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

Abstract

Described herein are methods and compositions for selectively activating cells using variant cytokine receptors and cytokine pairs, wherein the cytokine receptor comprises an extracellular domain (ECD) of a granulocyte colony stimulating factor receptor (G-CSFR). In certain embodiments, the methods and compositions described herein can be used to specifically activate cells for adoptive cell transfer therapy. Thus, included herein are methods for producing cells expressing variant receptors that are selectively activated by cytokines that do not bind to their natural receptors. Also disclosed herein are methods for treating subjects in need thereof, comprising administering to the subject a cell expressing a variant receptor comprising an extracellular domain of G-CSFR and co-administering a variant cytokine that activates the variant receptor.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 912,318, filed October 8, 2019, which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy was created on October 7, 2020, is named IKE-068WO_US_SL.txt, and is 85,109 bytes in size. Background Art Technical Field

[0005] The present invention relates to methods and compositions for selectively activating cells using variant cytokine receptors and cytokine pairs, wherein the cytokine receptor comprises a variant extracellular domain (ECD) of a granulocyte colony stimulating factor receptor (G-CSFR). Also disclosed herein are methods for treating a subject by adoptive cell transfer, comprising administering to the subject cells expressing the variant receptor and administering a variant cytokine to signal the cells expressing the variant receptor. The present disclosure includes nucleic acids, expression vectors, and kits for producing cells expressing variant cytokines and receptors, as well as kits for cytokines that bind to variant receptors.

[0006] Related technical description

[0007] Over the past two decades, great progress has been made in the treatment of cancer through adoptive cell therapy (ACT). ACT performed with naturally occurring tumor-infiltrating T cells (TIL) is now reproducible, producing an objective clinical response rate of >50% in advanced melanoma. ACT performed with T cells engineered to recognize B-lineage leukemia (using CD19-directed chimeric antigen receptors or CD19 CARs) produces a complete response rate of up to 90%, with most patients achieving a durable response. Driven by these remarkable results, several companies are commercializing TIL and CD19 CAR T cell approaches.

[0008] The engraftment, expansion, and persistence of T cells used for ACT are important determinants of clinical safety and efficacy. This is typically addressed by administering systemic IL-2 after ACT transfer and by expanding T cells with IL-2 in vitro before transfer. In addition to the intended immunostimulatory effects, systemic IL-2 therapy can also result in serious toxicities that require strict management to ensure patient safety, such as vascular leak syndrome. To manage these risks, patients typically require hospitalization for 2–3 weeks and admission to the ICU as a precaution. In addition, IL-2 induces the proliferation of both effector and regulatory (suppressor) T cells (5); thus, giving patients IL-2 is similar to pressing both the accelerator and brake pedals simultaneously. CAR T cells present the opposite problem, namely that T cell expansion and persistence may exceed safe levels. Furthermore, they generally eradicate normal B cells (which also express CD19), leaving some patients with lifelong immunodeficiency. Ideally, one would like to have precise control over the number of tumor-reactive T cells after adoptive transfer, including the ability to eliminate transferred cells once the cancer has been eradicated. Other cell-based therapies, such as stem cell therapies, would also benefit from improved control over the expansion, differentiation, and persistence of infused cells.

[0009] Human G-CSF is an approved treatment ( Filgrastim is used to treat neutropenia in cancer patients. G-CSF is a four-helix bundle (Hill, CP et al. Proc Natl Acad Sci US A. 1993 Jun 1;90(11):5167-71), and the structure of the complex between G-CSF and its receptor G-CSFR has been well characterized (Tamada, T et al. Proc Natl Acad Sci US A. 2006 Feb 28;103(9):3135-40). 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; it is the larger interface between G-CSF and the cytokine receptor homology (CRH) domain of G-CSFR. The second interface is called site III; it is the smaller interface between G-CSF and the N-terminal Ig-like domain of G-CSFR. Summary of the Invention

[0010] In certain embodiments, disclosed herein is a receptor comprising a variant extracellular domain (ECD) of a 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 some aspects, at least one mutation in the Site II interface region is selected from the group of mutations in the G-CSFR ECD consisting of: R141E, R167D, K168D, K168E, L171E, L172E, Y173K, Q174E, D197K, D197R, M199D, D200K, D200R, V202D, R288D, and R288E. In some aspects, at least one mutation in the Site III interface region is selected from the group of mutations in the G-CSFR ECD consisting of: amino acid positions 30, 41, 73, 75, 79, 86, 87, 88, 89, 91, and 93 of SEQ ID NO. 2. In some aspects, at least one mutation in the Site III interface region is selected from the group of mutations in the G-CSFR ECD consisting of: S30D, R41E, Q73W, F75KF, S79D, L86D, Q87D, I88E, L89A, Q91D, Q91K, and E93K. In some aspects, the G-CSFR ECD comprises a combination of mutations having design numbers in Table 6; wherein the mutations correspond to amino acid positions of SEQ ID NO. 2. In some aspects, the G-CSFR ECD comprises the following mutations: R41E, R141E, and R167D.

[0011] In some aspects, the receptors disclosed herein are chimeric receptors. In some aspects, the receptors are expressed on cells. In some aspects, the receptors are expressed on immune cells. In some aspects, the immune cells are optionally T cells, and optionally NK cells, and optionally NKT cells, and optionally B cells, and optionally plasma cells, and optionally macrophages, and optionally dendritic cells, and optionally the cells are stem cells, and optionally the cells are primary cells, and optionally the cells are human cells.

[0012] In certain aspects, activation of the receptor by variant G-CSF results in a cellular response selected from the group consisting of: increased proliferation, viability, and activity of cells expressing the receptor.

[0013] In certain embodiments, disclosed herein is a nucleic acid encoding any receptor 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, and optionally an NK cell, and optionally an NKT cell, and optionally a B cell, and optionally a plasma cell, and optionally a macrophage, and optionally a dendritic cell, and optionally, the cell is a stem cell, and optionally, the cell is a primary cell, and optionally, the cell is a human cell.

[0014] 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, the 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, the at least one mutation in the Site II interface region of the variant G-CSF is selected from the group consisting of mutations at positions 38, 39, 40, 41, 46, 47, 48, 49, and 147 of SEQ ID NO. 1. In certain aspects, at least one mutation in the Site III interface region of the variant G-CSF is selected from the group consisting of: T38R, Y39E, K40D, K40F, L41D, L41E, L41K, E46R, L47D, V48K, V48R, L49K, and R147E. In certain aspects, the variant G-CSF comprises a combination of mutations having designation numbers in Table 6; wherein the mutations correspond to amino acid positions of SEQ ID NO. 1. In certain aspects, the variant G-CSF comprises the following mutations: E46R, L108K, and D112R. In certain aspects, the variant G-CSF selectively binds to a receptor disclosed herein. In certain aspects, the receptor is expressed on a cell. In certain aspects, the cell is an immune cell. In certain aspects, the immune cell is optionally a T cell, and optionally a NK cell, and optionally a NKT cell, and optionally a B cell, and optionally a plasma cell, and optionally a macrophage, and optionally a dendritic cell, and optionally the cell is a stem cell, and optionally the cell is a primary cell, and optionally the cell is a human cell. In certain aspects, selective binding of variant G-CSF to the receptor results in a cellular response selected from the group consisting of: increased proliferation, viability, and activity of immune cells.

[0015] In certain embodiments, disclosed herein is a nucleic acid encoding a variant G-CSF. In certain aspects, 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 aspects, the cell is an immune cell.

[0016] In certain embodiments, disclosed herein is a system for selectively activating a receptor expressed on the surface of a cell, the system comprising a receptor and a variant G-CSF, wherein the receptor comprises at least one mutation in the Site II interface region, the Site III interface region, or a combination thereof, and the variant G-CSF comprises at least one mutation in the amino acid sequence of the Site II interface region, the Site III interface region, or a combination thereof that binds to G-CSF; and wherein the variant G-CSF preferentially binds to the receptor compared to wild-type G-CSFR ECD, and the receptor preferentially binds to the variant G-CSF compared to wild-type G-CSF. In certain aspects, the receptor and variant G-CSF comprise a combination of mutations in the Site II interface having design numbers as shown in Table 2; wherein the receptor mutations correspond to amino acid positions of SEQ ID NO. 2 and the variant G-CSF mutations correspond to amino acid positions of SEQ ID NO. 1. In certain aspects, the receptor and variant G-CSF comprise a combination of mutations in the Site III interface having design numbers as shown in Table 4; wherein the receptor mutations correspond to amino acid positions of SEQ ID NO. 2 and the variant G-CSF mutations correspond to amino acid positions of SEQ ID NO. 1. In certain aspects, the receptor and variant G-CSF comprise a combination of mutations at the Site II interface and the Site III interface having the design numbers of Table 6; wherein the receptor mutations correspond to amino acid positions of SEQ ID NO. 2 and the variant G-CSF mutations correspond to amino acid positions of SEQ ID NO. 1. In certain aspects, the combination of mutations comprises a mutation having design number 106; wherein the variant G-CSF comprises E46R and D104K mutations corresponding to amino acid positions of SEQ ID NO. 1; and wherein the receptor comprises R41E and K168D mutations corresponding to amino acid positions of SEQ ID NO. 2. In certain aspects, the combination of mutations comprises a mutation having design number 117; wherein the variant G-CSF comprises E46R, E122R, and E123R mutations corresponding to amino acid positions of SEQ ID NO. 1; and wherein the receptor comprises R41E and R141E mutations corresponding to amino acid positions of SEQ ID NO. 2. In certain aspects, the combination of mutations comprises a mutation with design number 130; wherein the variant G-CSF comprises E46R, L108K, and D112R mutations corresponding to amino acid positions of SEQ ID NO. 1; and wherein the receptor comprises R41E and R167D mutations corresponding to amino acid positions of SEQ ID NO. 2.In certain aspects, the combination of mutations comprises a mutation with design number 134; wherein the variant G-CSF comprises E46R, L108K, D112R, E122R, and E123R mutations corresponding to amino acid positions of SEQ ID NO. 1; and wherein the receptor comprises R41E, R141E, and R167D mutations corresponding to amino acid positions of SEQ ID NO. 2. In certain aspects, the combination of mutations comprises a mutation with design number 135; wherein the variant G-CSF comprises E46R, T115K, E122R, and E123R mutations corresponding to amino acid positions of SEQ ID NO. 1; and wherein the receptor comprises R41E, R141E, L171E, and Q174E mutations corresponding to amino acid positions of SEQ ID NO. 2. In certain aspects, the combination of mutations comprises a mutation with design number 137; wherein the variant G-CSF comprises E46R, L108K, and D112R mutations corresponding to amino acid positions of SEQ ID NO. 1; and wherein the receptor comprises R41E, R141E, and R167D mutations corresponding to amino acid positions of SEQ ID NO. 2. In certain aspects, the combination of mutations comprises a mutation with design number 300; wherein the variant G-CSF comprises K40D, L41D, L108K, and D112R mutations corresponding to amino acid positions of SEQ ID NO. 1; and wherein the receptor comprises F75K, Q91K, and R167D mutations corresponding to amino acid positions of SEQ ID NO. 2. In certain aspects, the combination of mutations comprises a mutation with design number 301, wherein the variant G-CSF comprises T38R, E46R, L108K, and D112R mutations corresponding to amino acid positions of SEQ ID NO. 1, and wherein the receptor comprises R41E, Q73E, and R167D mutations corresponding to amino acid positions of SEQ ID NO. 2. In certain aspects, the combination of mutations comprises a mutation with design number 302, wherein the variant G-CSF comprises E46R, L108K, and D112R mutations corresponding to amino acid positions of SEQ ID NO. 1, and wherein the receptor comprises R41E, L86D, and R167D mutations corresponding to amino acid positions of SEQ ID NO. 2. In certain aspects, the combination of mutations comprises a mutation with design number 303; wherein the variant G-CSF comprises L108K, D112R, and R147E mutations corresponding to amino acid positions of SEQ ID NO. 1; and wherein the receptor comprises E93K and R167D mutations corresponding to amino acid positions of SEQ ID NO. 2.In certain aspects, the combination of mutations comprises the mutation with design number 304, wherein the variant G-CSF comprises E46R, L108K, D112R, and R147E mutations corresponding to amino acid positions of SEQ ID NO. 1, and wherein the receptor comprises R41E, E93K, and R167D mutations corresponding to amino acid positions of SEQ ID NO. 2. In certain aspects, the combination comprises the mutation with design number 305, wherein the variant G-CSF comprises E19K, E46R, L108K, and D112R mutations corresponding to amino acid positions of SEQ ID NO. 1, and wherein the receptor comprises R41E, R167D, and R288E mutations corresponding to amino acid positions of SEQ ID NO. 2. In certain aspects, the combination of mutations comprises a mutation with design number 307; wherein the variant G-CSF comprises S12E, K16D, E19K, and E46R mutations corresponding to amino acid positions of SEQ ID NO. 1; and wherein the receptor comprises R41E, D197K, D200K, and R288E mutations corresponding to amino acid positions of SEQ ID NO. 2. In certain aspects, the combination of mutations comprises a mutation with design number 308; wherein the variant G-CSF comprises E19R, E46R, and D112K mutations corresponding to amino acid positions of SEQ ID NO. 1; and wherein the receptor comprises R41E, R167D, V202D, and R288E mutations corresponding to amino acid positions of SEQ ID NO. 2. In certain aspects, the combination of mutations comprises a mutation with design number 400; wherein the variant G-CSF comprises E19K, E46R, D109R, and D112R mutations corresponding to amino acid positions of SEQ ID NO. 1; and wherein the receptor comprises R41E, R167D, M199D, and R288D mutations corresponding to amino acid positions of SEQ ID NO. 2. In certain aspects, the combination of mutations comprises a mutation with design number 401; wherein the variant G-CSF comprises E19K, E46R, L108K, and D112R mutations corresponding to amino acid positions of SEQ ID NO. 1; and wherein the receptor comprises R41E, R167D, and R288D mutations corresponding to amino acid positions of SEQ ID NO. 2. In certain aspects, the combination of mutations comprises a mutation with design number 402; wherein the variant G-CSF comprises E19K, E46R, D112K, and T115K mutations corresponding to amino acid positions of SEQ ID NO. 1; and wherein the receptor comprises R41E, R167E, Q174E, and R288E mutations corresponding to amino acid positions of SEQ ID NO. 2.In certain aspects, the combination of mutations comprises a mutation with design number 403; wherein the variant G-CSF comprises E19R, E46R, and D112K mutations corresponding to amino acid positions of SEQ ID NO. 1; and wherein the receptor comprises R41E, R167D, and R288E mutations corresponding to amino acid positions of SEQ ID NO. 2.

[0017] In certain embodiments, disclosed herein is a method for selectively activating a receptor expressed on the surface of a cell, the method comprising contacting the receptor with a variant G-CSF. In certain aspects, the receptor is expressed on a cell. In certain aspects, the receptor is expressed on an immune cell. In certain aspects, the receptor is expressed on a T cell or a NK cell. In certain aspects, the selective activation of an immune cell results in a cellular response selected from the group consisting of: increased proliferation, viability, and activity of the immune cell.

[0018] In certain embodiments, disclosed herein is a method of producing a cell expressing a receptor disclosed herein, the method comprising introducing into the cell a nucleic acid or expression vector disclosed herein.

[0019] In certain embodiments, disclosed herein is a method of treating a subject in need thereof, comprising infusing a cell (e.g., an immune cell) disclosed herein into the subject. In certain aspects, the method further comprises administering a variant G-CSF to the subject.

[0020] In certain embodiments, disclosed herein is a kit for producing a system for selectively activating a receptor expressed on the surface of a cell, the kit comprising a nucleic acid or expression vector disclosed herein; a variant G-CSF disclosed herein; and instructions for use.

[0021] In certain embodiments, described herein are chimeric receptors 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 an 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), and optionally, the ICD IL-2R is selected from the group consisting of IL-2Rβ and IL-2Rγc, and optionally, the second domain comprises at least a portion of the C-terminal region of IL-2Rβ, IL-7Rα, IL-12Rβ2 or IL-21R; wherein at least a portion of the ICD of the cytokine receptor comprises at least one signaling molecule binding site from the intracellular domain of the cytokine receptor, and optionally, the at least one signaling molecule binding site is selected from the group consisting of: a STAT3 binding site of G-CSFR; a STAT3 binding site of gp130; Binding site; SHP-2 binding site of gp130; SHC binding site of IL-2Rβ; STAT5 binding site of IL-2Rβ; STAT3 binding site of IL-2Rβ; STAT1 binding site of IL-2Rβ; STAT5 binding site of IL-7Rα; phosphatidylinositol 3-kinase (PI3K) binding site of IL-7Rα; STAT4 binding site of IL-12Rβ2; STAT5 binding site of IL-12Rβ2; STAT3 binding site of IL-12Rβ2; IL-21 R's STAT5 binding site; IL-21R's STAT3 binding site; and IL-21R's STAT1 binding site; and optionally, the ICD comprises box 1 and box 2 regions of a protein selected from the group consisting of G-CSFR and gp130; and optionally, the chimeric receptor comprises a third domain, said third domain comprising at least a portion of a transmembrane domain of a protein selected from the group consisting of G-CSFR, gp130 (glycoprotein 130) and IL-2Rβ, and optionally, the transmembrane domain is a wild-type transmembrane domain.

[0022] In certain aspects, described herein are chimeric receptors comprising: an ECD of G-CSFR operably linked to a second domain; said second domain comprising:

[0023] (i)

[0024] (a) Transmembrane domain of gp130;

[0025] (b) Box 1 and Box 2 regions of gp130; and

[0026] (c) the C-terminal region of IL-2Rβ; or

[0027] (ii)

[0028] (a) Transmembrane domain of G-CSFR;

[0029] (b) Box 1 and Box 2 of G-CSFR; and

[0030] (c) the C-terminal region of IL-2Rβ; or

[0031] (iii)

[0032] (a) Transmembrane domain of G-CSFR;

[0033] (b) Box 1 and Box 2 of G-CSFR; and

[0034] (c) the C-terminal region of IL-12Rβ2; or

[0035] (iv)

[0036] (a) Transmembrane domain of G-CSFR;

[0037] (b) Box 1 and Box 2 of G-CSFR; and

[0038] (c) the C-terminal region of IL-21R; or

[0039] (v)

[0040] (a) Transmembrane domain of IL-2Rβ+γc;

[0041] (b) Box 1 and Box 2 regions of IL-2Rβ+γc; and

[0042] (c) the C-terminal region of IL-2Rβ+γc; or

[0043] (vi)

[0044] (a) Transmembrane domain of G-CSFR

[0045] (b) Box 1 and Box 2 of G-CSFR; and

[0046] (c) C-terminal region of IL-7Rα.

[0047] In certain embodiments, the activated chimeric receptor forms a homodimer, and optionally, activation of the chimeric receptor results in a cellular response selected from the group consisting of: increased proliferation, viability, and activity of cells expressing the chimeric receptor, and optionally, the chimeric receptor is activated upon contact with G-CSF, and optionally, the G-CSF is wild-type G-CSF, and optionally, the extracellular domain of G-CSFR is a wild-type extracellular domain. In certain embodiments, the activated chimeric receptor forms a homodimer, and optionally, activation of the chimeric receptor results in a cellular response selected from the group consisting of: increased proliferation, viability, and activity of cells expressing the chimeric receptor, and optionally, the chimeric receptor is activated upon contact with G-CSF, and optionally, the G-CSF is wild-type G-CSF, and optionally, the extracellular domain of G-CSFR is a wild-type extracellular domain.

[0048] In certain embodiments, the chimeric receptor is expressed in a cell, and optionally is an immune cell, and optionally is a T cell, and optionally is a NK cell, and optionally is a NKT cell, and optionally is a B cell, and optionally is a plasma cell, and optionally is a macrophage, and optionally is a dendritic cell, and optionally the cell is a stem cell, and optionally the cell is a primary cell, and optionally the cell is a human cell.

[0049] In certain embodiments, the ICD comprises: (a) at least a portion of an ICD of IL-2Rβ having an amino acid sequence of SEQ ID NO. 16, 19, 21, 29, 31, 33, 35, 37, or 39; or (b) at least a portion of an ICD of IL-7Rα having an amino acid sequence of SEQ ID NO. 41; or (c) at least a portion of an ICD of IL-21R having an amino acid sequence of SEQ ID NO. 25 or 27; or (d) at least a portion of an ICD of IL-12Rβ2 having an amino acid sequence of SEQ ID NO. 23, 32, or 26; or (e) at least a portion of an ICD of G-CSFR having an amino acid sequence of SEQ ID NO. 20, 22, 24, 26, 28, 30, 34, 40, or 42; or (f) at least a portion of an ICD of gp130 having an amino acid sequence of SEQ ID NO. 18 or 38; or (g) at least a portion of an ICD of SEQ ID NO. or (h) at least a portion of the ICD of IL-2RG having the amino acid sequence of SEQ ID NO. 17.

[0050] In certain embodiments, the transmembrane domain comprises the sequence shown below:

[0051] (a) SEQ ID NO.8; or (b) SEQ ID NO.9; or (c) SEQ ID NO.10; or (d) SEQ ID NO.11.

[0052] In certain aspects, described herein is 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 an 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), and either Optionally, the IL-2R is selected from the group consisting of IL-2Rβ and IL-2Rγc, and optionally, the second domain comprises at least a portion of the C-terminal region of IL-2Rβ, IL-7Rα, IL-12Rβ2 or IL-21R; wherein at least a portion of the ICD of the cytokine receptor comprises at least one signaling molecule binding site from the intracellular domain of the cytokine receptor, and optionally, the ICD comprises at least one signaling molecule binding site selected from the group consisting of: a STAT3 binding site of G-CSFR; gp1 30's STAT3 binding site; gp130's SHP-2 binding site; IL-2Rβ's SHC binding site; IL-2Rβ's STAT5 binding site; IL-2Rβ's STAT3 binding site; IL-2Rβ's STAT1 binding site; IL-7Rα's STAT5 binding site; IL-7Rα's phosphatidylinositol 3-kinase (PI3K) binding site; IL-12Rβ2's STAT4 binding site; IL-12Rβ2's STAT5 binding site; IL-12Rβ2's STAT3 binding site; I The STAT5 binding site of IL-21R; The STAT3 binding site of IL-21R; And the STAT1 binding site of IL-21R; And optionally, the ICD comprises frame 1 district and frame 2 district of the protein selected from the group consisting of G-CSFR and gp130; And optionally, the chimeric receptor comprises a third domain, and the third domain comprises at least a portion of the transmembrane domain of the protein selected from the group consisting of G-CSFR, gp130 (glycoprotein 130) and IL-2R β, and optionally, the transmembrane domain is a wild-type transmembrane domain. In certain embodiments, the ECD of G-CSFR is encoded by the nucleic acid sequence shown in SEQ ID NO.5 or 6. In certain embodiments, the nucleic acid comprises:

[0053] (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

[0054] (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

[0055] (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

[0056] (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

[0057] (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

[0058] (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

[0059] (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

[0060] (h) A sequence encoding at least a portion of the ICD of IL-2Rγc having the amino acid sequence of SEQ ID NO. 17.

[0061] In certain embodiments, the present disclosure describes an expression vector comprising a nucleic acid encoding a chimeric receptor described herein. In certain embodiments, the vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and a plasmid.

