1 and instructions for use.

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