[0062] 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; the second domain comprising:

[0063] (i)

[0064] (a) Transmembrane domain of gp130;

[0065] (b) Box 1 and Box 2 regions of gp130; and

[0066] (c) the C-terminal region of IL-2Rβ; or

[0067] (ii)

[0068] (a) Transmembrane domain of G-CSFR;

[0069] (b) Box 1 and Box 2 of G-CSFR; and

[0070] (c) the C-terminal region of IL-2Rβ; or

[0071] (iii)

[0072] (a) Transmembrane domain of G-CSFR;

[0073] (b) Box 1 and Box 2 of G-CSFR; and

[0074] (c) the C-terminal region of IL-12Rβ2; or

[0075] (iv)

[0076] (a) Transmembrane domain of G-CSFR;

[0077] (b) Box 1 and Box 2 of G-CSFR; and

[0078] (c) the C-terminal region of IL-21R; or

[0079] (v)

[0080] (a) Transmembrane domain of IL-2Rβ+γc;

[0081] (b) Box 1 and Box 2 regions of IL-2Rβ+γc; and

[0082] (c) the C-terminal region of IL-2Rβ+γc; or

[0083] (vi)

[0084] (a) Transmembrane domain of G-CSFR

[0085] (b) Box 1 and Box 2 of G-CSFR; and

[0086] (c) C-terminal region of IL-7Rα.

[0087] In certain embodiments, the ECD of G-CSFR is encoded by the nucleic acid sequence shown in SEQ ID NO. 5 or 6. In certain embodiments, the nucleic acid comprises:

[0088] (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

[0089] (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

[0090] (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

[0091] (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

[0092] (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

[0093] (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

[0094] (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

[0095] (h) A sequence encoding at least a portion of the ICD of IL-2Rγc having the amino acid sequence of SEQ ID NO. 17.

[0096] In certain aspects, described herein is an expression vector comprising a nucleic acid described herein. In certain embodiments, the vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and a plasmid.

[0097] In certain aspects, described herein is a cell 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 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), and optionally, the IL-2R is selected from the group consisting of IL-2Rβ and IL-2Rγc, and optionally, the second domain comprises at least a portion of the C-terminal region of IL-2Rβ, IL-7Rα, IL-12Rβ2 or IL-21R; wherein at least a portion of the ICD of the cytokine receptor comprises at least one signaling molecule binding site from the intracellular domain of the cytokine receptor, and optionally, the ICD comprises at least one signaling molecule binding site selected from the group consisting of: a STAT3 binding site of G-CSFR; STAT3 binding site of gp130; SHP-2 binding site of gp130; SHC binding site of IL-2Rβ; STAT5 binding site of IL-2Rβ; STAT3 binding site of IL-2Rβ; STAT1 binding site of IL-2Rβ; STAT5 binding site of IL-7Rα; phosphatidylinositol 3-kinase (PI3K) binding site of IL-7Rα; STAT4 binding site of IL-12Rβ2; STAT5 binding site of IL-12Rβ2; STAT3 binding site of IL-12Rβ2; I IL-21R STAT5 binding site; IL-21R STAT3 binding site; and IL-21R STAT1 binding site; and optionally, the ICD comprises box 1 and box 2 regions of a protein selected from the group consisting of G-CSFR and gp130; and optionally, the chimeric receptor comprises a third domain comprising at least a portion of a transmembrane domain of a protein selected from the group consisting of G-CSFR, gp130 (glycoprotein 130) and IL-2Rβ, and optionally, the transmembrane domain is a wild-type transmembrane domain; and optionally,

[0098] The cell is optionally an immune cell, and optionally a T cell, and optionally a NK cell, and optionally a NKT cell, and optionally a B cell, and optionally a plasma cell, and optionally a macrophage, and optionally a dendritic cell, and optionally the cell is a stem cell, and optionally the cell is a primary cell, and optionally the cell is a human cell.

[0099] In certain aspects, described herein is a cell comprising a nucleic acid encoding a chimeric receptor; wherein the chimeric receptor comprises: an ECD of G-CSFR operably linked to a second domain; the second domain comprising:

[0100] (i)

[0101] (a) Transmembrane domain of gp130;

[0102] (b) Box 1 and Box 2 regions of gp130; and

[0103] (c) the C-terminal region of IL-2Rβ; or

[0104] (ii)

[0105] (a) Transmembrane domain of G-CSFR;

[0106] (b) Box 1 and Box 2 of G-CSFR; and

[0107] (c) the C-terminal region of IL-2Rβ; or

[0108] (iii)

[0109] (a) Transmembrane domain of G-CSFR;

[0110] (b) Box 1 and Box 2 of G-CSFR; and

[0111] (c) the C-terminal region of IL-12Rβ2; or

[0112] (iv)

[0113] (a) Transmembrane domain of G-CSFR;

[0114] (b) Box 1 and Box 2 of G-CSFR; and

[0115] (c) the C-terminal region of IL-21R; or

[0116] (v)

[0117] (a) Transmembrane domain of IL-2Rβ+γc;

[0118] (b) Box 1 and Box 2 regions of IL-2Rβ+γc; and

[0119] (c) the C-terminal region of IL-2Rβ+γc; or

[0120] (vi)

[0121] (a) Transmembrane domain of G-CSFR

[0122] (b) Box 1 and Box 2 of G-CSFR; and

[0123] (c) the C-terminal region of IL-7Rα; and optionally,

[0124] The cell is an immune cell; and optionally is a T cell, and optionally is a NK cell, and optionally is a NKT cell, and optionally is a B cell, and optionally is a plasma cell, and optionally is a macrophage, and optionally is a dendritic cell, and optionally the cell is a stem cell, and optionally the cell is a primary cell, and optionally the cell is a human cell.

[0125] In certain embodiments, the ECD of G-CSFR is encoded by a nucleic acid contained in the cell having the sequence shown in SEQ ID NO. 5 or 6. In certain embodiments, the nucleic acid contained in the cell comprises:

[0126] (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

[0127] (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

[0128] (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

[0129] (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

[0130] (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

[0131] (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

[0132] (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

[0133] (h) A sequence encoding at least a portion of the ICD of IL-2Rγc having the amino acid sequence of SEQ ID NO. 17.

[0134] In some aspects, described herein is a cell comprising an expression vector described herein, and optionally, the cell is an immune cell, and optionally a T cell or NK cell. In some aspects, described herein is a cell comprising a chimeric receptor as described in claim 1, and optionally a cell in an immune cell, and optionally a T cell, and optionally a NK cell, and optionally a NKT cell, and optionally a B cell, and optionally a plasma cell, and optionally a macrophage, and optionally a dendritic cell, and optionally, the cell is a stem cell, and optionally, the cell is a primary cell, and optionally, the cell is a human cell.

[0135] In certain aspects, described herein are cells comprising a chimeric receptor described herein, and optionally are cells in an immune cell, and optionally are T cells, and optionally are NK cells, and optionally are NKT cells, and optionally are B cells, and optionally are plasma cells, and optionally are macrophages, and optionally are dendritic cells, and optionally the cells are stem cells, and optionally the cells are primary cells, and optionally the cells are human cells.

[0136] In certain aspects, 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 that selectively activates the 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; wherein the second domain comprises (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-1 receptor), IL-23R (interleukin-2 receptor), IL-7R (interleukin-7 receptor), IL-12R (interleukin-1 receptor), IL-13R (interleukin-1 receptor), IL-14R (interleukin-1 receptor), IL-15R (interleukin-1 receptor), IL-16R (interleukin-1 receptor), IL-17R (interleukin-1 receptor), IL-18R (interleukin-1 receptor), IL-19R (interleukin-2 receptor), IL-20R (interleukin-2 receptor), IL-21R (interleukin-2 receptor), IL-22R (interleukin-2 receptor), IL-23R (interleukin-2 receptor), IL-24R (interleukin-2 receptor), IL-25R (interleukin-2 receptor), IL-26R (interleukin-2 receptor), IL-27R (interleukin-2 receptor), IL-28R (interleukin-2 receptor), IL-29R (interleukin-2 receptor), IL-30R (interleukin-3 receptor), IL-31R (interleukin-3 receptor), IL-32R ( R (interleukin-12 receptor) and IL-21R (interleukin-21 receptor), and optionally, the IL-2R is selected from the group consisting of IL-2Rβ and IL-2Rγc, and optionally, the second domain comprises at least a portion of the C-terminal region of IL-2Rβ, IL-7Rα, IL-12Rβ2 or IL-21R; wherein at least a portion of the ICD of the cytokine receptor comprises at least one signaling molecule binding site from the intracellular domain of the cytokine receptor, and optionally, the at least one signaling molecule binding site is selected from the group consisting of: G-C STAT3 binding site of SFR; STAT3 binding site of gp130; SHP-2 binding site of gp130; SHC binding site of IL-2Rβ; STAT5 binding site of IL-2Rβ; STAT3 binding site of IL-2Rβ; STAT1 binding site of IL-2Rβ; STAT5 binding site of IL-7Rα; phosphatidylinositol 3-kinase (PI3K) binding site of IL-7Rα; STAT4 binding site of IL-12Rβ2; STAT5 binding site of IL-12Rβ2; ST binding site of IL-12Rβ2 AT3 binding site; STAT5 binding site of IL-21R; STAT3 binding site of IL-21R; and STAT1 binding site of IL-21R, and optionally, the ICD comprises box 1 and box 2 regions of a protein selected from the group consisting of G-CSFR and gp130; and optionally, the chimeric receptor comprises a third domain, said third domain comprising at least a portion of a transmembrane domain of a protein selected from the group consisting of G-CSFR, gp130 (glycoprotein 130) and IL-2Rβ, and optionally, the transmembrane domain is a wild-type transmembrane domain.

[0137] In certain aspects, 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 that selectively activates the chimeric receptor; wherein the chimeric receptor comprises an ECD of G-CSFR operably linked to a second domain; the second domain comprising:

[0138] (i)

[0139] (a) Transmembrane domain of gp130;

[0140] (b) Box 1 and Box 2 regions of gp130; and

[0141] (c) the C-terminal region of IL-2Rβ; or

[0142] (ii)

[0143] (a) Transmembrane domain of G-CSFR;

[0144] (b) Box 1 and Box 2 of G-CSFR; and

[0145] (c) the C-terminal region of IL-2Rβ; or

[0146] (iii)

[0147] (a) Transmembrane domain of G-CSFR;

[0148] (b) Box 1 and Box 2 of G-CSFR; and

[0149] (c) the C-terminal region of IL-12Rβ2; or

[0150] (iv)

[0151] (a) Transmembrane domain of G-CSFR;

[0152] (b) Box 1 and Box 2 of G-CSFR; and

[0153] (c) the C-terminal region of IL-21R; or

[0154] (v)

[0155] (a) Transmembrane domain of IL-2Rβ+γc;

[0156] (b) Box 1 and Box 2 regions of IL-2Rβ+γc; and

[0157] (c) the C-terminal region of IL-2Rβ+γc; or

[0158] (vi)

[0159] (a) Transmembrane domain of G-CSFR

[0160] (b) Box 1 and Box 2 of G-CSFR; and

[0161] (c) C-terminal region of IL-7Rα.

[0162] In certain embodiments of the methods described herein, the activated chimeric receptor forms homodimers, and optionally, activation of the chimeric receptor results in a cellular response selected from the group consisting of: increased proliferation, viability, and activity of cells expressing the chimeric receptor; and optionally, the chimeric receptor is activated upon contact with G-CSF, and optionally, the G-CSF is wild-type G-CSF, and optionally, the extracellular domain of G-CSFR is a wild-type extracellular domain; wherein the chimeric receptor is expressed in a cell, and optionally is an immune cell, and optionally is a T cell, and optionally is a NK cell, and optionally is a NKT cell, and optionally is a B cell, and optionally is a plasma cell, and optionally is a macrophage, and optionally is a dendritic cell, and optionally is a stem cell, and optionally is a primary cell, and optionally is a human cell.

[0163] In certain embodiments of the methods described herein, the chimeric receptor comprises

[0164] (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

[0165] (b) at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO. 41; or

[0166] (c) at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO. 25 or 27; or

[0167] (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

[0168] (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

[0169] (f) at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO. 18 or 38; or

[0170] (g) at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO. 43; or

[0171] (h) at least a portion of the ICD of IL-2Rγc having the amino acid sequence of SEQ ID NO. 17; and wherein the transmembrane domain comprises the sequence shown by: (a) SEQ ID NO. 8; or

[0172] (b) SEQ ID NO.9; or (c) SEQ ID NO.10; or (d) SEQ ID NO.11.

[0173] In certain aspects, described herein is a method of producing a chimeric receptor in a cell, comprising: introducing a nucleic acid as described in any one of claims 13-16 or 19-22 or an expression vector as described in any one of claims 17, 18, 23 or 24 into a cell; and optionally, the method comprises gene editing; and optionally, the cell is an immune cell, and optionally a T cell, and optionally a NK cell, and optionally a NKT cell, and optionally a B cell, and optionally a plasma cell, and optionally a macrophage, and optionally a dendritic cell, and optionally, the cell is a primary cell, and optionally, the cell is a human cell.

[0174] In certain embodiments, described herein is a method of treating a subject in need thereof, comprising: infusing into the subject a cell expressing a chimeric receptor, and administering a cytokine that binds to the 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 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-1 and optionally, the IL-2R is selected from the group consisting of IL-2Rβ and IL-2Rγc; and optionally, the second domain comprises at least a portion of the C-terminal region of IL-2Rβ, IL-7Rα, IL-12Rβ2 or IL-21R; wherein at least a portion of the ICD of the cytokine receptor comprises at least one signaling molecule binding site from the intracellular domain of the cytokine receptor; and optionally, the ICD comprises at least one signaling molecule binding site selected from the group consisting of Points: STAT3 binding site of G-CSFR; STAT3 binding site of gp130; SHP-2 binding site of gp130; SHC binding site of IL-2Rβ; STAT5 binding site of IL-2Rβ; STAT3 binding site of IL-2Rβ; STAT1 binding site of IL-2Rβ; STAT5 binding site of IL-7Rα; phosphatidylinositol 3-kinase (PI3K) binding site of IL-7Rα; STAT4 binding site of IL-12Rβ2; STAT5 binding site of IL-12Rβ2; S TAT3 binding site; STAT5 binding site of IL-21R; STAT3 binding site of IL-21R; and STAT1 binding site of IL-21R; and optionally, the ICD comprises box 1 and box 2 regions of a protein selected from the group consisting of G-CSFR and gp130; and optionally, the chimeric receptor comprises a third domain comprising at least a portion of a transmembrane domain of a protein selected from the group consisting of G-CSFR, gp130 (glycoprotein 130) and IL-2Rβ, and optionally, the transmembrane domain is a wild-type transmembrane domain.

[0175] In certain aspects, described herein is a method of treating a subject in need thereof, comprising: infusing into the subject a cell expressing a chimeric receptor, and administering a cytokine that binds to the chimeric receptor; wherein the chimeric receptor comprises: an ECD of G-CSFR operably linked to a second domain; the second domain comprising:

[0176] (i)

[0177] (a) Transmembrane domain of gp130;

[0178] (b) Box 1 and Box 2 regions of gp130; and

[0179] (c) the C-terminal region of IL-2Rβ; or

[0180] (ii)

[0181] (a) Transmembrane domain of G-CSFR;

[0182] (b) Box 1 and Box 2 of G-CSFR; and

[0183] (c) the C-terminal region of IL-2Rβ; or

[0184] (iii)

[0185] (a) Transmembrane domain of G-CSFR;

[0186] (b) Box 1 and Box 2 of G-CSFR; and

[0187] (c) the C-terminal region of IL-12Rβ2; or

[0188] (iv)

[0189] (a) Transmembrane domain of G-CSFR;

[0190] (b) Box 1 and Box 2 of G-CSFR; and

[0191] (c) the C-terminal region of IL-21R; or

[0192] (v)

[0193] (a) Transmembrane domain of IL-2Rβ+γc;

[0194] (b) Box 1 and Box 2 regions of IL-2Rβ+γc; and

[0195] (c) the C-terminal region of IL-2Rβ+γc; or

[0196] (vi)

[0197] (a) Transmembrane domain of G-CSFR

[0198] (b) Box 1 and Box 2 of G-CSFR; and

[0199] (c) C-terminal region of IL-7Rα.

[0200] In certain embodiments of the method, the activated chimeric receptor forms a homodimer; and optionally, activation of the chimeric receptor causes a cellular response selected from the group consisting of: increased proliferation, viability, and activity of cells expressing the chimeric receptor; and optionally, the chimeric receptor is activated upon contact with G-CSF; and optionally, the G-CSF is wild-type G-CSF; and optionally, the extracellular domain of G-CSFR is a wild-type extracellular domain; wherein the chimeric receptor is expressed in a cell; and optionally, the cell is an immune cell, and optionally a T cell, and optionally a NK cell, and optionally a NKT cell, and optionally a B cell, and optionally a plasma cell, and optionally a macrophage, and optionally a dendritic cell, and optionally, the cell is a stem cell, and optionally, the cell is a primary cell, and optionally, the cell is a human cell. In certain embodiments, the chimeric receptor optionally comprises:

[0201] (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

[0202] (b) at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO. 41; or

[0203] (c) at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO. 25 or 27; or

[0204] (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

[0205] (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

[0206] (f) at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO. 18 or 38; or

[0207] (g) at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO. 43; or

[0208] (h) at least a portion of the ICD of IL-2Rγc having the amino acid sequence of SEQ ID NO.17; and wherein the transmembrane domain comprises the sequence shown by: (a) SEQ ID NO.8; or (b) SEQ ID NO.9; or (c) SEQ ID NO.10; or (d) SEQ ID NO.11.

[0209] In certain embodiments, the methods described herein are used to treat cancer. In certain embodiments, the methods are used to treat autoimmune diseases. In certain embodiments, the methods are used to treat inflammatory conditions. In certain embodiments, the methods are used to prevent and treat transplant rejection. In certain embodiments, the methods are used to treat infectious diseases. In certain embodiments, the methods further comprise administering at least one additional active agent; and optionally, the additional active agent is another cytokine.

[0210] In certain embodiments, the methods described herein include: i) isolating a sample containing immune cells; (ii) transducing or transfecting immune cells with a nucleic acid sequence encoding a chimeric cytokine receptor; (iii) administering or infusing the immune cells from (ii) to the subject; and (iv) contacting immune cells with cytokines that bind to chimeric receptors. In certain embodiments, before administering or infusing the cells to the subject, the subject has undergone immunodepletion therapy. In certain embodiments, the sample containing immune cells is separated from the subject to whom the cells will be administered or infused. In certain embodiments, before administering or infusing the cells to the subject, the immune cells are contacted with cytokines in vitro. In certain embodiments, immune cells are contacted with cytokines that bind to chimeric receptors for a sufficient time to activate signal transduction from the chimeric receptor.

[0211] Described herein is a kit for treating a subject in need thereof, comprising: a cell encoding a chimeric receptor described herein, and optionally, the cell is an immune cell; and instructions for use; and optionally, the kit includes a cytokine that binds the chimeric receptor. Described herein is a kit for producing a chimeric receptor expressed on a cell, comprising: an expression vector encoding a chimeric receptor described herein and instructions for use; and optionally, the kit includes a cytokine that binds the chimeric receptor.

[0212] Described herein is a kit for producing a chimeric receptor expressed on a cell, comprising: a cell comprising an expression vector encoding the chimeric receptor described herein, and optionally, the cell is a bacterial cell; and instructions for use; and optionally, the kit comprises a cytokine that binds the chimeric receptor. BRIEF DESCRIPTION OF THE DRAWINGS

[0213] These and other features, aspects and advantages of the present invention will be better understood with reference to the following description and accompanying drawings, in which:

[0214] Figure 1 is a diagram showing the structure of the Site II and III interface of the 2:2 G-CSF:G-CSFR heterodimer complex.

[0215] Figure 2 is a diagram outlining the strategy used for G-CSF:G-CSFR interface design.

[0216] Figure 3 is a graph showing the energy components of the Site II interface interaction.

[0217] Figure 4 Diagram showing the structure of the Site II interface and the interactions of Arg167 (left) and Arg141 (right) of G-CSFR (CRH) with G-CSF residues at the Site II interface.

[0218] Figure 5 Figure 3 shows triplicate ZymeCAD images of wild-type G-CSF at site II (left) and site II design #35. TM Overlay (right) of the same region of the mean field package.

[0219] Figure 6 are images of SDS-PAGE showing the results of G-CSF pulldown assays of G-CSF designs #6, 7, 8, 9, 15, 17, 30, 34, 35, and 36 (upper panel) and co-expressed and purified Site II design complexes #6, 7, 8, 9, 15, 17, 30, 34, 35, and 36 (lower panel), WT G-CSF control in the first lane, and WT G-CSF:G-CSFR (CRH) control in the second lane.

[0220] Figure 7 are images of SDS-PAGE showing the results of G-CSF pull-down assays of G-CSF designs #6, 7, 8, 9, 15, 17, 30, 34, 35, and 36 co-expressed with WT-G-CSFR (upper panel) and WT G-CSF co-expressed with G-CSFR designs #6, 7, 8, 9, 15, 17, 30, 34, 35, and 36 (lower panel, first lane WT:WT G-CSF:G-CSFR pull-down control).

[0221] Figure 8 is a graph showing the energy components of the Site III interface interaction.

[0222] Figure 9Diagram showing the structure of the Site III interface and the interactions of R41 (left) and E93 (right) of G-CSFR(Ig) with G-CSF residues at the Site III interface.

[0223] Figure 10 are images of SDS-PAGE showing the results of G-CSF pull-down assays of Designs #401 and 402 (top panel) and co-expressed and purified Site II / III Design complexes #401 and 402 and WT G-CSFR, and pull-down of WT G-CSF co-expressed with Designs #401 and 402 G-CSFR (bottom panel), WT G-CSF control in the first lane, and WT G-CSF:G-CSFR(Ig-CRH) control in the second lane.

[0224] Figure 11 : Figure 3 shows the expression of WT (SX75) G-CSF and Design 130 (SX75), 303 (SX200) and 401 (SX200) G-CSF after TEV cleavage. E Graph of the size exclusion chromatographic profiles of the mutants.

[0225] Figure 12 Figure 3: WT (SX75) purified after TEV cleavage and purified designs 401 (SX200) and 402 (SX200) G-CSFR (Ig-CRH) E Graph of the size exclusion chromatographic profiles of the mutants.

[0226] Figure 13 Figures 1 and 2 are graphs showing binding SPR sensorgrams of WT, Design #130, #401, and #402 G-CSF binding to their cognate or mismatched G-CSFR (Ig-CRH). Each G-CSF to G-CSFR pair is labeled at the bottom of each graph. Representative steady-state fits used to derive the KD for the cognate pairs of Designs #401 and #402 are shown below their respective sensorgrams.

[0227] Figure 14 : are graphs showing: Upper left: WT G-CSF, Design #130 and #134 G-CSF E DSC thermograms; upper right: WT G-CSFR(Ig-CRH), design #130 and #134 G-CSFR(Ig-CRH) E DSC thermograms; bottom left: Design #401 and #402 G-CSF E DSC thermograms; bottom right: Designs #300, #303, #304, and #307 G-CSF E DSC thermogram of .

[0228] Figure 15 : is a graph showing the results of bromodeoxyuridine (BrdU) assay, showing that the proliferating 32D-IL-2RβIL2Rb cells express the following: A) G-CSFR WT -ICD IL-2Rb (homodimer) or B) G-CSFR WT -ICD IL-2Rb +G-CSFR WT -ICD gc (heterodimer). Cells were not stimulated with cytokines, with IL-2 (300 IU / ml) or G-CSF WT (100 ng / ml in A or 30 ng / ml in B) to stimulate the cells.

[0229] Figure 16 : is a graph showing the results of BrdU assay, showing the proliferation of 32D-IL-2Rβ cells expressing the following: A) G-CSFR 137 -ICD gp130-IL-2Rb (homodimer); or B) G-CSFR 137 -ICD IL-2Rb +G-CSFR 137 -ICD gc (heterodimer). Cells were not stimulated with cytokines, and were stimulated with IL-2 (300 IUIUIU / ml), G-CSF WT (30ngngng / ml) or G-CSFR 137 (30 ng / ml) to stimulate the cells.

[0230] Figure 17 : is a graph showing the results of BrdU assay, showing the proliferation of 32D-IL-Rβ cells expressing the following: A) G-CSFR WT -ICD gp130-IL-2Rb (homodimer); or B) G-CSFR WT -ICD IL-2Rb +G-CSFR WT -ICD gc (heterodimer). Cells were not stimulated with cytokines, and were stimulated with IL-2 (300 IU / ml), G-CSF WT (30 ng / ml) or G-CSFR 137 (30 ng / ml) to stimulate the cells.

[0231] Figure 18 : is a Western blot showing signaling in 32D-IL-2Rβ cells expressing the following: G-CSFR WT -ICD gp130-IL-2Rb (homodimer), G-CSFR 137 -ICDgp130-IL-2Rb (homodimer), G-CSFR WT -ICD IL-2Rb +G-CSFR WT -ICD gc (heterodimer) or G-CSFR 137 -ICD IL-2Rb +G-CSFR 137 -ICD gc (heterodimer). Cells were not stimulated with cytokines, and were stimulated with IL-2 (300 IU / ml), G-CSF WT (30 ng / ml) or G-CSFR 137 (30 ng / ml) to stimulate the cells.

[0232] Figure 19 Graphs are presented showing the results of BrdU incorporation assays to assess cell cycle progression of primary murine T cells (or untransduced cells) expressing the indicated chimeric receptors in response to no cytokine stimulation, stimulation with IL-2, or WT, 130, 304, or 307 cytokines. A and B represent experimental replicates.

[0233] Figure 20 Schematic diagram of the designed natural IL-2Rβ, IL-2Rγc and G-CSFR subunits and G2R-1 receptor subunits.

[0234] Figure 21 Presented are graphs showing the expansion (fold change in cell number) of 32D-IL-2Rβ cells (a 32D cell line stably expressing the human IL-2Rβ subunit) expressing the indicated G-CSFR chimeric receptor subunits and stimulated with WT G-CSF, IL-2, or without cytokine stimulation. G / γc was tagged with the Myc epitope at its N-terminus (Myc / G / γc), and G / IL-2Rβ was tagged with the Flag epitope at its N-terminus (Flag / G / IL-2Rβ); these epitope tags facilitate detection by flow cytometry and do not affect the function of the receptor. In addition, the lower panel in BD shows the percentage of cells expressing G-CSFR ECD (% G-CSFR+) under each culture condition. Squares represent cells stimulated with IL-2. Triangles represent cells stimulated with G-CSF. Circles represent cells not stimulated with cytokines.

[0235] Figure 22Presented are graphs showing the expansion (fold change in cell number) of human T cells expressing only the Flag-tagged G / IL-2Rβ subunit, only the Myc-tagged G / γc subunit, or full-length G-CSFR. A-D) PBMC-derived T cells; E-H) Tumor-associated lymphocytes (TAL). Squares represent cells stimulated with IL-2. Triangles represent cells stimulated with G-CSF. Circles represent cells not stimulated with cytokines.

[0236] Figure 23 is a schematic diagram of the native and chimeric receptors showing JAK, STAT, She, SHP-2, and PI3K binding sites. The shaded scheme includes Figure 1 receptors.

[0237] Figure 24 is a schematic diagram of the chimeric receptor showing the Jak, STAT, She, SHP-2, and PI3K binding sites. The shaded scheme includes Figure 1 and 4 receptors.

[0238] Figure 25 is a diagram of a lentiviral plasmid containing a G2R-2 cDNA insert.

[0239] Figure 26 Graphs showing G-CSFR ECD expression assessed by flow cytometry in cells transduced with G2R-2 are presented. A) 32D-IL-2Rβ cell line; B) PBMC-derived human T cells and human tumor-associated lymphocytes (TAL).

[0240] Figure 27 Presented are graphs showing the expansion of G2R-2-expressing cells (fold change in cell number) compared to untransduced cells. A) Human PBMC-derived T cells; B, C) Human tumor-associated lymphocytes (TAL) from two independent experiments. Squares represent cells stimulated with IL-2. Triangles represent cells stimulated with G-CSF. Circles represent cells not stimulated with cytokines.

[0241] Figure 28 Presented are graphs showing the expansion (fold change in cell number) of CD4- or CD8-selected human tumor-associated lymphocytes expressing G2R-2 compared to untransduced cells. A) Untransduced CD4-selected cells; B) Untransduced CD8-selected cells; C) CD4-selected cells transduced with G2R-2; D) CD8-selected cells transduced with G2R-2. The gray dashed line represents cells stimulated with IL-2. The black solid line represents cells stimulated with G-CSF. The gray dashed line represents cells not stimulated with cytokines.

[0242] Figure 29 A graph showing the expansion (fold change in cell number) of CD4+ or CD8+ tumor-associated lymphocytes expressing G2R-2 is presented. As shown, cells were initially expanded in G-CSF or IL-2. Cells were then plated in IL-2, G-CSF, or culture medium alone. The gray solid line represents cells stimulated with IL-2. The gray solid line represents cells stimulated with IL-2. The black solid line represents cells stimulated with G-CSF. The light gray dotted line represents cells expanded in IL-2 and then stimulated with culture medium alone. The dark gray dotted line represents cells expanded in G-CSF and then stimulated with culture medium alone.

[0243] Figure 30 Graphs are presented showing the immunophenotype (by flow cytometry) of CD4- or CD8-selected tumor-associated lymphocytes (TALs) expressing the G2R-2 chimeric receptor construct following expansion in G-CSF or IL-2 relative to untransduced cells. A) Percentage of viable cells showing the CD4+, CD8+, or CD3-CD56+ cell surface phenotype. B) Percentage of viable cells showing the indicated cell surface phenotype based on CD45RA and CCR7 expression.

[0244] Figure 31 Graphs showing the results of BrdU incorporation assays to assess the proliferation of primary human T cells expressing G2R-2 relative to untransduced cells are presented. Prior to the assay, T cells were selected by culturing in IL-2 or G-CSF, as indicated. A) Tumor-associated lymphocytes; B) PBMC-derived T cells.

[0245] Figure 32 Presented are graphs showing the results of BrdU incorporation assays to assess the proliferation of primary murine T cells expressing G2R-2 or single-chain G / IL-2Rβ (a component of G2R-1) relative to mock-transduced cells. A) Transduction efficiency, as reflected by the percentage of cells expressing G-CSFR ECD (by flow cytometry) after culture in the indicated cytokines; B) Percentage of BrdU incorporation in all viable cells in response to the indicated cytokines; C) Percentage of BrdU incorporation in cells expressing G-CSFR ECD (G-CSFR+ cells). All cells were expanded in IL-2 for 3 days prior to the assay. Squares represent cells stimulated with IL-2. Triangles represent cells stimulated with G-CSF. Circles represent cells not stimulated with cytokines.

[0246] Figure 33Western blots are shown to detect the indicated cytokine signaling events in human primary T cells expressing G2R-2 relative to untransduced cells. β-actin, total Akt, and histone H3 served as protein loading controls. A, B) Tumor-associated lymphocytes (TAL); C) PBMC-derived T cells.

[0247] Figure 34 Western blots are presented to detect the indicated cytokine signaling events in primary murine T cells expressing G2R-2 or single-chain G / IL-2Rβ (from G2R-1) relative to mock-transduced cells. Arrows indicate specific phospho-Jak2 bands, and other larger bands are presumed to be the result of cross-reaction of the primary anti-phospho-Jak2 antibody with phospho-Jak1. β-Actin and histone H3 served as protein loading controls.

[0248] Figure 35 is a graph showing the results of BrdU incorporation assays to assess cell cycle progression of 32D-IL-2Rβ cells expressing the indicated chimeric receptors (or untransduced cells) in response to no cytokine stimulation, stimulation with IL-2 (300 IU / mL), WT G-CSF (30 ng / mL), or 130G-CSF (30 ng / mL).

[0249] Figure 36 Graphs are presented showing the results of BrdU incorporation assays to assess cell cycle progression of primary murine T cells (or untransduced cells) expressing the indicated chimeric receptors in response to no cytokine stimulation, stimulation with IL-2, or WT, 130, 304, or 307 cytokines. A and B represent experimental replicates.

[0250] Figure 37 Western blots are presented to detect the indicated cytokine signaling events in 32D-IL-2Rβ cells (or untransduced cells) expressing the indicated chimeric receptor subunits in response to no cytokine stimulation, stimulation with IL-2, WT G-CSF, or 130G-CSF. β-Actin and histone H3 served as protein loading controls.

[0251] Figure 38 Presented are A) Western blots detecting the indicated cytokine signaling events in primary murine T cells expressing the indicated chimeric receptor subunits in response to no cytokine stimulation, stimulation with IL-2, WT G-CSF, 130G-CSF, or 304G-CSF. β-Actin and histone H3 served as protein loading controls. B) Transduction efficiency of the cells used in panel A, as assessed by flow cytometry using an antibody specific for the extracellular domain of the human G-CSF receptor.

[0252] Figure 39Figures are presented showing G-CSFR ECD expression by flow cytometry in primary human tumor-associated lymphocytes (TAL) transduced with the indicated chimeric receptor constructs. Live CD3+, CD56- cells were gated for CD8 or CD4, and G-CSFR ECD expression for each population is shown.

[0253] Figure 40 Presented are graphs and images showing the expansion, proliferation, and signaling of primary human tumor-associated lymphocytes (TALs) expressing G2R-3 relative to untransduced cells. A) Graph showing the results of a T cell expansion assay in which cells were transduced with G2R-3-encoding lentivirus, washed, and replated in IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokines. Viable cells were counted every 3-4 days. Squares represent cells stimulated with IL-2. Triangles represent cells stimulated with G-CSF. Circles represent cells not stimulated with cytokines. B) Western blots assessing intracellular signaling events. Cells were harvested from the expansion assay and stimulated with IL-2 (300 IU / ml) or wild-type G-CSF (100 ng / ml). Arrows indicate a specific phospho-Jak2 band at 125 kDa; the larger band is presumably the result of cross-reaction of the primary anti-phospho-Jak2 antibody with phospho-Jak1. β-actin and histone H3 served as protein loading controls. C) Graph showing the results of a BrdU incorporation assay to assess T cell proliferation. Cells were harvested from the expansion assay, washed, and replated in IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine.

[0254] Figure 41Figure 20 is a graph showing the expression of primary human PBMC-derived T cells with G2R-3 having WT ECD relative to the multiple amplification of non-transduced cells and the expression of G-CSFR ECD. A) A graph showing the results of T cell amplification assays is shown, in which cells are transduced with a lentiviral vector encoding G2R-3. On the 1st day, WT G-CSF (100 ng / ml) was added to the culture or cytokines (only culture medium) were not added. Thereafter, by the 21st day, cells were supplemented with culture medium containing WT G-CSF or cytokine-free culture medium. On the 21st day of amplification, cells were washed and re-plated in WT G-CSF (100 ng / mL), IL-7 (20 ng / mL) and IL-15 (20 ng / mL) or cytokine-free culture medium. Viable cells were counted every 2-4 days. Squares represent cells stimulated with G-CSF. Triangles represent cells stimulated with G-CSF and re-plated in IL-7 and IL-15 at the 21st day. Circles represent cells not stimulated with cytokines. Diamonds represent cells stimulated with G-CSF and replated in medium only on day 21. B) Graph showing expression of G-CSFR ECD as determined by flow cytometry on day 21 or day 42 of expansion.

[0255] Figure 42 Figures are presented showing the intracellular signaling and immunophenotype of primary human PBMC-derived T cells expressing G2R-3 relative to untransduced cells. A) Western blot to assess intracellular signaling events. Cells were harvested from the amplification assay and stimulated with IL-2 (300 IU / ml) or wild-type G-CSF (100 ng / ml). β-actin served as a protein loading control. B, C) Representative flow cytometry graphs and charts showing the immunophenotype of G2R-3-expressing cells relative to untransduced cells assessed by flow cytometry on day 42 of amplification.

[0256] Figure 43Present a chart showing the amplification times of primary human PBMC-derived T cells expressing G2R-3 with 304 or 307 ECD relative to untransduced cells. A) shows a chart of T cell amplification assay results, wherein cells are transduced with a lentivirus encoding G2R-3304 ECD. B) shows a chart of T cell amplification assay results, wherein cells are transduced with a lentivirus encoding G2R-3307 ECD. C) shows a chart of T cell amplification assay results of untransduced cells. As shown, IL-2 (300 IU / mL), 304 G-CSF (100 ng / ml), 307 G-CSF (100 ng / mL) or no cytokine (medium only) was added to the culture on day 2, and supplemented every two days thereafter. Viable cells were counted every 3-4 days. Diamonds represent cells stimulated with 304 G-CSF. Squares represent cells stimulated with 307 G-CSF. Triangles represent cells stimulated with IL-2. Inverted triangles indicate cells not stimulated with cytokines.

[0257] Figure 44 Present the chart showing BrdU incorporation assay results to assess the proliferation of primary human PBMC-derived T cells expressing G2R-3 with 304 or 307 ECD relative to non-transduced cells. Cells were transduced with a lentivirus encoding G2R-3 304 ECD or 307 ECD and amplified in 304 or 307 G-CSF (100 ng / mL). Non-transduced cells were amplified in IL-2 (300 IU / mL). Cells were washed at day 12 of amplification and re-plated in IL-2 (300 IU / ml), 130 G-CSF (100 ng / ml), 304 G-CSF (100 ng / ml), 307 G-CSF (100 ng / ml) or without cytokines.

[0258] Figure 45 Graphs showing G-CSFR ECD expression by flow cytometry in primary murine T cells transduced with the indicated chimeric receptor constructs are presented.

[0259] Figure 46 Shown is G-CSF-induced STAT3 phosphorylation (detected by flow cytometry) in primary PBMC-derived human T cells expressing G21R-1 or G21R-2. Cells are subdivided (i.e., gated) into G-CSFR positive (upper panel) or G-CSFR negative (lower panel) populations.

[0260] Figure 47Figure 24 is a graph showing the biochemical signaling events induced by G-CSF in primary mouse T cells expressing G21R-1 or G12R-1. A) shows a graph of the phosphorylation of STAT3 (detected by flow cytometry) in CD4+ or CD8+ cells that were transduced with G21R-1 and not stimulated by cytokines, stimulated by IL-21 or G-CSF. B) shows a graph showing the percentage of cells that are positive for phospho-STAT3 staining after stimulation with IL-21 (squares) or WT G-CSF (grey circles) without cytokines (black circles). Live cells were gated for CD8 or CD4, and the percentage of phospho-STAT3-positive cells in each colony was shown. C) Western blot was performed to assess the indicated cytokine signaling events in cells expressing G21R-1 or G12R-1 and stimulated by IL-21, IL-12 or WT G-CSF. Beta actin and histone H3 served as protein loading controls.

[0261] Figure 48 Presented are graphs and images showing proliferation, G-CSFR ECD expression, and WT G-CSF-induced intracellular signaling events in primary murine T cells expressing G2R-2, G2R-3, G7R-1, G21 / 7R-1, and G27 / 2R-1 or mock-transduced T cells. A, B) Graphs showing the results of BrdU incorporation assays to assess T cell proliferation. Cells were harvested, washed, and replated in IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. Panels A and B are replicates of the experiment. C) Graph showing G-CSFR ECD expression by flow cytometry in primary murine T cells transduced with the indicated chimeric receptor constructs. D) Western blot to assess the indicated cytokine signaling events in cells expressing G2R-2, G2R-3, G7R-1, G21 / 7R-1, and G27 / 2R-1 or mock-transduced T cells. Cells were stimulated with IL-2 (300 IU / mL), IL-7 (10 ng / mL), IL-21 (10 ng / mL), IL-27 (50 ng / mL), or G-CSF (100 ng / mL). β-actin and histone H3 served as protein loading controls.

[0262] Figure 49Presented are graphs and images showing proliferation, G-CSFR ECD expression, and G-CSF-induced biochemical signaling events in primary murine T cells expressing G21 / 2R-1, G12 / 2R-1, and 21 / 12 / 2R-1, or mock-transduced T cells. A, B) Graphs showing the results of BrdU incorporation assays to assess T cell proliferation. Cells were harvested, washed, and replated in IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. Panels A and B are replicates of the experiment. C) Graph showing G-CSFR ECD expression by flow cytometry in primary murine T cells transduced with the indicated chimeric receptor constructs. D) Western blots to assess the indicated cytokine signaling events in cells expressing G21 / 2R-1, G12 / 2R-1, and 21 / 12 / 2R-1, or mock-transduced T cells. Cells were stimulated with IL-2 (300 IU / mL), IL-21 (10 ng / mL), IL-12 (10 ng / mL), or G-CSF (100 ng / mL). β-actin and histone H3 served as protein loading controls.

[0263] Figure 50 Figure 1 shows a graph of the amplification times and G-CSFR ECD expression of primary human PBMC-derived T cells expressing G12 / 2R-1 with 134 ECD relative to non-transduced cells. A) A graph showing the results of T cell amplification assays, wherein cells were transduced with a lentivirus encoding G12 / 2R-1_134-ECD and amplified in IL-2 (300 IU / mL), 130G-CSF (100 ng / ml) or culture medium. Viable cells were counted every 4-5 days. Squares represent cells not stimulated with cytokines. Triangles represent cells stimulated with 130G-CSF. Diamonds represent cells stimulated with IL-2. B) A graph showing the results of T cell amplification assays, wherein cells were transduced as in Figure A. On the 19th day of amplification, cells were washed and re-plated in IL-2, 130G-CSF or culture medium only. Viable cells were counted every 4-5 days. Squares represent cells not stimulated with cytokines. Light gray diamonds represent cells stimulated with 130G-CSF. Dark gray diamonds represent cells stimulated with IL-2. Light gray inverted triangles represent cells initially stimulated with IL-2 and then replated in culture medium only on day 19. Dark gray triangles represent cells initially stimulated with 130G-CSF and then replated in culture medium only on day 19. C) Graph showing expression of G-CSFR ECD, as determined by flow cytometry on day 4 or day 16 of expansion.

[0264] Figure 51Figure 1 shows a graph of the proliferation and immunophenotype of primary human PBMC-derived T cells expressing G12 / 2R-1 with 134 ECD relative to untransduced cells. A) A graph showing the results of BrdU incorporation assays to assess T cell proliferation. Cells were harvested, washed, and re-plated in IL-2 (300 IU / ml), IL-2+IL-12 (300 IU / ml and 10 ng / mL, respectively), 130G-CSF (300 ng / ml), or culture medium alone. B, C) Representative flow cytometry graphs and graphs showing the immunophenotype of cells expressing G12 / 2R-1 with 134 ECD relative to untransduced cells assessed by flow cytometry at day 16 of amplification.

[0265] Figure 52 Presented is a chart showing the expansion fold and proliferation of primary human PBMC-derived T cells expressing G12 / 2R-1 with 304 ECD relative to untransduced cells. A) A chart showing the results of a T cell expansion assay in which cells were transduced with a lentivirus encoding G12 / 2R-1_134-ECD and expanded in IL-2 (300 IU / mL), 130G-CSF (100 ng / ml), 304G-CSF (100 ng / ml), or culture medium alone. Untransduced cells were cultured in IL-2, 130G-CSF, 304G-CSF, or culture medium alone. Viable cells were counted every 3-4 days. Inverted triangles represent cells not stimulated with cytokines. Triangles represent cells stimulated with IL-2. Circles represent cells stimulated with 130G-CSF. Diamonds represent cells stimulated with 304G-CSF. B) Cells were harvested from expansion assays on day 12 and washed and replated in IL-2 (300 IU / ml), 130G-CSF (300 ng / ml), 304G-CSF (100 ng / ml), 307G-CSF (100 ng / ml), or medium alone.

[0266] Figure 53 Western blots are presented to detect the indicated cytokine signaling events in primary PBMC-derived T cells expressing G2R-3 with 304 ECD, G12 / 2R-1 with 304 ECD, or untransduced T cells. Cells were harvested from amplification assays and stimulated with 304G-CSF (100 ng / mL), IL-2 (300 IU / mL), IL-2 and IL-12 (10 ng / mL), or culture medium alone as indicated. The black arrows and the right outset indicate molecular weight markers at 115 kDa and 140 kDa from the protein ladder. β-actin and histone H3 served as protein loading controls. DETAILED DESCRIPTION

[0267] In brief, and as described in more detail below, methods and compositions for selectively activating cells using variant cytokine receptors and cytokine pairs are described herein, wherein the cytokine receptor comprises a variant extracellular domain (ECD) of a granulocyte colony stimulating factor receptor (G-CSFR). In certain embodiments, the methods and compositions described herein can be used to specifically activate cells for adoptive cell transfer therapy. Thus, methods are included herein for producing cells expressing variant receptors that are selectively activated by cytokines that do not bind to their native receptors. Also disclosed herein are methods for treating subjects in need thereof, comprising administering to the subject a cell expressing a receptor comprising a variant ECD of G-CSFR, and co-administering a variant of G-CSF bound to the variant ECD of G-CSFR. In some aspects, the compositions and methods described herein address the urgent need for selective activation of cells for adoptive cell transfer methods, and can reduce or eliminate the need for immunodepletion of the subject or administration of broad-acting stimulatory cytokines such as IL-2 prior to adoptive cell transfer.

[0268] definition

[0269] Unless otherwise stated, the terms used in the claims and the specification are defined as set out below.

[0270] The term "treat" refers to any therapeutically beneficial result in treating a disease state (eg, a cancer disease state), including prevention thereof, lessening of its severity or progression, remission thereof, or cure thereof.

[0271] The term "in vivo" refers to processes that occur in a living organism.

[0272] As used herein, the term "mammal" includes humans and non-humans, and includes, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0273] The term "sufficient amount" refers to an amount sufficient to produce a desired effect, such as an amount sufficient to selectively activate a receptor expressed on a cell.

[0274] The term "therapeutically effective amount" is an amount effective to ameliorate symptoms of a disease. A therapeutically effective amount can be a "prophylactically effective amount" since prophylaxis can be considered treatment.

[0275] The term "operably linked" refers to a nucleic acid or amino acid sequence that is placed into a functional relationship with another nucleic acid or amino acid sequence, respectively. Generally, "operably linked" means that the nucleic acid or amino acid sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase.

[0276] As used herein, the term "extracellular domain" (ECD) refers to the domain of a receptor (e.g., G-CSFR) that is located outside the plasma membrane when expressed on the cell surface. In certain embodiments, the ECD of G-CSFR comprises at least a portion of SEQ ID NO. 2 or SEQ ID NO. 7.

[0277] As used herein, the term "intracellular domain" (ICD) refers to the domain of a receptor that is located inside a cell when the receptor is expressed on the surface of a cell.

[0278] As used herein, the term "transmembrane domain" (TMD or TM) refers to the domain or region of a cell surface receptor that is located within the plasma membrane when the receptor is expressed on the cell surface.

[0279] The term "cytokine" refers to a small protein (about 5-20 kDa) that binds to a cytokine receptor and can induce cell signaling after binding to and activating a cytokine receptor expressed on a cell. Examples of cytokines include, but are not limited to, interleukins, lymphokines, colony-stimulating factors, and chemokines.

[0280] The term "cytokine receptor" refers to a receptor that binds to a cytokine, including type 1 and type 2 cytokine receptors. 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).

[0281] As used herein, the term "chimeric receptor" refers to a transmembrane receptor that has been engineered to have at least a portion of at least one domain (e.g., ECD, ICD, TMD, or C-terminal region) derived from the sequence of one or more different transmembrane proteins or receptors.

[0282] As used herein, the terms "Site II interface," "Site II region," "Site II interface region," or "Site II" refer to the larger of the G-CSF:G-CSFR 2:2 heterodimer binding interfaces of G-CSF and G-CSFR, located at the interface between G-CSF and the cytokine receptor homology (CRH) domain of G-CSFR.

[0283] As used herein, the terms "Site III interface," "Site III region," "Site III interface region," or "Site III" refer to the smaller of the G-CSF:G-CSFR 2:2 heterodimer binding interfaces of G-CSF and G-CSFR, and are located between G-CSF and the N-terminal Ig-like domain of G-CSFR.

[0284] As used herein, the term "at least a portion" or "a portion" refers in certain aspects to greater than 75%, greater than 80%, greater than 90%, greater than 95%, or greater than 99% of the contiguous nucleic acid bases or amino acid length of a SEQ ID NO described herein. In certain aspects, 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 may have greater than 75%, greater than 80%, greater than 90%, greater than 95%, or greater than 99% identity to a SEQ ID NO described herein.

[0285] The term "wild type" refers to the natural amino acid sequence of a polypeptide or the natural 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 the species of the protein or gene.

[0286] The terms "variant cytokine-receptor pair," "variant cytokine and receptor pair," "variant cytokine and receptor design," "variant cytokine-receptor switch," or "orthogonal cytokine-receptor pair" refer to a genetically engineered protein pair that has been modified by amino acid changes to (a) lack binding to a native cytokine or cognate receptor; and (b) specifically bind to the corresponding engineered (variant) ligand or receptor.

[0287] 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.

[0288] As used herein, the term "variant ECD" refers to a genetically engineered extracellular domain of a receptor of a variant cytokine-receptor pair (eg, G-CSFR).

[0289] 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.

[0290] As used herein, "do not bind," "does not bind," or "cannot bind" means no detectable binding or insignificant binding, ie, with a binding affinity much lower than that of the natural ligand.

[0291] As used herein, the term "selectively activates" or "selective activation" when referring to a cytokine and a variant receptor refers to a cytokine that preferentially binds to a variant receptor, and the receptor is activated after the cytokine binds to the variant receptor. In some aspects, the cytokine selectively activates a chimeric receptor that has co-evolved to specifically bind to the cytokine. In some aspects, the cytokine is a wild-type cytokine, and it selectively activates a chimeric receptor expressed on a cell, while the native wild-type receptor for the cytokine is not expressed in the cell.

[0292] As used herein, the term "activity enhancement" refers to an increase in the activity of a variant receptor expressed on a cell when stimulated with a variant cytokine, wherein the activity is the activity observed for the native receptor when stimulated with the native cytokine.

[0293] The term "immune cell" refers to any cell known to support the function of the immune system (including innate immune response and adaptive immune response) of an organism, and includes but is not limited to lymphocytes (e.g., B cells, plasma cells and T cells), natural killer cells (NK cells), macrophages, monocytes, dendritic cells, neutrophils and granulocytes. Immune cells include stem cells, immature immune cells and differentiated cells. Immune cells also include any cell subset, no matter how rare or abundant in an organism. In certain embodiments, immune cells are identified by carrying known markers (e.g., cell surface markers) of immune cell types and subpopulations.

[0294] The term "T cell" refers to a mammalian immune effector cell that can be characterized by the expression of CD3 and / or T cell antigen receptor, and the cell can be engineered to express an orthologous cytokine receptor. In some embodiments, the T cell is selected from naive CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, helper T cells, such as T H 2. T H 9. T H 11. T H 22. T FH Regulatory T cells, such as T R 1. Natural T Reg , inducible T Reg ; Memory T cells, such as central memory T cells, effector memory T cells, NKT cells and γδ T cells.

[0295] 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 the gene with Ensembl identification number ENSG00000119535. Human G-CSFR is encoded by the cDNA sequence corresponding to GeneBank accession number NM_156039.3.

[0296] 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 the gene with Ensembl identification number ENSG00000108342. Human G-CSF is encoded by the cDNA sequence corresponding to GeneBank accession number KP271008.1.

[0297] "JAK" may also be referred to as Janus kinase. JAK is a family of intracellular non-receptor tyrosine kinases that transduce cytokine-mediated signals through the Jak-STAT pathway and includes JAK1, JAK2, JAK3, and TYK2. Human JAK1 is encoded by the gene with Ensembl identification number ENSG00000162434. Human JAK1 is encoded by the cDNA sequence corresponding to GeneBank accession number NM_002227. Human JAK2 is encoded by the gene with Ensembl identification number ENSG00000096968. Human JAK2 is encoded by the cDNA sequence corresponding to GeneBank accession number NM_001322194. Human JAK3 is encoded by the gene with Ensembl identification number ENSG00000105639. Human JAK3 is encoded by the cDNA sequence corresponding to GeneBank accession number NM_000215. Human TYK2 is encoded by the gene with Ensembl identification number ENSG00000105397. Human TYK2 is encoded by the cDNA sequence corresponding to GeneBank accession number NM_001385197.

[0298] STATs can also be referred to as signal transducers and activators of transcription. STATs are a family of seven STAT proteins: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. Human STAT1 is encoded by the gene with Ensembl identification number ENSG00000115415. Human STAT1 is encoded by the cDNA sequence corresponding to GeneBank accession number NM_007315. Human STAT2 is encoded by the gene with Ensembl identification number ENSG00000170581. Human STAT2 is encoded by the cDNA sequence corresponding to GeneBank accession number NM_005419. Human STAT3 is encoded by the gene with Ensembl identification number ENSG00000168610. Human STAT3 is encoded by the cDNA sequence corresponding to GeneBank accession number NM_139276. Human STAT4 is encoded by the gene with Ensembl identification number ENSG00000138378. Human STAT4 is encoded by the cDNA sequence corresponding to GeneBank Accession No. NM_003151. Human STAT5A is encoded by the gene with Ensembl identification No. ENSG00000126561. Human STAT5A is encoded by the cDNA sequence corresponding to GeneBank Accession No. NM_003152. Human STAT5B is encoded by the gene with Ensembl identification No. ENSG00000173757. Human STAT5B is encoded by the cDNA sequence corresponding to GeneBank Accession No. NM_012448. Human STAT6 is encoded by the gene with Ensembl identification No. ENSG00000166888. Human STAT6 is encoded by the cDNA sequence corresponding to GeneBank Accession No. NM_003153.

[0299] SHC can also be referred to as Src homology 2 domain-containing transforming proteins. The Shc family consists of three isoforms, including p66Shc, p52Shc, and p46Shc, SHC1, SHC2, and SHC3. Human SHC1 is encoded by the gene with Ensembl identification number ENSG00000160691. Human SHC1 is encoded by the cDNA sequence corresponding to GeneBank accession number NM_183001. Human SHC2 is encoded by the gene with Ensembl identification number ENSG00000129946. Human SHC2 is encoded by the cDNA sequence corresponding to GeneBank accession number NM_012435. Human SHC3 is encoded by the gene with Ensembl identification number ENSG00000148082. Human SHC3 is encoded by the cDNA sequence corresponding to GeneBank accession number NM_016848.

[0300] SHP-2 is also known as protein tyrosine phosphatase non-receptor type 11 (PTPN11) and protein tyrosine phosphatase 1D (PTP-1D). Human SHP-2 is encoded by the gene with Ensembl identification number ENSG00000179295. Human SHP-2 is encoded by the cDNA sequence corresponding to GeneBank accession number NM_001330437.

[0301] PI3K may also be referred to as phosphatidylinositol-4,5-bisphosphate 3-kinase. The catalytic subunit of PI3K may be referred to as PIK3CA. Human PIK3CA is encoded by the gene with Ensembl identification number ENSG00000121879. Human PIK3CA is encoded by the cDNA sequence corresponding to GeneBank accession number NM_006218.

[0302] Abbreviations used in this application include the following: 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 137 ECD"; and "G-CSFR137-ICDIL-2Rb GCSFR137-ICDgc" is also referred to herein as "G2R-1 with 137 ECD." IL-2Rγ (i.e., IL-2RG, IL-2Rgc, γc, or IL-2Rγc).

[0303] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0304] Where a numerical range is provided, it is understood that each intermediate value between the upper and lower limits of the range is also explicitly disclosed, wherein the lower limit is accurate to the tenth place unless the context explicitly dictates otherwise. Each smaller range between any stated value or intermediate value in a stated range and any other stated value or intermediate value in that stated range is encompassed within the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded within the range, and each range in which either, no, or both limits are included within the smaller range is also encompassed within the present invention and is subject to any explicitly excluded limits in the stated range. When an express range includes one or both of the stated limits, ranges excluding one or both of those included limits are also encompassed within the present invention.

[0305] Variant cytokine and receptor design

[0306] Variant cytokines and receptor pairs for selectively activating variant receptors are described herein. The variant receptors disclosed herein comprise the extracellular domain of G-CSFR; and the variant cytokine comprises G-CSF (granulocyte colony stimulating factor), which binds to and activates the variant receptor. In certain embodiments, the variant receptor is a chimeric receptor comprising the ECD of G-CSFR and at least a portion of the ICD of a receptor different from G-CSFR.

[0307] Variant G-CSF and variant G-CSFR ECD

[0308] In certain aspects, the variant G-CSF and receptor designs described herein comprise 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 comprise at least one Site II or Site III interface region mutation listed in Tables 2, 4, or 6.

[0309] In certain aspects, at least one mutation on the variant receptor in 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).

[0310] In certain aspects, at least one mutation in the Site II interface region of the variant G-CSF is 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).

[0311] In certain aspects, at least one mutation in the variant receptor site II interface region is selected from the group of mutations in the extracellular domain of G-CSFR consisting of: R141E, R167D, K168D, K168E, L171E, L172E, Y173K, Q174E, D197K, D197R, M199D, D200K, D200R, V202D, R288D, and R288E.

[0312] 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 in G-CSF 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.

[0313] In certain aspects, the at least one mutation in the variant site III interface region is selected from the group of mutations in the G-CSFR extracellular domain consisting of amino acid positions 30, 41, 73, 75, 79, 86, 87, 88, 89, 91 and 93 of SEQ ID NO. 2.

[0314] In certain aspects, at least one mutation in the variant Site III interface region is selected from the group of mutations of G-CSF consisting of amino acid positions 38, 39, 40, 41, 46, 47, 48, 49 and 147 of SEQ ID NO. 1.

[0315] In certain aspects, at least one mutation in the variant receptor site III interface region is selected from the group of mutations in the extracellular domain of G-CSFR consisting of: S30D, R41E, Q73W, F75K, S79D, L86D, Q87D, I88E, L89A, Q91D, Q91K, and E93K.

[0316] 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 of G-CSF consisting of: T38R, Y39E, K40D, K40F, L41D, L41E, L41K, E46R, L47D, V48K, V48R, L49K, and R147E.

[0317] The variant cytokine and receptor pairs described herein may comprise mutations in only the Site II region, only the Site III region, or both the Site II and Site III regions.

[0318] The variant cytokine and receptor pairs described herein may have any number of site II mutations and / or site III mutations described herein. In certain aspects, the variant G-CSF and receptor have the mutations listed in Table 6. In certain aspects, the variant receptor and / or variant G-CSF may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations described herein.

[0319] In certain aspects, 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 SEQ ID NO. described herein. In certain aspects, the chimeric receptor comprises an ECD of a G-CSFR having the amino acid sequence of SEQ ID NO. 2, 3, 6, or 8.

[0320] In certain aspects, the variant G-CSF described herein comprises 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 G-CSFR ECD SEQ ID NO. 1.

[0321] In certain aspects, the ECD of G-CSFR comprises at least one amino acid substitution selected from the group consisting of R41E, R141E, and R167D.

[0322] Variant cytokines and / or receptors can be produced not only by direct recombinant production, but also as fusion polypeptides with heterologous polypeptides (e.g., signal sequences or other polypeptides with specific cleavage sites at the N-terminus of mature proteins or polypeptides). Typically, the signal sequence can be a component of the vector, or it can also be a part of the coding sequence inserted into the vector. The selected heterologous signal sequence is preferably a sequence that is recognized and processed (i.e., cut by a signal peptidase) by the host cell. In mammalian cell expression, natural signal sequences can be used, or other mammalian signal sequences can be suitable, such as signal sequences of secretory polypeptides from the same or related species and viral secretory leader sequences. 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.

[0323] In certain aspects, the variant receptor and / or variant G-CSF is modified naturally or synthetically (e.g., glycosyl, 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 its half-life, for example, by pegylation, glycosylation, etc., as known in the art. Fc-fusion can also promote properties mediated by alternative Fc receptors in vivo. An "Fc region" can be a naturally occurring or synthetic polypeptide that is homologous to the C-terminal domain of IgG produced by digestion of IgG with papain. The molecular weight of IgG Fc is approximately 50 kDa. The variant cytokine can comprise the entire Fc region or a smaller portion that retains the ability to extend the circulating half-life of the chimeric polypeptide of which it is a part. In addition, the full-length or fragmented Fc region can be a variant of the wild-type molecule.

[0324] Upon binding of the variant cytokine to the variant receptor, the variant receptor activates signaling transduced through native cellular elements to provide a biological activity that mimics the native response, but is specific for cells engineered to express the variant receptor. In certain aspects, the variant receptor and G-CSF pair does not bind to its native wild-type G-CSF or native wild-type G-CSFR. Thus, in certain embodiments, the variant receptor does not bind to an endogenous counterpart cytokine, including the native counterpart of the variant cytokine, and 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 times, less than 100 times, less than 1,000 times, or less than 10,000 times 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 D Binding to natural receptors: greater than 1X10 -4 M, 1X10 -5 M, greater than 1X10 -6 M; larger than 1X10 -7 M, greater than 1X10 -8 M or larger than 1X10 -9 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 receptor less than 10 times, less than 100 times, less than 1,000 times, or less than 10,000 times the binding of the native cytokine to the native cytokine receptor. In certain embodiments, the variant cytokine receptor binds to the native cytokine receptor with a K of less than 10 times, less than 100 times, less than 1,000 times, or less than 10,000 times the binding of the native cytokine to the native cytokine receptor. DBinding natural cytokines: greater than 1X10 -4 M, 1X10 -5 M, greater than 1X10 -6 M; or larger than 1x10 -7 M, larger than 1x10 -8 M, or larger than 1x10 -9 M. In some embodiments, the affinity of the variant cytokine for the variant receptor can be comparable to the affinity of the native cytokine for the native receptor, for example, having an affinity that is at least about 1%, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 100% of the affinity of the native cytokine receptor for the native receptor, and can be higher, for example, 2 times, 3 times, 4 times, 5 times, 10 times or more the affinity of the native cytokine for the native receptor. Affinity can be determined by any number of assays well known to those skilled in the art. For example, affinity can be determined by competitive binding experiments that measure binding of the receptor using a single concentration of labeled ligand in the presence of various concentrations of unlabeled ligand. Typically, the concentration of unlabeled ligand is varied over at least six orders of magnitude. By competitive binding experiments, the IC 50 As used herein, “IC 50 IC is the concentration of unlabeled ligand required to inhibit the association between receptor and labeled ligand by 50%. 50 It is an indicator of ligand-receptor binding affinity. Low IC 50 indicates high affinity, while high IC 50 Indicates low affinity.

[0325] The binding of variant cytokines to variant cytokine receptors expressed on the cell surface may or may not affect the function of the variant cytokine receptor (compared to native cytokine receptor activity); native activity is not required or desired in all cases. In certain embodiments, the binding of variant cytokines to variant cytokine receptors will induce one or more aspects of native cytokine signaling. In certain embodiments, the binding of variant cytokines to variant cytokine receptors expressed on the cell surface causes a cellular response selected from the group consisting of: proliferation, viability, and activity enhancement.

[0326] Table 1: Sequences of human WT G-CSF and human WT G-CSFR Ig-CRH domains

[0327]

[0328]

[0329] Table 2: With G-CSF E and G-CSFRE Site II design of mutation.

[0330]

[0331]

[0332] Table 4: Site III design.

[0333] design# <![CDATA[Mutated G-CSF E > <![CDATA[Mutant G-CSFR E <!-- 28 -->]]> 51 E46R_L49K S30D_R41E 52 K40D_L41D F75K_Q91K 53 L41K_E46R R41E_Q91D 54 L41E_L47D I88K 55 T38R_E46R R41E_Q73E 56 K40D_R147E F75K_E93K 57 E46R_V48R R41E_Q87E 58 E46R R41E_L86D 59 K40D_E46R R41E_F75K 60 E46R R41E_I88D 61 E46R_L49F R41E_L89A 62 L41D Q91K 63 T38R Q73E 64 Y39E_K40D F75K 65 L41K I88E_Q91D 66 E46R R41E 67 E46R R41E_S79D 68 K40D F75K 69 V48K Q87D 70 R147E E93K 71 L41K Q91D 72 K40F F75K

[0334] Table 6: Example designs generated by combining Site II and III designs.

[0335]

[0336]

[0337] Chimeric receptors

[0338] In some aspects, variant receptors as described herein are chimeric receptors.Chimeric receptors may include any variant G-CSFR ECD domains as described herein. In some aspects, chimeric receptors also include at least a portion of the intracellular domain (ICD) of different cytokine receptors. The intracellular domain of different cytokine receptors can be selected from the group consisting of: gp130 (glycoprotein 130), IL-2R β or IL-2Rb (interleukin-2 receptor β), IL-2R γ or γc or IL-2RG (interleukin-2 receptor γ), IL-7R α (interleukin-7 receptor α), IL-12R β2 (interleukin-12 receptor β2) and IL-21R (interleukin-21 receptor). In certain aspects, 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 a cytokine receptor ICD described herein. In certain aspects, at least a portion of a 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.

[0339] In certain embodiments, described herein are chimeric cytokine receptors comprising an extracellular domain (ECD) of a G-CSFR (granulocyte colony stimulating factor receptor) operably linked to a second domain; said second domain comprising at least a portion of an intracellular domain (ICD) of a multisubunit cytokine receptor (e.g., IL-2R). In certain aspects, the chimeric cytokine receptor comprises a portion of an ICD from Table 15A and Table 15B. In certain aspects, the chimeric cytokine receptor comprises a transmembrane domain selected from Table 15A and Table 15B. In certain aspects, the chimeric cytokine receptor ICD comprises box 1 and box 2 districts from Table 15A, Table 15B, and Table 16. In certain aspects, the chimeric cytokine receptor comprises at least one signaling molecule binding site from Table 15A, Table 15B, and Table 16.

[0340] In certain aspects, the chimeric receptors described herein comprise an amino acid sequence from the N-terminus to the C-terminus of each of the sequences disclosed in Tables 17-20. In certain aspects, the sequence of the chimeric receptors described herein comprises a nucleic acid sequence from the 5' to the 3' sequence of each of the sequences disclosed in Tables 17-20. In certain aspects, the chimeric cytokine receptors share at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity with the amino acid sequence from the N-terminus to the C-terminus of each of the amino acid sequences disclosed in 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' to 3' order of each of the disclosed nucleic acid sequences in Tables 17-20.

[0341] In certain aspects, the chimeric receptors described herein comprise at least a portion of an 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 an ICD described herein as SEQ ID NO. In certain aspects, the chimeric receptor comprises at least a portion of an 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 aspects, the chimeric receptor comprises at least a portion of an 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 aspects, the chimeric receptor comprises at least a portion of an 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 aspects, 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 aspects, 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 aspects, 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 aspects, 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 aspects, 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 aspects, 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 aspects, 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 aspects, 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 aspects, 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 aspects, the chimeric receptor comprises at least a portion of the ICD of IL-2Rγ (i.e., IL-2RG, IL-2Rgc, γc, or IL-2Rγc) having the amino acid sequence of SEQ ID NO: 27.In certain aspects, the chimeric receptor comprises at least a portion of the ICD of IL-2Rγ (ie, IL-2RG, IL-2Rgc, γc, or IL-2Rγc) having the nucleic acid sequence of SEQ ID NO. 55.

[0342] In some aspects, at least a portion of the ICD described herein comprises at least one signaling molecule binding site. In some aspects, at least one signaling molecule binding site is a STAT3 binding site for G-CSFR; a STAT3 binding site for gp130; a SHP-2 binding site for gp130; a Shc binding site for IL-2Rβ; a STAT5 binding site for IL-2Rβ; a STAT3 binding site for IL-2Rβ; a STAT1 binding site for IL-2Rβ; a STAT5 binding site for IL-7Rα; a phosphatidylinositol 3-kinase (PI3K) binding site for IL-7Rα; a STAT5 binding site for IL-12Rβ2; a STAT4 binding site for IL-12Rβ2; a STAT3 binding site for IL-12Rβ2; a STAT5 binding site for IL-21R; a STAT3 binding site for IL-21R; and a STAT1 binding site for IL-21R. In some aspects, at least one signaling molecule binding site comprises a sequence further comprising the amino acids listed in Table 16.

[0343] In some aspects, at least a portion of an ICD described herein comprises the Box 1 region and the Box 1 region of gp130 or G-CSFR. In some aspects, the Box 1 region comprises an amino acid sequence listed in Table 2. In some aspects, the Box 1 region comprises an amino acid sequence that is greater than 50% identical to a Box 1 sequence listed in Table 16.

[0344] In certain aspects, the intracellular domain of the different cytokine receptor is a wild-type intracellular domain.

[0345] In some aspects, chimeric variant receptors as described herein also include at least a portion of a transmembrane domain (TMD) of a different cytokine receptor. The TMD of a different cytokine receptor 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 some aspects, at least a portion of a TMD includes an amino acid sequence sharing 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 a cytokine receptor TMD as described herein. In certain aspects, 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.

[0346] In some respects, such as Figure 20 、 23 As shown in the chimeric receptor designs of and 24, the chimeric receptors described herein comprise a G-CSFR ECD domain, a transmembrane domain (TMD), and at least a portion of an ICD arranged in N-terminal to C-terminal order.

[0347] In some aspects, the chimeric receptor comprises the G-CSFR ECD of SEQ ID NO. 3, the gp130 TMD and a portion of the ICD of SEQ ID NO. 4, and a portion of the IL-2Rβ ICD of SEQ ID NO. 5. In some aspects, 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 some aspects, 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.

[0348] The binding of a variant or wild-type cytokine to a chimeric cytokine receptor expressed on the cell surface may or may not affect the function of the variant cytokine receptor (compared to the activity of the native cytokine receptor); the native activity is not required or desired in all cases. In certain embodiments, the binding of a variant cytokine to a chimeric cytokine receptor will induce one or more aspects of native cytokine signaling. In certain embodiments, the binding of a variant cytokine to a chimeric cytokine receptor expressed on the cell surface causes a cellular response selected from the group consisting of: proliferation, viability, and enhanced activity.

[0349] Nucleic acids encoding variant cytokines and receptors

[0350] Included in the present disclosure are nucleic acids encoding any of the receptors and variant G-CSFs described herein.

[0351] Variant receptors or variant G-CSF can be produced not only directly recombinantly, but also as fusion polypeptides with heterologous polypeptides (e.g., signal sequences or other polypeptides having a specific cleavage site at the N-terminus of the mature protein or polypeptide). Generally, the signal sequence can be a component of the vector, or it can also be 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 a signal peptidase). In mammalian cell expression, the native signal sequence can be used, or other mammalian signal sequences may be suitable, such as signal sequences of secreted polypeptides from the same or related species and viral secretory leaders. In certain aspects, the signal sequence can be an amino acid sequence comprising a signal sequence at the N-terminal region of SEQ ID NO. 2, 3, 6, or 8. In certain aspects, the signal sequence can be the amino acid sequence MARLGNCSLTWAALIILLLPGSLE (SEQ ID NO. 11).

[0352] Expression vectors encoding variant cytokines or receptors

[0353] Also described herein are expression vectors and kits of expression vectors comprising one or more nucleic acid sequences encoding one or more of the variant receptors or variant G-CSFs described herein.

[0354] In certain embodiments, a nucleic acid encoding a variant receptor or variant G-CSF is inserted into a replicable vector for expression. Such a vector can be used to introduce a nucleic acid sequence into a host cell such that it expresses a variant receptor or cytokine described herein. Many such vectors are available. Vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Vectors include viral vectors, plasmid vectors, integrating vectors, 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 capable of transfecting or transducing cells (e.g., T cells, NK cells, or other cells).

[0355] Expression vectors typically contain a selection gene, also known as a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with a vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) compensate for nutritional deficiencies, or (c) provide key nutrients that are not available in complex culture media.

[0356] In some aspects, expression vector contains the promoter that is recognized by host organism and is operably connected to variant protein coding sequence.Promoter is the non-translated sequence (usually in about 100 to 1000bp) that is positioned at the upstream (5') of the start codon of structural gene, and it controls the transcription and translation of the specific nucleic acid sequence that they are operably connected. Such promoter is usually divided into inducible and constitutive two classes. Inducible promoter is the promoter that starts the level that is raised by DNA transcription in response to some variation (for example, the presence or absence of nutrients or temperature change) of culture condition under its control. A large amount of promoters identified by a variety of potential host cells are well known.

[0357] Transcription of the vector in a mammalian host cell can be controlled, for example, by a promoter, provided that such a promoter is compatible with the host cell system, obtained from the genome of a virus such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus (e.g., murine stem cell virus), hepatitis B virus, and most preferably Simian Virus 40 (SV40); a promoter from a heterologous mammal, such as the actin promoter, PGK (phosphoglycerate kinase), or an immunoglobulin promoter; or from a heat shock promoter. The early and late promoters of the SV40 virus can be conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.

[0358] Generally, the transcription by higher eukaryotic cells is increased by inserting enhancer sequences into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300bp, which act on the promoter to increase its transcription. Enhancers are relatively directional and position-independent, found at the 5' and 3' positions of the transcription unit in introns and in the coding sequence itself. Many enhancer sequences from mammalian genes (globin, elastase, albumin, fetoprotein and insulin) are known. However, enhancers from eukaryotic cell viruses are generally used. Examples include the SV40 enhancer, cytomegalovirus early promoter enhancer, the polyoma enhancer and adenovirus enhancer located at the rear side of the replication initiation point (late side) . Enhancers can be spliced ​​into the 5' or 3' positions of the coding sequence in the expression vector, but are preferably located at the 5' site of the promoter.

[0359] The expression vector used in the eukaryotic host cell will also contain sequences required for termination of transcription and stabilization of the mRNA. Such sequences are generally available from the 5' (sometimes 3') untranslated regions of eukaryotic or viral DNA or cDNA. The construction of a suitable vector comprising one or more of the components listed above adopts standard techniques.

[0360] In certain aspects, disclosed herein are lentiviral vectors encoding the chimeric receptors disclosed herein. In certain aspects, the lentiviral vector comprises HIV-1 5'LTR and 3'LTR. In certain aspects, the lentiviral vector comprises an EF1a promoter. In certain aspects, the lentiviral vector comprises an SV40 poly a terminator sequence. In certain aspects, the vector is psPAX2, 12260, pCMV-VSV-G or 8454.

[0361] In some embodiments, the nucleic acid and peptide sequence with high sequence identity (such as 95%, 96%, 97%, 98%, 99% or more sequence identity) with sequence described herein are also described.In the context of two or more nucleic acid or peptide sequences, the term sequence " identity " percentage refers to two or more sequences or subsequences of identical nucleotides or amino acid residues when comparing and comparing for maximum correspondence with a specified percentage, as measured using one of the sequence comparison algorithms described below (such as, BLASTP and BLASTN or the available algorithm of other technical personnel) or by visual inspection.According to application, " identity " percentage can be present in the district of the sequence compared, for example, is present in a functional domain, or alternatively is present in the total length of two sequences to be compared.

[0362] For sequence comparison, typically a sequence serves as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test sequence and reference sequence are input into a computer, subsequence coordinates are specified, if necessary, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the specified program parameters.

[0363] For example, optimal alignment of sequences for comparison can be 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 search similarity 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 Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.); or by visual inspection (see generally, Ausubel et al., infra).

[0364] An example of a suitable algorithm 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 / ).

[0365] Cells expressing variant receptors and variant cytokines

[0366] Cells expressing variant receptors are also described herein.Host cells (including engineered immune cells) can be transfected or transduced with the above-described expression vectors for variant cytokine or receptor expression.

[0367] In certain embodiments, the present disclosure provides a kind of cell, which comprises one or more of the variant receptors or variant cytokines described herein. The cell may comprise a nucleic acid or a vector encoding the variant receptors or variant cytokines described herein. The present disclosure also provides a method for producing a cell expressing a variant receptor. In some aspects, cells are produced by introducing nucleic acids or expression vectors as described herein into cells. Nucleic acids or expression vectors can be introduced into cells by any method, including but not limited to transfection, transduction, transposition or gene editing of viral vectors. Any gene editing technology known in the art can be used, including but not limited to clustered regularly interspaced short palindromic repeats (CRISPR-Cas) systems, zinc finger nucleases, nucleases based on transcription activator-like effectors and technologies of meganucleases.

[0368] The host cell can be any cell in the body. In certain embodiments, the cell is an immune cell. In some embodiments, the cell is a T cell, including but not limited to a naive CD8 T cell. + T cells, cytotoxic CD8 + T cells, naive CD4 T cells, helper T cells, such as T H 1. T H 2. T H 9. T H 11. T H 22. T FH Regulatory T cells, such as T R 1. Natural T Reg , inducible T Reg ; Memory T cells, such as central memory T cells, effector memory T cells, NKT cells, γδT cells; Etc. In certain embodiments, the cell is a B cell, including but not limited to naive B cells, germinal center B cells, memory B cells, cytotoxic B cells, cytokine-producing B cells, regulatory B cells (Breg), centroblasts, centrocytes, antibody-secreting cells, plasma cells, etc. In certain embodiments, the cell is an innate lymphoid cell, including but not limited to NK cells, etc. In certain embodiments, the cell is a myeloid cell, including but not limited to macrophages, dendritic cells, myeloid-derived suppressor cells, etc.

[0369] In certain embodiments, the cells are stem cells, including but not limited to hematopoietic stem cells, mesenchymal stem cells, neural stem cells, and the like.

[0370] In some embodiments, cells are genetically modified in an ex vivo procedure prior to transfer into a subject.The cells can be provided in unit doses for treatment and can be allogeneic, autologous, etc., relative to the intended recipient.

[0371] T cells, or T lymphocytes, are a type of lymphocyte that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of T cell receptors (TCRs) on their cell surface. There are several types of T cells, summarized below.

[0372] Helper T helper cells (Th cells) assist other white blood cells in immune processes, including maturing B cells into plasma cells and memory B cells and activating cytotoxic T cells and macrophages. Th cells express CD4 on their surface. Th cells become activated when they are presented with peptide antigens 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.

[0373] Cytolytic 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 their targets by binding to antigens associated with MHC class I present on the surface of all nucleated cells.

[0374] Memory T cells are a small group of antigen-specific T cells that persist long after an infection has resolved. Upon re-exposure to their cognate antigen, they rapidly expand into a large number of effector T cells, 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+. Memory T cells typically express the cell surface protein CD45RO.

[0375] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are crucial for maintaining immune tolerance. Their primary role is to shut down T cell-mediated immunity at the end of an immune response and to suppress autoreactive T cells that escape the negative selection process in the thymus. Two major categories of CD4+ Treg cells have been described: naturally occurring Treg cells and adaptive Treg cells.

[0376] Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) appear in the thymus and are associated with interactions between developing T cells and myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells that have been activated by TSLP. Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3.

[0377] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can originate from normal immune responses. 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 signals from virally infected cells in an MHC-independent manner.

[0378] In some aspects, the cells expressing the variant receptors or variant cytokines described herein are tumor infiltrating lymphocytes (TIL) or tumor associated lymphocytes (TAL). In some aspects, TIL or TAL include CD4+T cells, CD8+T cells, natural killer (NK) cells, and combinations thereof.

[0379] In certain embodiments, the T cells described herein are chimeric antigen receptor T cells (CAR-T cells), which have been genetically engineered to produce artificial T cell receptors for immunotherapy. In some aspects, CAR-T cells are derived from T cells in the patient's own blood (i.e., autologous). In some aspects, CAR-T cells are derived from T cells (i.e., allogeneic) from another healthy donor.

[0380] In certain embodiments, the T cells described herein are engineered T cell receptors (eTCR-T cells) that have been genetically engineered to produce specific T cell receptors for immunotherapy. In some aspects, eTCR-T cells are derived from T cells in the patient's own blood (i.e., autologous). In some aspects, eTCR-T cells are derived from T cells of a donor (i.e., allogeneic).

[0381] NK cells (belonging to the innate lymphoid cell group) are defined as large granular lymphocytes (LGL) and constitute the third cell type to differentiate from common lymphocyte progenitor cells that give rise to B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus before entering the circulation.

[0382] In some aspects, the cells expressing chimeric cytokine receptors as 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 (Breg), centroblasts, centrocytes, antibody-secreting cells, plasma cells, etc.

[0383] In certain aspects, the cells expressing the chimeric cytokine receptor described herein are myeloid cells, including but not limited to macrophages, dendritic cells, myeloid-derived suppressor cells, and the like.

[0384] The cell of expression variant receptor or variant cytokine as described herein can be any cell type. In some aspects, the cell of expression variant receptor or variant cytokine as described herein is the cell of hematopoietic system. According to the immune cell of the present invention (for example, T cell or NK cell) can be produced in vitro by the peripheral blood (the 1st party) of the patient itself, or produced in the case of hematopoietic stem cell transplantation from donor peripheral blood (the 2nd party), or produced by the peripheral blood from unconnected donor (the 3rd party). Alternatively, immune cell as described herein can be derived from the in vitro differentiation of induced progenitor cells or embryonic progenitor cells to immune cells. Alternatively, it is possible to use the immortalized immune cell line (for example, T cell or NK cell line that keeps its lysis function; The plasma cell line that keeps its antibody production function, or the dendritic cell line or macrophage that keeps its phagocytosis and antigen presentation function) that can be used as therapeutic agent. In all these embodiments, the cell expressing variant receptor is produced by introducing the DNA or RNA encoding each variant receptor in one of a variety of ways, and the a variety of ways include transduction with viral vectors or transfection with DNA or RNA.

[0385] Cells as described herein can be immune cells derived from a subject that are engineered in vitro to express variant receptors and / or variant cytokines. Immune cells can be from peripheral blood mononuclear cell (PBMC) samples or tumor samples. Immune cells can be activated and / or amplified before transduction with nucleic acid encoding a molecule of a variant receptor or variant cytokine provided according to a first aspect of the present invention, for example, by processing with anti-CD3 monoclonal antibodies and / or IL-2. The immune cells of the present invention can be prepared in the following manner: (i) a sample containing immune cells is separated from a subject or other sources listed above; and (ii) immune cells are transduced or transfected with one or more nucleic acid sequences encoding variant receptors or variant cytokines.

[0386] Cells can be cultured in conventional nutrient media that are changed when appropriate to induce promoters, select transformants, or amplify genes encoding the desired sequence. Mammalian host cells can be cultured in a variety of culture media. Commercially available culture media, such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM, (Sigma)), RPMI 1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM, Sigma), are suitable for culturing host cells. Any of these culture media can be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source. Any other necessary supplements of appropriate concentrations well known to those skilled in the art can also be included. Culture conditions, such as temperature, pH, etc., are those previously used with the host cells selected for expression, and will be apparent to those of ordinary skill in the art.

[0387] Immune cells can then be selected by purification, e.g., based on expression of an antigen binding domain of an antigen binding polypeptide. In certain embodiments, cells are selected by expressing a selectable marker (e.g., a protein, fluorescent marker, or epitope tag) or by any method known in the art for selecting, separating, and / or purifying cells.

[0388] Reagent test kit

[0389] The present disclosure also describes kits for producing cells expressing at least one of any of the variant receptors or variant G-CSFs described herein. In certain embodiments, the kits include at least one expression vector encoding at least one variant receptor and instructions for use. In certain aspects, the kits also include at least one variant cytokine or an expression vector encoding a variant G-CSF that binds to at least one of the variant receptors described herein in a pharmaceutical formulation. In certain embodiments, the kits include cells comprising an expression vector encoding a variant receptor described herein.

[0390] In certain embodiments, the kit includes cells comprising expression vectors encoding chimeric antigen receptors (CAR) / T cell receptors (TCR) and the like. (CAR) / engineered T cell receptors (eTCR) and the like (e.g., engineered non-natural TCR receptors). In certain embodiments, the kit includes expression vectors encoding chimeric antigen receptors (CAR) / engineered T cell receptors (eTCR) and the like. In certain embodiments, the kit includes expression vectors encoding variant receptors described herein and chimeric antigen receptors (CAR) / engineered T cell receptors (eTCR) and the like.

[0391] In some aspects, the kits described herein further include variant cytokines. In certain embodiments, the kits further include at least one additional variant cytokine. In certain aspects, the kits further include at least one variant cytokine in a pharmaceutical formulation. In certain embodiments, the components are provided in any convenient packaging in a dosage form, liquid or solid form.

[0392] Additional reagents can be provided for the growth, selection and preparation of cells provided or generated as described herein. For example, the kit can include components for cell culture, growth factors, differentiation agents, reagents for transfection or transduction, etc.

[0393] In certain embodiments, in addition to the above components, the kit may also include instructions for use. The instructions may be provided in any convenient form. For example, the instructions may be provided as printed information, in the packaging of the kit, on a package insert, etc. The instructions may also be provided as a computer-readable medium having information recorded thereon. In addition, the instructions may be provided on a website address that can be used to access the information.

[0394] Methods for selectively activating variant receptors

[0395] The present disclosure provides methods for selectively activating a variant receptor expressed on a cell surface, the methods comprising contacting a variant receptor described herein with a cytokine that selectively activates the chimeric receptor. In certain aspects, the cytokine that selectively activates the chimeric receptor is a variant G-CSF. The G-CSF can be wild-type G-CSF or a G-CSF that comprises one or more mutations that confer preferential binding and activation of the variant receptor to G-CSF compared to the native (wild-type) cytokine receptor.

[0396] In certain aspects, selective activation of the variant receptor by cytokine binding to the variant receptor results in homodimerization, heterodimerization, or a combination thereof.

[0397] In some aspects, the activation of variant receptors leads to the activation of downstream signaling molecules. In some aspects, variant receptors activate signaling molecules or pathways transduced by natural cell signaling molecules to provide biological activity that simulates the natural response, but it is specific to cells engineered to express variant receptors. In some aspects, the activation of downstream signaling molecules includes activating cell signaling pathways that stimulate cell cycle progression, proliferation, vigor and / or enhance activity. In some aspects, the activated signaling pathways or molecules are but are not limited to Jak1, Jak2, Jak3, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6, Shc, ERK1 / 2 and Akt. In some aspects, after administering a cytokine that binds to the receptor, the activation of variant receptors leads to increased cell proliferation. In some aspects, the degree of proliferation is between 0.1-10 times the proliferation observed when the cells are stimulated by IL-2.

[0398] Adoptive cell transfer method

[0399] The present invention provides a method for treating and / or preventing a disease, comprising the step of administering to a subject cells expressing a variant receptor and / or variant cytokine described herein (eg, in a pharmaceutical composition as described below).

[0400] Methods for treating diseases relate to the therapeutic use of cells described herein, such as T cells, NK cells, or any other immune or non-immune cells expressing variant receptors. Cells can be administered to subjects with existing diseases or conditions to alleviate, reduce, or improve at least one symptom associated with the disease and / or to slow, reduce, or block the progression of the disease. Methods for preventing diseases relate to the prophylactic use of cells disclosed herein. Such cells can be administered to subjects who have not yet contracted the disease and / or do not exhibit any symptoms of the disease to prevent or impair the cause of the disease or to reduce or prevent the development of at least one symptom associated with the disease. The subject may have a susceptibility to the disease or be considered at risk of developing the disease.

[0401] In some embodiments, the compositions, methods and kits of the present invention are used to enhance immune responses. In some embodiments, the immune response is directed against conditions where it is desired to deplete or modulate target cells (e.g., cancer cells, infected cells, immune cells involved in autoimmune diseases, etc.) by systemic administration of cytokines (e.g., intramuscularly, intraperitoneally, intravenously, etc.).

[0402] Method may include the following steps: (i) separating a sample containing immune cells; (ii) transducing or transfecting such cells with a nucleic acid sequence or a vector (e.g., expressing variant receptors); (iii) administering (i.e., infusing) cells from (ii) to the subject, and (iv) administering the variant cytokine that stimulates the infused cells. In some aspects, before administering the cells to the subject, the subject has undergone immunodepletion therapy. In some aspects, before administering the cells to the subject, the subject has not undergone immunodepletion therapy. In some aspects, before administering the cells to the subject, the subject has undergone immunodepletion therapy that reduces the severity, dosage, and / or duration necessary without using variant receptors as described herein.

[0403] The sample containing immune cells can be isolated from the subject or other sources, such as described above. Immune cells can be isolated from the subject's own peripheral blood (Part 1), or produced in the case of hematopoietic stem cell transplantation from donor peripheral blood (Part 2), or produced by peripheral blood from an unrelated donor (Part 3). Immune cells can also be derived from in vitro methods, such as by inducing differentiation from stem cells or other forms of precursor cells.

[0404] In some embodiments, immune cells are contacted with variant cytokines in vivo, that is, wherein the immune cells are transferred to a recipient, and an effective dose of the variant cytokine is administered to the recipient and the variant cytokine is allowed to contact the immune cells at its natural location (e.g., in a lymph node, etc.). In some embodiments, the contact is carried out in vitro. When cells are contacted with variant cytokines in vitro, the cytokine is added to the cells with a dosage and time period sufficient to activate the signal transduction from the receptor, and the signal transduction can utilize aspects of natural cell mechanisms, such as auxiliary proteins, co-receptors, etc. Activated cells can be used for any purpose, including but not limited to experimental purposes relevant to antigen-specific determination, cytokine profile analysis, and in vivo delivery.

[0405] In some aspects, a therapeutically effective number of cells is administered to a subject. In some aspects, cells expressing a variant receptor are administered or infused to a subject on multiple different occasions. In some embodiments, at least 1 x 10 6 cells / kg, at least 1x10 7 cells / kg, at least 1x10 8 cells / kg, at least 1x10 9 cells / kg, at least 1x10 10Cells / kg or more are sometimes limited by the number of cells obtained during the collection process, such as transfected T cells. The transfected cells can be infused into the subject in any physiologically acceptable medium (usually intravascularly), although they can also be introduced into any other convenient site where the cells can find a suitable site for growth.

[0406] In some aspects, a variant cytokine of a therapeutically effective amount is administered to a subject. In some aspects, a variant cytokine is administered to a subject under a plurality of different situations. In some aspects, the amount of the variant cytokine administered is an amount sufficient to achieve the desired result (e.g., alleviating the symptoms of the disease in the subject) for treatment. In some aspects, the amount of the variant cytokine administered is an amount sufficient to stimulate the cell cycle progression, proliferation, vigor and / or functional activity of the cells expressing variant cytokine receptors as described herein. In some aspects, the variant cytokine is administered with the dosage and / or duration necessary to achieve the desired result for treatment. In some aspects, the variant cytokine is administered with the dosage and / or duration sufficient to stimulate the cell cycle progression, proliferation, vigor and / or functional activity of the cells expressing variant cytokine receptors as described herein. Dosage and frequency may vary depending on the agent; mode of administration; the nature of the cytokine; etc. Those skilled in the art will appreciate that such guidelines will be adjusted for individual situations. For topical administration (e.g., intranasal, inhalation, etc.), for systemic administration (e.g., intramuscular, intraperitoneal, intravascular, etc.), dosage can also be changed.

[0407] Indications for adoptive cell transfer

[0408] The present invention provides a cell expressing the variant receptor described herein, which is used for treating and / or preventing a disease. The present invention also relates to the use of the cell expressing the variant receptor described herein in the manufacture of a medicament for treating and / or preventing a disease.

[0409] The disease treated and / or prevented by the method of the present invention can be a cancerous disease, such as, but not limited to, bile duct cancer, bladder cancer, breast cancer, cervical cancer, ovarian cancer, colon cancer, endometrial cancer, hematological malignancies, kidney cancer (renal cell), leukemia, lymphoma, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, sarcoma and thyroid cancer.

[0410] Disease to be treated and / or prevented can be an autoimmune disease. The feature of autoimmune disease is that T lymphocytes and B lymphocytes abnormally target self proteins, polypeptides, peptides and / or other self molecules, thereby causing damage and or disorder to organs, tissues or cell types (for example, pancreas, brain, thyroid or gastrointestinal tract) in the body, to cause the clinical manifestations of the disease. Autoimmune disease includes the disease that affects specific tissues and the disease that can affect multiple tissues, which can depend in part on whether the reaction is directed to antigens that are confined to specific tissues or to antigens that are widely distributed in the body. Autoimmune diseases include but are not limited to type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, autoimmune thyroid disease and Graves' disease.

[0411] Disease to be treated and / or prevented can be an inflammatory condition, such as cardiac fibrosis. Typically, inflammatory conditions or illnesses typically result in the immune system attacking cells or tissues of the human body itself, and may result in abnormal inflammation, thereby causing chronic pain, redness, swelling, stiffness, and damage to normal tissue. Inflammatory conditions are characterized by or caused by inflammation, and include but are not limited to celiac disease, vasculitis, lupus, chronic obstructive pulmonary disease (COPD), irritable bowel disease, atherosclerosis, arthritis, myositis, scleroderma, gout, Sjorgren's syndrome, ankylosing spondylitis, antiphospholipid antibody syndrome, and psoriasis.

[0412] In certain embodiments, the methods are used to treat infectious diseases.

[0413] In certain embodiments, the condition to be treated is the prevention and treatment of transplant rejection. In certain embodiments, the condition to be treated and / or prevented is allograft rejection. In certain aspects, allograft rejection is acute allograft rejection.

[0414] The disease to be treated and / or prevented may involve transplanting cells, tissues, organs or other anatomical structures into an affected individual. The cells, tissues, organs or other anatomical structures may be from the same individual (autologous or "autologous" transplant) or from a different individual (allogeneic or "allogeneic" transplant). The cells, tissues, organs or other anatomical structures may also be produced using in vitro methods, including cell cloning, inducing cell differentiation or manufacturing with synthetic biomaterials.

[0415] The present invention provides a method for treating and / or preventing a disease comprising one or more steps of administering to a subject a variant cytokine and / or cell as described herein (eg, in a pharmaceutical composition as described above).

[0416] Methods for treating and / or preventing diseases relate to the therapeutic use of cells disclosed herein. In this article, cells can be administered to subjects with existing diseases or conditions to alleviate, reduce or improve at least one symptom associated with the disease and / or slow down, reduce or block the progression of the disease. Methods for preventing diseases relate to the prophylactic use of cells disclosed herein. Such cells can be administered to subjects who have not yet contracted the disease and / or do not show any symptoms of the disease to prevent or damage the cause of the disease or reduce or prevent the development of at least one symptom associated with the disease. The subject may have a susceptibility to the disease or be considered to be at risk of developing the disease. The method may comprise the following steps: (i) isolating a sample containing immune cells; (ii) transducing or transfecting such cells with a nucleic acid sequence or vector provided by the present invention; (iii) administering cells from (ii) to the subject, and (iv) administering a variant cytokine that stimulates the infused cells. Samples containing immune cells can be isolated from subjects or other sources, for example as described above. Immune cells can be isolated from the subject's own peripheral blood (Part 1), or generated in the case of hematopoietic stem cell transplantation from donor peripheral blood (Part 2), or from peripheral blood from an unrelated donor (Part 3).

[0417] Treatment can be combined with other active agents, such as, but not limited to, antibiotics, anticancer agents, antiviral agents, and other immunomodulators (e.g., antibodies against the programmed cell death protein-1 [PD-1] pathway or antibodies against CTLA-4). Other cytokines (e.g., interferon gamma, tumor necrosis factor alpha, interleukin 12, etc.) may also be included.

[0418] Methods using stem cells expressing variant cytokine receptors

[0419] The present invention provides a method for treating and / or preventing a condition or disease, comprising the step of administering stem cells expressing a variant receptor and / or variant cytokine as described herein. In certain embodiments, the stem cells expressing a variant cytokine receptor and / or variant cytokine as described herein are used for regenerative medicine, cell / tissue / organ transplantation, tissue reconstruction, or tissue repair.

[0420] Pharmaceutical composition of the present invention

[0421] The present disclosure also relates to a pharmaceutical composition comprising a plurality of cells expressing the variant receptors described herein and / or the cytokines described herein. The present invention also relates to a pharmaceutical composition comprising the variant cytokines described herein. The cells of the present invention can be formulated into pharmaceutical compositions. In addition to one or more of the cells expressing the variant receptors described herein, these compositions may also contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other substances known to those skilled in the art. The substances should be non-toxic and should not interfere with the efficacy of the active ingredient. The pharmaceutical composition may optionally contain one or more additional pharmaceutically active polypeptides and / or compounds. Such formulations may be in a form suitable for intravenous infusion, for example.

[0422] For cells expressing variant receptors and variant cytokines as described herein to be administered to an individual according to the present invention, it is preferred to administer a "therapeutically effective amount" sufficient to show benefit to the individual. When sufficient to show benefit to the individual, a "prophylactic effective amount" may also be administered. The actual amount of the cytokine or the number of cells administered, as well as the rate and time course of administration, will depend on the nature and severity of the disease being treated. The designation of treatment, such as decisions about dosage, is within the purview of ordinary practitioners and other physicians, and typically takes into account the disease to be treated, the condition of the individual patient, the delivery site, the method of administration, and other factors known to practitioners. Examples of the above-mentioned techniques and protocols can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.), 1980.

[0423] Depending on the condition to be treated, the pharmaceutical composition may be administered alone or in combination with other treatments, either simultaneously or sequentially.

[0424] Example

[0425] The following are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to the values ​​used (e.g., amounts, temperatures, etc.), but of course some experimental errors and deviations should be allowed for.

[0426] Unless otherwise indicated, the practice of the present invention will utilize conventional methods of protein chemistry, biochemistry, recombinant DNA technology, cell culture, adoptive cell transfer, and pharmacology within the skill of the art. Such techniques are fully described in the literature. See, for example, T. Creighton, Proteins: Structures and Molecular Properties (W. H. Freeman and Company, 1993); A. Lehninger, Biochemistry (Worth Publishers, Inc., new in this issue); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., 1989); Methods in Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th ed. (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry, 3rd ed. (Plenum Press) Vols. A and B (1992).

[0427] Example 1: Rational design of the dedicated site II interface of G-CSF:G-CSFR(CRH)

[0428] 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 ).

[0429] To design co-evolved, engineered (E)G-CSF E :G-CSFR EThe cytokine:receptor pair, which separates the 2:2 complex between WT G-CSF and the Ig-CRH extracellular domain of G-CSFR (Protein Data Bank ID 2D9Q, Tamada et al. PNAS 2006), is separated into two distinct subcomplexes comprising the Site II and Site III interfaces, which are composed of G-CSF:G-CSFR(CRH) and G-CSF:G-CSFR(Ig), respectively (see Figure 1 ), the sequence is shown in Table 1.

[0430] method

[0431] To create a co-evolved, specialized G-CSF E :G-CSFR E Mutant pairs (designed), using a computational design workflow (see Figure 2 ). First, a dedicated design was created at site II.

[0432] G-CSF WT :G-CSFR(CRH) WT Computer structure analysis of the site II interface interactions of α-β ... Figure 3 ). In-depth inspection revealed mainly electrostatic and hydrogen bonding interactions, such as the interaction between R167 of G-CSFR(CRH) and D112 / D109 of G-CSF, which contributed 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 contributes 21.4% of the total attractive AMBER energy at site II (see Figure 3 Likewise, the salt bridge between E19 of the cytokine and R288 of the receptor CRH domain contributes 17.3%, further stabilizing the arginine through electrostatic and hydrogen bonding interactions with D200 of the receptor CRH domain.

[0433] Each design of site II is composed of G-CSF E and G-CSFR(CRH) E First, a positive design is created at site II, for example by reversing the charge, by mutating basic residues to acidic residues and vice versa on the binding partner, while maintaining packing and hydrophobic interactions by additional mutations when necessary. E :G-CSFR(CRH) E By ZymeCADTM The computer model designed for packing at site II was visually inspected for structural integrity and analyzed using the ZymeCAD TM Specifically, the goal was to design G-CSF E Mutants with ZymeCAD of <10 kcal / mol to their corresponding G-CSFR(CRH)E mutants TM Computer dAMBER binding affinity (paired interaction). This metric compares the sum of Lennard Jones affinity and electrostatic affinity, i.e., AMBER affinity, to the AMBER affinity of the WT:WT cytokine receptor pair. Designs with dAMBER_fold > 80 kcal / mol were excluded. The dAMBER_fold metric scores the change in the sum of Lennard Jones bond fold and electrostatic fold after mutation. Designs with a dDDRW apo stability (apostability) score above 400 kcal / mol were also excluded. This metric describes the knowledge-based change in potential stability of a protein after mutation from its apo form.

[0434] Next, use ZymeCAD TM G-CSF for each design E The mutants were packaged in complex with WT G-CSFR (and vice versa G-CSFR E With G-CSF WT ) to evaluate the metric of each positive design under mismatch conditions when forming the following two complexes: GC SF WT :G-CSFR(CRH) E and G-CSF E :G-CSFR(CRH) WT . Using ZymeCAD TM Computer-calculated ddAMBER metrics for mismatch orientation (DdAMBER_Affinity_Awt_Bmut is the AMBER affinity of the paired engineered complex minus the mismatched complex G-CSF W T :G-CSFR(CRH) E AMBER affinity, ddAMBER_Affinity_Amut_Bwt is the AMBER affinity of the paired engineered complex minus the mismatched complex G-CSF E :G-CSFR(CRH) WT Designs that minimize the magnitude of mismatched ddAMBER affinity are believed to be more selective for binding to their paired binding partners than to binding to wild-type cytokines or receptors.

[0435] All site II designs were clustered, and G-CSF E :G-CSFR(CRH) E , G-CSF WT :G-CSFR(CRH) E , G-CSF E :G-CSFR(CRH) WT The packaging metrics were considered to evaluate the strength of the pairing (positive design) and selectivity against mismatches with WT G-CSF and G-CSFR(CRH) (negative design) and ranked designs.

[0436] result

[0437] The designs listed in Table 2 were created in ZymeCAD. TM There are packaging metrics that facilitate in silico G-CSF E :G-CSFR(CRH) E The pairs were selected (see Table 3) and showed satisfactory interactions in silico, such as the presence of salt bridges, hydrogen bonds and the absence of severe clashes (see Figure 5 These designs also showed high selectivity against mismatches with WT G-CSF or G-CSFR (CRH) (see Table 3).

[0438] Therefore, the Site II mutations of the same variant G-CSF and receptor designs shown in Table 2 are predicted to exhibit preferential binding relative to wild-type G-CSFR and G-CSF, respectively.

[0439] Table 3: Features of ZymeCAD Tm Site II design with AMBER metrics in kcal / mol from triplicate computer mean-field packages.

[0440]

[0441]

[0442] Example 2: In vitro screening of site II designs

[0443] In pull-down experiments, G-CSF E :G-CSFR(CRH) E The selected Site II designs were screened for their ability to form a Site II complex when co-expressed in a baculovirus-based insect cell system. The designs were also evaluated for their ability to form a Site II complex by incorporating each design into the G-CSF E Mutants and G-CSFR(CRH) WTCo-expression to form mismatched complexes with WT receptors or cytokines, and vice versa, each designed G-CSFR(CRH) E Mutants and GCSF WT Co-expression of G-CSF alone was confirmed by single infection of cytokine mutants E expression.

[0444] method

[0445] Briefly, site II G-CSF designs and corresponding paired G-CSFR (CRH) mutants were cloned separately into insect cell transfection vectors. G-CSF WT (residues 1-173, Table 1) and mutants were cloned in frame into a modified pAcGP67b transfer vector (Pharmingen) along with an N-terminal secretion signal and a C-terminal TEV-cleavable Twin Strep tag with the sequence AAAENLYFQ / GSAWSHPQFEKGGGSGGGSGGSAWSHPQF EK (SEQ ID NO. 82). In the receptor extracellular domain, only the CRH domain with site II designed mutations (residues 98-308, Table 1) was cloned in frame into a modified pAcGP67b transfer vector along with an N-terminal secretion signal and a TEV-cleavable hexahistidine (SEQ ID NO. 89) tag with the sequence HHHHHHSSGRENLYFQ / GSMG (SEQ ID NO. 83). All constructs were synthesized and codon optimized for insect cell expression (Genscript). Transfer vector DNA was prepared by Midi-prep (ThermoScientific, catalog K0481), endotoxin-free, and A 260 / 280 The absorbance ratio was 1.8-2.0. Recombinant virus production was achieved by co-transfection of recombinant linearized baculovirus DNA with vector DNA into Spodoptera frugiperda 9 (Sf9) cells using the adherence method as described by the manufacturer (Expression Systems, California). Approximately 1 hour before transfection, 0.46 x 10 6 cells ml -1Inoculate 2 mL of healthy logarithmic phase Sf9 cells. Prepare the transfection mixture as follows: Place 100 μl of transfection medium (Expression Systems, California, catalog 95-020-100) into each of two sterile 1.5 ml microcentrifuge tubes A and B. Add 0.4 μg of recombinant BestBac 2.0Δv-cath / chiA linearized DNA (Expression Systems, California, catalog 91-002) and 2 μg of vector DNA to tube A. Add 1.2 μl of 5X Express 2 TR transfection reagent (Expres2ION, catalogue S2-55A-001). Solution A and B were incubated at approximately 24°C for 5 minutes, then merged 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. Old ESF 921 culture medium was removed from the wells and replaced with 1 mL of transfection mixture that was applied dropwise so as not to interfere with the cell monolayer. The plate was gently shaken before and after and left and right to evenly distribute the transfection mixture, and incubated at 27°C for 4 hours. After 4 hours, the transfection mixture was removed from the co-transfection plate and dropwise added with 2 mL of fresh ESF 921 insect cell culture medium (Expression Systems, California, catalogue 96-001-01) containing 10 μg / ml gentamicin (catalogue 15750-060). To prevent evaporation, the plate was wrapped in saran wrap, placed in a sterile plastic box, and incubated at 27°C for 4-5 days. On day 4 or 5 after transfection, collect the P1 supernatant and clarify by centrifugation at 5000 rpm for 5 min, transfer to a new sterile tube, and store at 4 °C, protected from light.

[0446] The recombinant P1 stock generated as described above was further amplified into a high titer, low passage P2 stock for protein expression studies. The following workflow uses the P1 seed stock of virus harvested from the co-transfection as an inoculum to generate 50-100 mL of virus. 50 mL of logarithmic phase Sf9 cells were plated at 1.5 x 10 6 cells ml -1 Inoculate into 250mL shake flask (FisherScientific, catalog PBV 250), and add 0.5mL P1 virus stock solution. Incubate cells at 27 ℃, shake at 135rpm, and monitor infection. Harvest P2 virus supernatant 5-7 days after infection and clarify by centrifugation at 4000rpm for 10 minutes. In order to minimize titer loss, add 10% heat-inactivated FBS (VWR, catalog 97068-085), and store P2 virus at 4 ℃ in the dark.

[0447] The P2 virus stock was tested for protein expression on a small scale. P2 virus was used to co-infect G-CSF in Trichoplusia ni (Tni) cells in 12-well plates. E Mutants and their corresponding G-CSFR E Mutants. Using G-CSF WT and GCSFR(CRH) WT Each designed mutant was co-infected with a separate P2 stock solution and for G-CSF only E For each reaction, 20 μl of P2 virus was used at 2 x 10 6 cells ml -1 2 mL of healthy logarithmic phase Tni was inoculated. The plate was incubated at 27°C for approximately 70 h with shaking at 135 rpm. The supernatant was clarified by centrifugation at 5000 rpm for 3 minutes and the supernatant was purified by G-CSF in batch mode using the streptactin-XT workflow platform (IBA Lifesciences, catalog 2-4010-010). E Mutants and G-CSF WT The secreted proteins were pulled down using a double-streptavidin tag (TST) on the lysate. Briefly, 0.2 mL of 10x HEPES-buffered saline (HBS: 20 mM HEPES pH 8, 150 mM NaCl) was added to each 1.8 mL reaction supernatant to 1x, 20 μl bed volume (bv) of purification beads was added, and the reaction was incubated for 30 minutes at 24°C with inversion mixing. An additional 20 μl bv of purification beads was added, followed by a second incubation for 30 minutes. The beads were precipitated by centrifugation at 2200 rpm for 3 minutes, the supernatant was removed, and the beads were washed with 1x HBS buffer. Proteins were 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, catalog 2-1042-025)), boiled with SDS-PAGE sample buffer, and analyzed on 12% Bolt Bis-Tris plus, 12-well gels (Thermo Fisher Scientific, catalog NW00122BOX) run under reducing conditions at 200 V for 30 minutes.

[0448] result

[0449] Site II designs #6, 7, 8, 9, 15, 17, 30, 34, 35, 36 show G-CSF only E expression and formed a sufficiently stable paired G-CSF E :G-CSFRE complexes, which are activated by G-CSF E The Twin Strep tab on the Figure 6 For example, Site II design #6 of the engineered complex showed G-CSF at approximately 22 kDa on SDS-PAGE after pull-down. E Mutants and the corresponding co-expressed G-CSFR (CRH) at approximately 33 kDa E The mutant bands (see Figure 6 ).

[0450] Site II designs #8, 9, 15, and 34 were also selective for mismatches with WT G-CSF and WT G-CSFR in co-expression assays (see Figure 7 ), as they were not pulled down by WT G-CSF (cf. Figure 7 lower panel), and WT receptors were not activated by G-CSF E Mutant pull-down (see Figure 7 (above). Site II G-CSFR E Designs 30 and 35 were selective for mismatches with WT G-CSF in the co-expression assay as they were not pulled down by WT G-CSF (see Figure 7 Lower panel), but in co-expression assays, reverse G-CSF E Designed to be non-selective against mismatches with WT G-CSFR, since WT G-CSFR is pulled down (see Figure 7 In co-expression assays, site II designs 6, 7, 17, and 36 had no selectivity for mismatches with WT G-CSF or WT G-CSFR (see Figure 7 ), as they pull down WT G-CSFR and are pulled down by WT G-CSF.

[0451] Site II receptor mutants with a mutation at residue R288 were not expressed in the G-CSFR (CRH) receptor chain form without the Ig domain. Paired complex formation was evaluated by SPR in combination with the Site III design on the Ig domain (see Example 6). Thus, several Site II designs were identified that formed sufficiently stable paired G-CSFR (CRH) receptors. E :G-CSFR E complex that is also selective for mismatches with WT G-CSF and WT G-CSFR.

[0452] Thus, various Site II mutations of the same variant G-CSF and receptor designs shown in Table 2 exhibit preferential binding compared to wild-type G-CSFR and G-CSF, respectively.

[0453] Example 3: Rational design of the dedicated site III interface of G-CSF:G-CSFR(Ig)

[0454] The avidity effect of site III receptor Ig domain binding to G-CSF favors the formation of a 2:2 heterodimeric G-CSF:G-CSFR(Ig-CRH) complex (see Figure 1 Selective design only at site II with the WT site III interface does not appear to be sufficient to create a fully specialized 2:2G-CSF E :G-CSFR(Ig-CRH) E To facilitate selective binding of paired, co-evolved designs over mismatched binding to WT cytokines or receptors, the computational design workflow described in Example 1 was applied to the Site III interface to create selective Site III designs (see Figure 2 ).

[0455] method

[0456] First, the structural analysis of the site III interface interaction was performed. The site III interface contributes 55.64 kcal / mol AMBER energy to the G-CSF:G-CSFR complex and has an interface area of Compared with site II, its overall interface area is smaller. In-depth inspection of the Site III interface revealed fewer electrostatic and hydrogen bonding interactions compared to the Site II interface (see Figure 8 Key interactions at site III are for example the salt bridge between E46 of G-CSF and R41 of the receptor Ig domain, and furthermore the salt bridge between R147 of G-CSF and E93 of the receptor Ig domain (see Figure 9 These two interactions contribute 15.9% and 15.4% of the total attractive AMBER energy at Site III, respectively. Further interactions occur, for example, through Q87 of the receptor Ig domain, which forms hydrogen bond interactions with side chain amides of the G-CSF Site III backbone and Lennard Jones interactions with surrounding side chains of the cytokine, such as E46 and L49. These interactions contribute 12.3% of the total attractive AMBER energy at Site III.

[0457] Next, a positive design was created at site III, where G-CSF E Mutants and their coevolved G-CSFR(Ig) EMutants were designed to have favorable AMBER binding affinities (pairwise interactions) in silico. This was accomplished, for example, by inverting charge or changing shape complementarity while maintaining favorable Lennard Jones and hydrogen bonding interactions. E :G-CSFR(Ig) E By ZymeCAD TM The computer model designed to pack on site III was visually inspected for structural integrity and analyzed by ZymeCAD. TM It was evaluated by metric analysis as described in Example 1. Next, ZymeCAD TM G-CSF for each design E The mutants were packaged with WT G-CSFR(Ig) (and vice versa, G-CSFR(Ig) E With G-CSF WT ) to assess the metrics under conditions of mismatch with WT cytokine and receptor. ZymeCAD was used as previously described for Site II in Example 1. TM (See Table 5) Calculate the ddAMBER affinity metrics for the Site III design (G-CSF WT :G-CSFR(Ig) E , G-CSF E :G-CSFR(Ig) WT ).

[0458] The site III design was clustered and 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 The packing metrics were considered together with visual inspection to assess the strength of pairing and selectivity against mismatches with WT in order to rank the designs.

[0459] result

[0460] The designs listed in Table 4 were constructed in ZymeCAD. TM Computerized packaging metrics are beneficial for G-CSF E :G-CSFR(Ig) E The pairing with WT G-CSF or G-CSFR(Ig) was highly selective.

[0461] Therefore, the various site III mutations of the same variant G-CSF and receptor designs shown in Table 4 are predicted to exhibit preferential binding compared to wild-type G-CSFR and G-CSF, respectively.

[0462] Table 5: Features in ZymeCAD Tm Site III design in kcal / mol from AMBER metrics of triplicate computer mean-field packing.

[0463]

[0464] Example 4: Combination of cytokine-receptor switches that co-evolve to create variants at Site II and Site III

[0465] To develop a fully selective design for G-CSF E :G-CSF(Ig-CRH) E , which are capable of binding and signaling through a 2:2 heterodimeric engineered complex and have low or completely eliminated cross-reactivity with wild-type cytokines or receptors, the selected sites II and III from Examples 1 and 3 were designed and combined (see Table 6) and tested in vitro.

[0466] In the same manner, combining the designs in Table 6, any other combination of the Site II design of Example 1 (Table 2) with the Site III design of Example 3 (Table 4) can produce a fully selective G-CSF that can achieve a combination of variant signaling. E :G-CSFR(Ig-CRH) E Design. Combination designs 401 and 402 were tested in the co-expression assay described in Example 2 to form engineered G:CSF E :G-CSFR(Ig-CRH) E The ability of the complexes to bind to WT cytokines or receptors.

[0467] method

[0468] Pull-down co-expression assays by cytokine TST tag were performed as described above in Example 2, except that the receptor construct contained both Ig and CRH domains (residues 2-308, Table 1) termed G-CSFR (Ig-CRH).

[0469] result

[0470] The combined designs 401 and 402 were completely selective in the co-expression assay, as the designer cytokines pulled down their co-evolved engineered receptors but not the WT receptor, and vice versa, WT G-CSF did not pull down the engineered receptors (see Figure 10 ).

[0471] These results demonstrate that variant G-CSF and receptors comprising variant G-CSF RECD designs combining select site II and site III mutations are able to specifically bind to engineered cytokine receptor pairs and do not bind to wild-type receptors or cytokines, respectively.

[0472] Example 5: Generation of G-CSF and G-CSFR wild-type and mutants

[0473] To generate wild-type and engineered cytokine and receptor variants and compare their biophysical properties, recombinant proteins were expressed and purified from insect cells.

[0474] method

[0475] G-CSF was cloned as described above E and G-CSF WT The preparative scale production of recombinant proteins was performed in 2-4 L of healthy logarithmic phase Tni cells as follows: 800 mL of 2 x 10 6 cells ml -1 The Tni of 50 μ m is added, and it is hatched 70h at 27 ℃ with 135rpm shaking.After hatching, by making cell precipitation with 5500rpm centrifugal 15 minutes, and supernatant is filtered twice, at first by the A / E type glass fiber filter (PALL, catalogue 61631) of 1 μ m, filter, subsequently by 0.45 μ m PVDF membrane filter (Sigma Aldrich, catalogue HVLP04700).Add protease inhibitor cocktail III (Sigma Aldrich, catalogue plus 539134) and supernatant buffer is exchanged in HBS (20mM HEPES pH8, 150mM NaCl) and on tangential flow, is concentrated into 300mL.With 3x 3mL b.vStreptactin-XT workflow platform with batch mode purified protein, and under agitation, hatch 2x 1h, and at 4 ℃, hatch 1x and spend the night.Before wash-out, use 10 CV HBS buffer wash resins. The protein was eluted in 4 x 5 mL of BXT elution buffer. The eluate was analyzed by nanodrop A280 and reducing SDS-PAGE, concentrated to approximately 2 mL, and the TST purification tag was cleaved by incubation with TEV at a TEV:protein ratio of 1:80 at 18°C ​​overnight with inversion mixing. The cleaved protein was confirmed by SDS-PAGE before loading onto an SX7516 / 600 or SX200 16 / 600 size exclusion column (GE Healthcare, catalog 28-9893-33 or 28-9893-35) equilibrated in 20 mM BisTris pH 6.5, 150 mM NaCl (see Figure 11). Protein-containing fractions were analyzed by reducing SDS-PAGE, pooled, and the concentration was measured by nanodrop A280 measurement.

[0476] For protein purification of receptor variants, wild-type and G-CSFR (Ig-CRH) were cloned as described above. E Mutant, the receptor construct for purification also comprises Ig domain (residue 3-308 of Uniprot ID Q99062, table 1) except CRH domain.As mentioned above, prepared virus stock solution and used for the infection of 2-4L scale.The supernatant solution buffer of clarification is exchanged into Ni-NTA binding buffer (20mM HEPES pH8, 1M NaCl, 30mM imidazoles), and is concentrated to 300mM as mentioned above.With 3x 3mL bv Ni-NTA workflow platform with batch binding mode purifying protein, and under agitation, hatch 2x 1h, and at 4 DEG C, hatch 1x and spend the night.Before eluting, use 10 CV binding buffer solution to wash resin.Elute protein in 4x5mL Ni-NTA elution buffer (20mM HEPES pH8, 1M NaCl, 250mM imidazoles). The eluate was analyzed, buffer exchanged into 20 mM Bis-Tris pH 6.5, 150 mM NaCl, concentrated and lysed overnight as described above. The cleaved protein was then loaded onto a SX 75 16 / 600 or SX200 16 / 600 size exclusion column (GE Healthcare) equilibrated in 20 mM BisTris pH 6.5, 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.

[0477] result

[0478] Wild type, G-CSF E and G-CSFR E The mutants were >90% pure after SEC as judged by reducing SDS-PAGE (see Figure 11 and 12 Design 401 and 402G-CSF E The yields of 401 and 402 G-CSFR per 1 L culture after SEC were 2.7 mg and 1.6 mg, respectively. E The yields of the products after SEC per 1 L were 1.7 mg and 1.5 mg, respectively. WT G-CSF was purified at a yield of 2.1 mg per 1 L of culture after SEC, and WT G-CSFR was purified at a yield of 3.1 mg per 1 L of culture after SEC.

[0479] These results demonstrate that the methods used to purify variant G-CSF and receptors can effectively produce purified proteins for in vitro analysis of biophysical properties.

[0480] Example 6: Determination of the affinity of designed binding partners to their matched and mismatched counterparts by SPR

[0481] To determine the affinity of the designed cytokines for their coevolved receptor mutants, G-CSF E G-CSFR E The affinity of G-CSF of the designed subset was also determined by SPR (mismatch). E G-CSFR WT affinity and G-CSF WT G-CSFR E affinity.

[0482] method

[0483] SPR binding assays were performed on a Biacore T200 instrument (GE Healthcare, Mississauga, ON, Canada) using PBS-T (PBS + 0.05% (v / v) Tween 20) running buffer at 25°C. CM5 Series S sensor chips, Biacore amine coupling kits (NHS, EDC, and 1 m ethanolamine), and 10 mM sodium acetate buffer were all purchased from GE Healthcare. PBS running buffer with 0.05% Tween 20 (PBS-T) was purchased from Teknova Inc. (Hollister, CA). The design was evaluated in three different fixed orientations.

[0484] To determine G-CSF E G-CSFR E The binding affinity of G-CSFR was determined by standard amine coupling as described by the manufacturer (GE LifeSciences). E Briefly, after EDC / NHS activation, G-CSFR was injected at a flow rate of 5 μL / min. E A 5 μg / mL solution in 10 mM NaOAc pH 5.0 was added until a receptor density of approximately 700-900 RU was reached. Remaining active groups were quenched by injecting 1 M ethanolamine hydrochloride-NaOH pH 8.5 at 10 μL / min for 420 s. Using single-cycle kinetics, two-fold serial dilutions of the corresponding G-CSF starting at 200 nM using blank buffer control were added. ESix concentrations of the mutant were sequentially injected at 25 μL / min for 300 s, followed by an 1800 s dissociation phase, generating a set of sensorgrams with a buffer blank reference. The same sample titration was also performed on a reference cell without captured variants. The chip was regenerated by a single pulse of 10 mM glycine / HCl pH 2.0 at 30 μL / min for 30 s in preparation for the next injection cycle.

[0485] To evaluate G-CSF WT G-CSFR E The binding affinity, as described above, of G-CSF E Using single cycle kinetics, each G-CSFR was captured on the chip at a density of approximately 700-900 RU. A two-fold serial dilution of each G-CSFR starting at 200 nM with a blank buffer control was used. WT Six concentrations of α-aminobutyric acid (α-aminobutyric acid) were sequentially injected at 25 μL / min for 300 s with a total dissociation time of 1800 s to generate a set of sensorgrams with a buffer blank reference. The same sample titration was also performed on reference cells without capture variants, and the chip was regenerated as described above.

[0486] To evaluate G-CSF E G-CSFR WT The binding affinity of the recombinant G-CSFR was captured as described above in this example and purified as described in Example 5. WT Using single-cycle kinetics, two-fold serial dilutions of each G-CSF starting at 200 mM with blank buffer were injected sequentially as described above. E Six concentrations of the mutant were obtained. The same sample titration was also performed on reference cells without captured variants. G-CSFR was regenerated as described above. WT surface.

[0487] As a control, binding of WT G-CSF to WT G-CSFR (Ig-CRH) was assessed in each experiment and used to calculate K in each independent measurement. D Fold change.

[0488] Using Biacore TM Dual-referenced sensorgrams from duplicate or triplicate injections were analyzed by T200 Evaluation Software v3.0 and fitted to a 1:1 Langmuir binding model.

[0489] result

[0490] Kinetically derived affinity constant (K D ), which is obtained by fitting the association and dissociation phases of the curve. K of WT G-CSF to WT G-CSFR (Ig-CRH) DThe range of K was between 1.8 and 2.5E-9. In cases where the kinetic parameters could not be fitted, an attempt was made to derive the steady-state affinity constant. In these cases, the steady-state derived K for the WT G-CSF:WT G-CSFR(Ig-CRH) pair was D KD fold changes were calculated and indicated in Table 7.

[0491] Designs 9, 130, 134, 137, 307, 401, and 402 showed affinities to their coevolved binding partners no greater than 2-fold the WT:WT KD (see Tables 7 and Figure 13 ).

[0492] Design 9, 30, and 34G-CSFR (Ig-CRH) E The mutant showed an affinity for WT G-CSF that was more than 700-fold weaker than WT G-CSFR(Ig-CRH). Design #35G-CSFR(Ig-CRH) E The selectivity for mismatches with WT cytokines is low, with affinity for WT G-CSF reduced by approximately 19-fold compared to WT:WT affinity. Designs 130, 134, 401, 402, 300, 3003, 304, and 307 G-CSFR (Ig-CRH) E The mutants were most selective for mismatches with WT G-CSF and showed no significant binding to WT G-CSF at the titrated concentrations (see Table 8, Figure 13 ).

[0493] Designs 124, 130, 401, 402, 300, 303, 304, and 307 G-CSF E The mutants did not show significant binding to WT G-CSFR (Ig-CRH) at the titrated concentrations. Design of 9, 30 and 34 G-CSF E The mutants showed K for WT G-CSFR (Ig-CRH) D At least about 20-fold weaker than WT:WT KD. Design #134G-CSF E The affinity for WT G-CSFR (Ig-CRH) was shown to be approximately 500-fold weaker (see Table 9, Figure 13 Design 35 and 117G-CSF E The mutants showed binding to WT G-CSFR (Ig-CRH) similar to the WT:WT KD.

[0494] These results indicate that the selected variant G-CSF designs either do not bind to wild-type G-CSFR ECD or bind with significantly reduced affinity (at least approximately 20-fold weaker KD) to wild-type G-CSFR ECD.

[0495] Table 7: G-CSF determined by SPR E Mutants to their corresponding G-CSFR (Ig-CRH) E Changes in binding affinity (KD) of mutants compared to WT:WT binding affinity

[0496]

[0497] ss represents the steady-state derived affinity constant.

[0498] Table 8: WT G-CSF and designed G-CSFR (Ig-CRH) determined by SPR E Changes in binding affinity compared to WT:WT binding affinity

[0499]

[0500]

[0501] ss represents the steady-state derived affinity constant

[0502] Table 9: G-CSF determined by SPR E Changes in binding affinity to wild-type G-CSFR (Ig-CRH) compared to WT:WT binding affinity

[0503]

[0504] Example 7: Design of G-CSF E Determination of thermal stability of mutants

[0505] To measure G-CSF E and G-CSFR E The thermal stability of the mutants compared to WT cytokines and receptors was determined by differential scanning calorimetry (DSC).

[0506] method

[0507] The thermal stability of the variants was evaluated by differential scanning calorimetry (DSC) as follows: 950 mL of purified samples at a concentration of 1-2 mg / mL were used for DSC analysis using a Nano DSC (TA instruments, New Castle, DE). At the beginning of each run, a buffer blank injection was performed to stabilize the baseline. Each sample was scanned from 25°C to 95°C at a rate of 60°C / h under 60 psi nitrogen pressure. The resulting thermograms were analyzed using NanoAnalyze software to determine the melting temperature (Tm) as an indicator of thermal stability.

[0508] result

[0509] The thermal stability of the engineered variants is reported as the difference between the most significant transition (highest enthalpy) of the engineered molecule and the equivalent wild-type molecule measured under the same conditions and experimental setup. In independent experiments, the measured Tm of WT GCSF varied between 52.2°C and 55.4°C, while WT G-CSFR showed a Tm of 50.5°C. Except for designs #15 and 34, the G-CSFRs tested E The mutants showed a Tm of less than 5°C, which was different from the Tm of WT G-CSF (see Tables 10 and Figure 14 All tested receptor mutants showed the same thermal stability as the WT receptor (see Tables 10 and Figure 14 ).

[0510] These results indicate that variant G-CSF and receptors with variant G-CSFR ECD designs have similar thermostabilities to wild-type G-CSF and G-CSFR; and that mutations at Site II and / or Site III do not disrupt the thermostability of G-CSF or G-CSFR ECD.

[0511] Table 10: Changes in melting temperature (Tm) of designed cytokine and receptor mutants compared to wild type as determined by DSC.

[0512]

[0513]

[0514] *Measured in 150 mM NaCl, 20 mM BisTris pH 6.5

[0515] Example 8: Determination of G-CSF by UPLC-SEC E Monodispersity of mutants

[0516] To measure G-CSF E The monodispersity of the mutants compared to WT G-CSF was analyzed by UPLC-SEC.

[0517] method

[0518] SEC-purified protein samples were subjected to UPLC-SEC using an Acquity BEH125 SEC column (4.6 x 150 mm, stainless steel, 1.7 μm particles) (Waters LTD, Mississauga, ON), which was set at 30°C and installed on an Agilent Technologies 1260infinity II system with a PDA detector. The run time consisted of 7 minutes with either 150 mM NaCl, 20 mM HEPES pH 8.0 or 150 mM NaCl, 20 mM BisTris pH 6.5 running buffer at a flow rate of 0.4 mL / min. Elution was monitored by UV absorbance in the range of 210-500 nm and chromatograms were extracted at 280 nm. HPLC was performed using OpenLAB TM CDS ChemStation TM The software performs peak integration.

[0519] result

[0520] WT G-CSF and Design 34, 35, and 130 G-CSF E The mutants were 100% monodisperse (see Table 11). Mutants 8, 9, 15, 117, and 135 showed lower monodispersities ranging from 65.3% to 79.5%. Design #134 Cytokine showed 57.3% monodispersity at pH 8.0, which improved to 86.6% monodispersity at pH 6.5. The improvement in mobile phase monodispersity at lower pH may be due to a shift in the pI, for example, from a calculated pI of 5.41 for WT G-CSF to a calculated pI of 5.41 for Design #134 G-CSF. E The calculated pI is 8.35.

[0521] These results demonstrate that certain variant G-CSF designs are 100% monodisperse compared to wild-type G-CSF, indicating that a subset of site II and / or site III mutations do not disrupt the monodispersity of G-CSF; whereas other variant G-CSF designs result in decreased monodispersity that increases at lower pH values.

[0522] Table 11: G-CSF assayed by UPLC-SEC E Design mutants for monodispersity.

[0523]

[0524]

[0525] *In 150 mM NaCl, BisTris pH 6.5

[0526] Example 9: Construction of a chimeric G-CSF receptor with an intracellular IL-2 receptor signaling domain

[0527] G-CSF for study design E Can cytokine mutants be engineered to produce G-CSFR (Ig-CRH) E The receptor mutants signaled and induced immune cell proliferation using G-CSFR ECD fused to the gp130 transmembrane (TM) domain and intracellular signaling domain (ICD) and the IL-2Rβ intracellular signaling domain (G-CSFR WT -ICD gp130-IL-2Rβ ) to construct a single-chain chimeric G-CSF receptor. A chimeric G-CSFR consisting of two subunits designed to be co-expressed as a heterodimeric receptor was also utilized: 1) G-CSFR WT -ICD IL-2Rβ The subunit consists of the G-CSFR ECD fused to the IL-2RβTM and ICD; and 2) the G-CSFR WT -ICD γc The subunits consist of the G-CSFR ECD fused to the common γ chain (γc, IL-2Rγ2Rγ)TM and ICD.

[0528] method

[0529] Single-chain chimeric receptor constructs were designed to contain the G-CSFR signal peptide and ECD, followed by the gp130 TM and partial ICD and the IL-2Rβ partial ICD (Table 12). Heterodimeric chimeric receptor constructs were designed to contain: 1) the G-CSFR signal peptide and ECD, followed by the IL-2Rβ TM and ICD (Table 13); and 2) the G-CSFR signal peptide and ECD, followed by the γc TM and ICD (Table 14). The chimeric receptor constructs were cloned into lentiviral transfer plasmids, and the construct sequences were verified by Sanger sequencing. The transfer plasmids and 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 before transfection. Plasmid DNA and water are mixed in a polypropylene tube and CaCl2 (0.25M) is added dropwise. After incubation for 2 to 5 minutes, the DNA is precipitated by mixing with 2x HEPES buffered saline (0.28M NaCl, 1.5mM Na2HPO4, 0.1M HEPES) 1:1. The precipitated DNA mixture is added to the cells and incubated overnight at 37°C, 5% CO2. HEK293T / 17 culture medium is replaced the next day, and the cells are incubated for another 24 hours. The next morning, cell supernatants are collected from the plates, centrifuged briefly to remove debris, and filtered through a 0.45 μm filter. In a Beckman Optima L-XP ultracentrifuge, a SW-32Ti rotor is used to spin the supernatant at 25,000 rpm for 90 minutes. The supernatant is removed, and the precipitation is resuspended in the Opti-MEM culture medium of an appropriate volume. Viral titers were determined by adding serial dilutions of virus to BAF3 cells (grown in RPMI containing 10% fetal bovine serum, penicillin, streptomycin, and 100 IU / ml hIL-2). 48–72 hours after transduction, cells were incubated with anti-human G-CSFR APC-conjugated antibody (1:50 dilution) and eBioscience TM FixableViability Dye eFluor TM450 (1:1000 dilution) were incubated together at 4°C for 15 minutes, washed and analyzed on a CytekAurora or BD FACS Calibur flow cytometer. Using the estimated titer determined by this method, the 32D-IL-2Rβ cell line (grown in RPMI containing 10% fetal bovine serum, penicillin, streptomycin and 300 IU / ml hIL-2) was transduced with a lentiviral supernatant encoding a chimeric receptor construct at an MOI of 0.5. Transduction was performed by adding the relevant amount of viral supernatant to the cells, incubating for 24 hours and replacing the cell culture medium. 3-4 days after transduction, the expression of human G-CSFR was verified by flow cytometry as described above. Before performing the BrdU assay, the cells were incubated in G-CSF WT The cells were expanded for about 14-28 days.

[0530] As described above, G-CSF WT 32D-IL-2Rβ cells expanded in the medium were washed three times in PBS and replated in fresh medium containing relevant assay cytokines (no cytokines, hIL-2 (300 IU / ml), G-CSF WT (30 ng / ml) or G-CSF E (30 ng / ml) for 48 hours. BrdU assay procedures were performed according to BD Pharmingen TM The instruction manual of the APC BrdU Flow Kit (557892) was added with the following: Cells were treated with BrdU and eBioscience TM FixableViability Dye eFluor TM The cells were incubated with 450 μg / ml (1:5000) for 30 min. Flow cytometry was performed using a Cytek Aurora instrument.

[0531] result

[0532] In the BrdU assay, the single-chain chimeric receptor construct G-CSFR was used in comparison with hIL-2 (300 IU / ml). WT -ICD gp130-IL-2Rβ or heterodimeric receptor construct G-CSFR WT -ICD IL-2Rβ Plus G-CSFR WT -ICD γc Transfected cells respond to G-CSF WT (30ng / ml) showed similar or superior proliferation. Cells did not proliferate in the absence of cytokines (see Figure 15 ).

[0533] These results indicate that both single-chain and heterodimeric chimeric receptor constructs can be activated to induce cell proliferation upon G-CSF stimulation.

[0534] Example 10: Design of 137G-CSFR by BrdU Detection E -ICD IL-2 Transduced with wild-type or engineered G- CSF 137 Proliferation of 32D-IL-2Rβ-treated cells

[0535] Site II / III combination designs that were sufficiently selective as judged by SPR or co-expression assays were tested in vitro for their ability to induce proliferation of 32D-IL-2Rβ cells.

[0536] method

[0537] The point mutations designed in 137 were introduced into the constructs described in Tables 12-14. 137 -ICD gp130-IL-2Rβ (homodimer) or G-CSFR 137 -ICD IL-2Rβ Plus G-CSFR 137 -ICD γc The cloning and expression of the (heterodimer) constructs followed the same procedures as described above. Before the BrdU assay, cells were cultured in G-CSF. 137 The cells were expanded for about 14-28 days.

[0538] The BrdU assay was performed using the G-CSF 137 32D-IL-2Rβ cells were expanded in G-CSF 137 (30ng / ml), G-CSF WT Proliferation in the presence of cytokines (30 ng / ml), hIL-2 (300 IU / ml), or no cytokines.

[0539] result

[0540] In the BrdU assay, the single-chain chimeric receptor construct G-CSFR was used in comparison with hIL-2 (300 IU / ml). 137 -ICD gp130-IL-2Rβ or heterodimeric receptor construct G-CSFR 137 -ICD IL-2Rβ Plus G-CSFR 137 -ICD γc Transfected cells were in the presence of G-CSF 137 The cells did not proliferate in the absence of cytokines and in the presence of G-CSF (30 ng / ml). WT (30 ng / ml) showed poor proliferation (see Figure 16 ).

[0541] These results indicate that variant G-CSF specifically activates the engineered receptor; conversely, the engineered receptor is activated by variant G-CSF, but significantly less than wild-type G-CSF. Therefore, variant G-CSF can specifically activate chimeric receptors with variant G-CSF RECD, thereby specifically inducing the proliferation of cells expressing the chimeric receptor.

[0542] Example 11: Wild-type G-CSFR-ICD detected by BrdU IL-2 Transduction and wild-type or engineered G- CSF E Proliferation of 32D-IL-2Rβ-treated cells

[0543] The ability of site II / III combinations to restore paired signaling in 32D-IL-2R2Rβ cells was subsequently tested by designing single-chain chimeric receptor constructs for G-CSFR. WT -ICD gp130-IL-2Rβ or heterodimeric receptor construct G-CSFR WT -ICD IL-2Rβ Plus G-CSFR WT -ICD γc Proliferative capacity of transduced 32D-IL-2R2Rβ cells.

[0544] method

[0545] G-CSFR WT -ICD gp130-IL-2Rβ (homodimer) or G-CSFR WT -ICD IL-2Rβ Plus G-CSFR WT -ICD γc The cloning and expression of the (heterodimer) constructs followed the same procedures as described above. Before the BrdU assay, cells were cultured in G-CSF. WT The cells were expanded for about 14-28 days.

[0546] The BrdU assay was performed using the G-CSF WT 32D-IL-2Rβ cells were expanded in G-CSF 137 (30ng / ml), G-CSF WT Proliferation in the presence of cytokines (30 ng / ml), hIL-2 (300 IU / ml), or no cytokines.

[0547] result

[0548] In the BrdU assay, G-CSF WT (30 ng / ml) compared to the single-chain chimeric receptor construct G-CSFR WT -ICD gp130-IL-2Rβ or heterodimeric receptor construct G-CSFR WT -ICD IL-2Rβ and G-CSFR WT-ICD γc Transduced cells in G-CSF 137 (30 ng / ml) showed poor proliferation. Cells did not proliferate in the absence of cytokines (see Figure 17 ).

[0549] These results indicate that variant G-CSF cannot effectively bind to wild-type G-CSFR and that variant G-CSF specifically activates the engineered receptor, rather than wild-type G-CSFR, to induce cell proliferation.

[0550] Example 12: Western blot analysis of WT or engineered 137G-CSFR E -ICD IL-2 Transduction and wild-type Genetic or engineered G-CSF 137 Signaling in treated 32D-IL-2Rβ cells

[0551] The site II / III combination design was able to restore proliferative signaling through the engineered cytokine-receptor complex in 32D-IL-2Rβ cells and to inhibit the proliferation of IL-2Rβ cells by binding G-CSF. WT or WT-GCSFR-ICD-IL2 did not significantly transmit signaling, as assessed by Western blot. WT or G-CSF 137 And the ability to activate downstream signal transduction molecules.

[0552] method

[0553] G-CSFR WT -ICD gp130-IL-2Rβ (homodimer) or G-CSFR WT -ICD IL-2Rβ Plus G-CSFR WT -ICD γc The cloning and expression of the (heterodimer) constructs followed the same procedures as described above. Before Western blot analysis, cells were cultured in G-CSF. 137 or G-CSF WT Untransduced cells were maintained in IL-2 for approximately 14-28 days.

[0554] For Western blotting, cells were washed three times in PBS and placed in cytokine-free medium for 16-20 hours. Cells were not stimulated with cytokines, with IL-2 (300 IU / ml), G-CSF 137 (30 ng / ml) or G-CSF WTThe cells were stimulated with 10 mM HEPES (pH 777.9), 1 mM MgCl₂, 0.05 mM EGTA, 0.5 mM EDTA pH 8.0, 1 mM DTT, and 1 x Pierce protease and phosphatase inhibitor mini tablets (A32961) at 37°C for 20 minutes. The cells were washed once in a wash buffer containing 10 mM HEPES pH 777.9, 1 mM MgCl₂, 0.05 mM EGTA, 0.5 mM EDTA pH 8.0, 1 mM DTT, and 1 x Pierce protease and phosphatase inhibitor mini tablets (A32961). The cells were lysed in the wash buffer, 0.2% Igepal CA630 (Sigma) was added, lysed on ice for 10 minutes, and centrifuged at 13,000 rpm for 10 minutes at 4°C before collecting the supernatant (cytoplasmic fraction). The pellet was resuspended and dissolved in the wash buffer, 0.42 M NaCl, and 20% glycerol was added. The cells were lysed on ice for 30 minutes, vortexed frequently, and centrifuged at 13,000 rpm for 20 minutes at 4°C before collecting the nuclear fraction (supernatant). The cytoplasmic and nuclear fractions were reduced (70°C) for 10 min and subjected to NuPAGE. TM Run on a 4-12% Bis-Tris protein gel. Transfer the gel to a nitrocellulose membrane (at 20 V in SD Semi-Dry Transfer Cell for 60 min), dried and Block in blocking buffer (927-50000) for 1 h. The blot was incubated overnight with primary antibodies (1:1,000) in blocking buffer. Primary antibodies used were obtained from Cell Signaling Technologies: Phospho-Shc (Tyr239 / 240) Antibody #2434, Phospho-Akt (Ser473) (D9E) 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) Rabbit mAb #9145, phospho-Stat5 (Tyr694) (C11C5) rabbit mAb #9359 and histone H3 (96C10) mouse mAb #3638. The blot was washed three times in TBS containing 0.1% Tween20 and incubated with secondary antibodies (1:10,000) in TBS buffer containing 0.1% Tween20 for 30-60 minutes at room temperature. Secondary antibodies were obtained from CellSignaling Technologies: anti-mouse IgG (H+L) (DyLight TM800 4X PEG conjugate) #5257 and anti-rabbit IgG (H+L) (DyLight TM 800 4X PEG conjugate) #5151. The blot was washed and exposed on a LI-COR Odyssey imager.

[0555] result

[0556] In non-transduced 32D-IL-2Rβ cells, only activated IL-2R-associated signaling molecules were detected in response to stimulation with IL-2. WT -ICD gp130-IL-2Rβ or G-CSFR WT -ICD IL-2Rβ Plus G-CSFR WT -ICD γc 32D-IL-2Rβ cells were observed to respond to IL-2 or G-CSF WT No IL-2R-associated signaling molecules were observed in response to expression of G-CSFR. WT -ICD gp130-IL-2Rβ 32D-IL-2Rβ cells or cells expressing G-CSFR WT -ICD IL-2Rβ Plus G-CSFR WT -ICD γc G-CSF in cells 137 Stimulation of activation. 137 -ICD gp130-IL-2Rβ or G-CSFR 137 -ICD IL-2Rβ Plus G-CSFR 137 -ICD γc 32D-IL-2Rβ cells were observed to respond to IL-2 or G-CSF 137 Similar patterns of activation of signaling molecules were observed in the G-CSFR 137 -ICD gp130-IL-2Rβ 32D-IL-2Rβ cells or cells expressing G-CSFR 137 -ICD IL-2Rβ Plus G-CSFR 137 -ICD γc cells respond to G-CSF WT Stimulated activation of IL-2R2R2R-related signaling molecules (see Figure 18 ).

[0557] These results demonstrate that variant G-CSF is able to activate chimeric receptors expressing variant G-CSFR ECD, thereby inducing aspects of native cytokine signaling in cells expressing the chimeric receptor.

[0558] Methods of Examples 13-30

[0559] 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 previously generated by stably transfecting the human IL-2Rβ subunit into the BAF3 cell line and were cultured in DMEM containing 10% fetal bovine serum, penicillin, streptomycin, and 100 IU / ml human IL-2 (hIL-2) ( Novartis Pharmaceuticals Canada). 32D-IL-2Rβ cells were previously generated by stably transfecting 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 hIL-2 or other cytokines as indicated. Human PBMC-derived T cells (Hemacare) were cultured in TexMACS containing 3% human AB serum (Sigma-Aldrich, H4522) and 300 IU / ml hIL-2 or other cytokines as indicated. TM Human tumor-associated lymphocytes (TAL) were generated by culturing primary ascites samples for 14 days in T cell culture medium, which was a 50:50 mixture of: 1) RPMI-1640 containing 10% fetal bovine serum, 50 μM β-mercaptoethanol, 10 mM HEPES, 2 mM L-glutamine, penicillin, and streptomycin; and 2) AIM V containing a final concentration of 3000 IU / ml hIL-2. TM Culture medium (ThermoFisher, 12055083). Following this high-dose IL-2 expansion, TALs were cultured in T cell culture medium containing 300 IU / ml hIL-2 or other cytokines as indicated. The retroviral packaging cell line Platinum-E (Cell Biolabs, RV-101) was cultured in DMEM containing 10% FBS, penicillin / streptomycin, puromycin (1 mcg / ml), and blasticidin (10 mcg / ml).

[0560] Lentiviral production and transduction of 32D-IL-2Rβ cells: The chimeric receptor construct was cloned into a lentiviral transfer plasmid, and the resulting sequence was verified by Sanger sequencing. The transfer plasmid and lentiviral packaging plasmid were co-transfected into HEK293T / 17 cells using the calcium phosphate transfection method as follows: 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 (0.25M) was added dropwise. After incubation for 2 to 5 minutes, the DNA was precipitated by mixing 1:1 with 2x HEPES-buffered saline (0.28M NaCl, 1.5mM Na2HPO4, 0.1M HEPES). The precipitated DNA mixture was added to the cells and incubated overnight at 37°C, 5% CO2. The HEK293T / 17 medium was changed the next day, and the cells were incubated for an additional 24 hours. The next morning, cell supernatants were collected from the plates, centrifuged briefly to remove debris, and the supernatants were filtered through a 0.45 μm filter. The supernatants were spun at 25,000 rpm for 90 minutes using a SW-32Ti rotor in a Beckman Optima L-XP ultracentrifuge. The supernatant was removed and the pellet was resuspended in an appropriate volume of Opti-MEM medium. Viral titer was determined by adding serial dilutions of the virus to BAF3-IL-2Rβ cells. 48-72 hours after transduction, cells were incubated with anti-human G-CSFR APC-conjugated antibody (1:50; Miltenyi Biotec, 130-097-308) and Fixable Viability Dye eFluor TM 450(1:1000,eBioscience TM ,65-0863-14) were incubated at 4 ° C for 15 minutes, washed, and analyzed on a Cytek Aurora or BD FACS Calibur flow cytometer. Using the estimated titer determined by this method, the 32D-IL-2Rβ cell line was transduced with lentiviral supernatant encoding the chimeric receptor construct at a multiplicity of infection (MOI) of 0.5. Transduction was performed by adding the relevant amount of viral supernatant to the cells, incubating for 24 hours, and then replacing the culture medium. 3-4 days after transduction, the expression of human G-CSFR was determined by flow cytometry as described above.

[0561] Lentiviral transduction of human primary T cells: To transduce PBMC-derived T cells and TAL, cells were thawed and transduced in the Human T Cell TransAct TM(Miltenyi Biotec, 130-111-160). 24 hours after activation, lentiviral supernatant was added at an MOI of 0.125-0.5. After 48 hours of activation, cells were divided into fresh medium to remove residual virus and activation reagent. Two to four days after transduction, the transduction efficiency was determined by flow cytometry as described above. For experiments in which the transduction efficiency of CD4+ and CD8+ fractions was determined separately, the transduction efficiency of the CD4+ and CD8+ fractions was determined using antibodies against human G-CSFR, CD4 (1:50, Alexa Fluor 500). 700 conjugate, BioLegend, 300526), ​​CD8 (1:50, PerCP conjugate, BioLegend, 301030), CD3 (1:50, Brilliant Violet 510 TM Conjugate, BioLegend, 300448) and CD56 (1:50, BrilliantViolet 711 TM Conjugate, BioLegend, 318336) antibody and Fixable Viability DyeeFluor TM 450(1:1000).

[0562] Human T cell and 32D-IL-2Rβ expansion assays: Human primary T cells expressing the indicated chimeric receptor constructs generated above or 32D-IL-2Rβ cells were washed three times in PBS and replated in fresh medium or had their medium changed gradually as indicated. Complete medium was replaced with medium containing wild-type human G-CSF (produced in-house or Cells were analyzed using a 400-well plate (Amgen, Canada), mutant G-CSF (produced in-house), hIL-2, or no cytokine. Cell viability and density were determined every 3-5 days by trypan blue exclusion, and fold expansion was calculated relative to the starting cell number. G-CSFR expression was assessed by flow cytometry as described above.

[0563] CD4+ and CD8+ human TAL expansion assay: To examine the expansion of the CD4+ and CD8+ fractions of TAL, ex vivo ascites samples were thawed and enriched for CD4+ and CD8+ fractions using a human CD4+ T cell isolation kit (Miltenyi Biotec, 130-096-533) and a human CD8+ T cell isolation kit (Miltenyi Biotec, 130-096-495). After expansion in cytokine-containing medium, the immunophenotype of the cells was assessed by flow cytometry using antibodies against human G-CSFR, CD4 (1:50, Alexa Fluor, 512). 700 conjugate, BioLegend, 300526), ​​CD8 (1:50, PerCP conjugate, BioLegend, 301030), CD3 (1:50, Brilliant Violet 510 TM Conjugate, BioLegend, 300448) and CD56 (1:50, Brilliant Violet 711 TM Conjugate, BioLegend, 318336) antibody and FixableViability Dye eFluor TM 450(1:1000).

[0564] Primary human T cell immunophenotyping assay: After expansion in cytokine-containing medium, the immunophenotype of T cells was assessed by flow cytometry using the following antibodies: 700 conjugate, BioLegend, 300526; or PE conjugate, eBioscience TM , 12-0048-42; or Brilliant Violet 570 TM conjugate, Biolegend, 317445), CD8 (1:100, PerCP conjugate, BioLegend, 301030), CD3 (1:100, Brilliant Violet 510 TM or Brilliant Violet 750 TM conjugate, BioLegend, 300448 or 344845), CD56 (1:100, Brilliant Violet 711 TM conjugate, BioLegend, 318336), CCR7 (1:50, APC / Fire TM 750 conjugate, Biolegend, 353246), CD62L (1:33, PE / Dazzle TM 594 conjugate, Biolegend, 304842), CD45RA (1:33, FITC conjugate, Biolegend, 304148), CD45RO (1:25, 710 conjugate, eBioscience TM , 46-0457-42), CD95 (1:33, PE-Cyanine7 conjugate, eBioscience TM,25-0959-42) and Fixable Viability Dye eFluor TM 450 or 5106 (1:1000).

[0565] Retroviral transduction: The pMIG transfer plasmid (plasmid #9044, Addgene) was modified by restriction endonuclease cloning to remove IRES-GFP (BglII to PacI site) and introduce an annealed primer encoding a custom multiple cloning site. The chimeric receptor construct was cloned into the custom transfer plasmid and the resulting sequence was verified by Sanger sequencing. As described above, the transfer plasmid was transfected into Platinum-E cells using the calcium phosphate transfection method. 24 hours after transfection, the culture medium was replaced with 5 ml of fresh complete medium. 48 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. The purified retroviral supernatant was used to transduce mouse lymphocytes as described below.

[0566] 48 hours before collecting retroviral supernatant, 24-well adhesive plates were coated with unconjugated anti-mouse CD3 (5mcg / ml, BD Biosciences, 553058) and anti-mouse CD28 (1mcg / ml, BD Biosciences, 553294) antibodies, diluted in PBS, and stored at 4 degrees Celsius. 24 hours before collecting retroviral supernatant, C57Bl / 6J mice (generated in-house) were euthanized according to the animal use protocol approved by the University of Victoria Animal Care Committee. Spleens were harvested and murine T cells were isolated as follows: spleens were manually isolated and filtered through a 100 micron filter. Red blood cells were lysed by incubation in ACK lysis buffer (Gibco, A1049201) for five minutes at room temperature, then washed once in serum-containing culture medium. CD8a-positive or Pan-T cells were isolated using a specific bead-based separation kit (Miltenyi Biotec, 130-104-075 or 130-095-130, respectively). Cells were added to a plate coated with anti-CD3 and anti-CD28 antibodies in mouse T cell expansion medium (containing 10% FBS, penicillin / streptomycin, 0.05 mM β-mercaptoethanol and 2 ng / ml mouse IL-2 (Peprotech, 212-12) or 300 IU / mL human IL-2 (Proleukin's RPMI-1640)) and incubated at 37 degrees Celsius, 5% CO2 for 24 hours. On the day of transduction, approximately half of the culture medium was replaced with the retroviral supernatant produced above. Cells were spin-fected with the retroviral supernatant at 30 degrees Celsius at 1000 g for 90 minutes. Return the plate to the incubator for 0-4 hours, then replace approximately half of the medium with fresh T cell expansion medium. Repeat retroviral transduction as described above 24 hours later for a total of two transductions. 24 hours after the final transduction, T cells were split into 6-well plates and removed from antibody stimulation.

[0567] 48–72 h after transduction, transduction efficiency was assessed by flow cytometry to detect human G-CSFR, CD4 (Alexa Fluor 532 conjugate, eBioscience TM , 58-0042-82), CD8a (PerCP-eFluor 710 conjugate, eBioscience TM , 46-0081-82) and Fixable Viability Dye eFluor TM 450 (1:1000 dilution).

[0568] BrdU incorporation assay: Human primary T cells, 32D-IL-2Rβ cells, or murine primary T cells generated as described above were washed three times in PBS and replated for 48 hours in fresh medium containing the following relevant assay cytokines: no cytokine, hIL-2 (300 IU / ml), wild-type or engineered G-CSF (at the concentrations indicated in individual experiments). The BrdU assay procedure followed the protocol described by BD Pharmingen. TM The instruction manual of the APC BrdU Flow Kit (BD Biosciences, 557892) was added with the following content: cells were incubated with BrdU and Fixable Viability Dye eFluor TM The cells were incubated with 450 (1:5000) at 37°C for 30 minutes to 4 hours. Flow cytometry was performed using a Cytek Aurora instrument. To specifically assess the proliferation of murine T cells expressing the chimeric receptor, additional staining for human G-CSFR (1:20 dilution), CD4 (1:50 dilution), and CD8 (1:50 dilution) was performed on ice for 15 minutes before fixation.

[0569] Western blot: Human primary T cells, 32D-IL-2Rβ cells, or murine primary T cells generated as described above were washed three times in PBS and placed in cytokine-free medium for 16-20 hours. Cells were not stimulated with cytokines, with IL-2 (300 IU / ml), wild-type G-CSF (at the concentrations indicated in individual experiments), or with G-CSF at 37°C. 137 The cells were stimulated with 10 mM HEPES pH 7.9 (30 ng / ml) for 20 minutes. The cells were washed once in a buffer containing 10 mM HEPES pH 7.9, 1 mM MgCl , 0.05 mM EGTA, 0.5 mM EDTA pH 8.0, 1 mM DTT and 1 x Pierce protease and phosphatase inhibitor mini tablets (A32961). The cells were lysed on ice for 10 minutes in the above-mentioned wash buffer, wherein 0.2% NP-40 (Sigma) was added. The lysate was centrifuged at 13,000 rpm for 10 minutes at 4 degrees Celsius, and the supernatant (cytoplasmic fraction) was collected. The pellet (containing nuclear protein) was resuspended in the above-mentioned wash buffer, wherein 0.42 M NaCl and 20% glycerol were added. The nucleus was incubated on ice for 30 minutes, vortexed frequently, and the supernatant (nuclear fraction) was collected after centrifugation at 13,000 rpm for 20 minutes at 4 degrees Celsius. The cytoplasmic and nuclear fractions were reduced (70°C) for 10 min and subjected to NuPAGE. TM Run on a 4-12% Bis-Tris protein gel. Transfer the gel to a nitrocellulose membrane (at 20 V in SD Semi-Dry Transfer Cell for 60 min), dried and Blocked in blocking buffer (927-50000) for 1 h. The blots were incubated with primary antibodies (1:1000) overnight in blocking buffer. 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) 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) Rabbit mAb #9145, phospho-STAT4 (Tyr693) antibody #5267, phospho-STAT5 (Tyr694) (C11C5) rabbit mAb #9359, and histone H3 (96C10) mouse mAb #3638. The blot was washed three times in 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 antibody obtained from Cell Signaling Technologies was anti-mouse IgG (H+L) (DyLight TM 800 4X PEG conjugate) #5257 and anti-rabbit IgG (H+L) (DyLight TM 800 4X PEG conjugate) #5151. The blot was washed and exposed on a LI-COR Odyssey imager.

[0570] Flow cytometry for detection of phosphorylated proteins: Human primary T cells, 32D-IL-2Rβ cells, or mouse primary T cells generated as described above were washed three times in PBS and placed in cytokine-free medium for 16-20 hours. TM Cells were not stimulated with cytokines, stimulated with IL-2 (300 IU / ml), or wild-type G-CSF (100 ng / ml) in the presence of IL-450 (1:1000) and the indicated anti-G-CSFR (1:20), anti-CD4 (1:50), and anti-CD8a (1:50) at 37 degrees Celsius for 20 minutes. The cells were pelleted and analyzed using BD Phosflow TM Fixation buffer I (BD Biosciences, 557870) was used for 15 minutes. The cells were washed and then lysed on ice using BD Phosflow TM The cells were permeabilized with permeabilization buffer III (BD Biosciences, 558050) for 15 minutes. The cells were washed twice and resuspended in 20 μl of BD Phosflow TM PE mouse anti-Stat3 (pY705) (BD Biosciences, 612569) or PE mouse IgG2aκ isotype control (BD Biosciences, 558595) were added to the buffer. The cells were washed and flow cytometry was performed using a Cytek Aurora instrument.

[0571] Example 13: Expression of G2R-1, G-CSFR / IL-2R only Β subunit, only MYC-tagged G-CSFR / γ -C subunit or whole Expansion of human T cells with long G-CSFR .

[0572] Use encoding Figure 1 PBMC-derived T cells or tumor-associated lymphocytes (TAL) were lentivirally transduced with the indicated chimeric receptor constructs, and the cells were washed and replated in the indicated cytokines. Cells were counted every 3-4 days. G / γc was tagged with the Myc epitope at its N-terminus (Myc / G / γc), and G / IL-2Rβ was tagged with the Flag epitope at its N-terminus (Flag / G / IL-2Rβ); these epitope tags facilitate detection by flow cytometry and do not affect the function of the receptors. 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 only observed for PBMC-derived T cells and TAL expressing the G2R-1 chimeric cytokine receptor ( Figure 22). Note that the lentiviral transduction efficiency was less than 100%, resulting in less than 100% of T cells expressing the indicated chimeric cytokine receptors, which may explain the lower proliferation rate mediated by G2R-1 relative to IL-2. Similarly, increased proliferation was observed in 32D-IL-2Rβ cells (stable expression of 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 observed in 32D-IL-2Rβ cells expressing only the G / γc chimeric receptor subunit ( Figure 2 ).

[0573] These results indicate that G-CSF can stimulate the proliferation and viability of PMBC-derived T cells and TAL cells 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.

[0574] Example 14: G-CSF RECD is expressed on the surface of cells transduced with G / IL-2Rβ, G2R-1, and G2R-2.

[0575] Flow cytometry was performed on the 32D-IL-2Rβ cell line, PBMC-derived human T cells, and human tumor-associated lymphocytes after transduction with a lentiviral vector encoding the G2R-2 chimeric cytokine receptor ( Figure 23 and 25 G-CSFR positive cells were detected in all transduced cell types ( Figure 26 In a separate experiment, 32D-IL-2Rβ cells expressing G / IL-2Rβ, G2R-1, and G2R-2 chimeric receptors were positive for G-CSFR ECD as measured by flow cytometry ( Figure 21 B-21D (below).

[0576] These results indicate that G / IL-2Rβ, G2R-1, and G2R-2 chimeric receptors are expressed on the cell surface.

[0577] Example 15: Expansion of cells expressing G2R-2 compared to non-transduced cells

[0578] Human PBMC-derived T cells and human tumor-associated lymphocytes were lentivirally transduced with G2R-2 receptor constructs ( Figure 4 and 6), washed, and re-plated 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. After stimulation with G-CSF (100 ng / ml), PMBC-derived T cells ( Figure 27 A) and tumor-associated lymphocytes ( Figure 27 B, proliferation of 27C (two independent experiments).

[0579] These results indicate that G-CSF-induced activation of the G2R-2 chimeric receptor is sufficient to induce the proliferation and viability of immune cells.

[0580] Example 16: CD4-selected or CD8-selected human tumor-associated lymphocytes expressing G2R-2 compared to untransduced cells Cell expansion and immunophenotyping

[0581] CD4-selected and CD8-selected human T cells were transduced with lentiviral vectors encoding G2R-2 ( Figure 25 ), or remained untransduced as indicated. Cells were washed and replated with the indicated cytokines and counted every 3-4 days. Proliferation of CD4-selected or CD8-selected TAL 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 (medium only). Figure 28 and 29 ).exist Figure 28 In , each line represents the results from one of five patient samples.

[0582] Immunophenotyping by flow cytometry showed that T cells cultured in G-CSF or IL-2 retained their CD4+ or CD8+ identity ( Figure 30 A), lacking NK cell phenotype (CD3-CD56+) ( Figure 30 A), and exhibited CD45RA-CCR7-T effector memory (T) under these culture conditions. EM ) phenotype( Figure 30 B).

[0583] BrdU assay was performed to confirm that cell cycle progression of G2R-2 expressing T cells was increased after stimulation with G-CSF ( Figure 31 As indicated, T cells were selected by culture in IL-2 or G-CSF before assay. Tumor-associated lymphocytes ( Figure 31 A) and PBMC-derived T cells ( Figure 31 B).

[0584] These results demonstrate that G-CSF can selectively activate cell cycle progression and long-term expansion of primary human TALs by activating the chimeric cytokine receptor G2R-2. These results also indicate that activation of the G2R-2 chimeric receptor through homodimer formation is sufficient to activate cytokine-like signaling and proliferation in TALs. Furthermore, TALs expressing G2R-2 remain cytokine-dependent as they undergo cell death upon G-CSF withdrawal, similar to the response to IL-2 withdrawal. TALs cultured in G-CSF maintain a similar immunophenotype to those cultured in IL-2.

[0585] Example 17: Primary murine T cells expressing G2R-2 proliferate in response to G-CSF.

[0586] BrdU incorporation assays were performed to assess the proliferation of primary murine T cells expressing G2R-2 or single-chain G / IL-2Rβ (a component of G2R-1) relative to mock-transduced cells when stimulated with G-CSF. All cells were expanded in 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 32 A). Cells were then plated in IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine, as indicated. Increased cell cycle progression following G-CSF stimulation was observed in cells expressing G2R-2 compared to untransduced cells or cells expressing single-chain G / IL-2Rβ ( Figure 32 Panels B and C show the results for all viable cells or G-CSFR+ cells, respectively.

[0587] These results indicate that the G2R-2 chimeric receptor activates cytokine-like signaling and proliferation in murine T cells more efficiently than the single-chain G / IL-2Rβ receptor in response to G-CSF-induced homodimerization.

[0588] Example 18: Cytokine activation in response to G-CSF or IL-2 in human primary T cells expressing G2R-2 Related intracellular signaling events

[0589] To confirm that the chimeric cytokine receptor is indeed able to activate cytokine signaling similar to IL-2, the ability of the cytokine receptor to activate various signaling molecules was assessed. Tumor-associated lymphocytes and PBMC-derived T cells expressing G2R-2 were previously expanded in G-CSF, while non-transduced cells were previously expanded in IL-2. The cells were washed and then stimulated with IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine, and cell lysates were subjected to Western blotting using antibodies against the indicated signaling molecules ( Figure 33). Panels A and B show the results of TAL, and Panel C shows the results of PBMC-derived T cells. T cells expressing G2R-2 activated IL-2-related signaling molecules when stimulated with G-CSF to a similar extent as that seen after IL-2 stimulation of untransduced or transduced cells, with the expected exception that G-CSF induced Jak2 phosphorylation, while IL-2 induced Jak3 phosphorylation.

[0590] These results demonstrate that the G2R-2 chimeric receptor is capable of activating IL-2 receptor-like cytokine receptor signaling upon stimulation with G-CSF.

[0591] Example 19: Activation of cytokine signaling in response to G-CSF in murine primary T cells expressing G2R-2.

[0592] To assess whether the chimeric cytokine receptor G2R-2 or single-chain G / IL-2Rβ (from G2R-1) could activate cytokine signaling, the ability of these cytokine receptors to activate various signaling molecules was assessed by Western blotting of cell lysates of murine primary T cells expressing G2R-2 or G / IL-2Rβ relative to mock-transduced cells. All cells were expanded in IL-2 for 3 days prior to the assay. The cells were then washed and stimulated with IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. Cells expressing G2R-2 activated IL-2-related signaling molecules upon stimulation with G-CSF to a similar extent as that seen after IL-2 stimulation of untransduced or transduced cells, with the expected exception that G-CSF induced phosphorylation of Jak2, while IL-2 induced phosphorylation of Jak3 ( Figure 34 In contrast, G / IL-2Rβ does not activate cytokine signaling upon exposure to G-CSF.

[0593] These results demonstrate in primary murine T cells that the G2R-2 chimeric receptor is able to activate IL-2 receptor-like cytokine receptor signaling through homodimerization following G-CSF stimulation, whereas single-chain G / IL-2Rβ alone is unable to activate cytokine signaling through homodimerization in response to G-CSF.

[0594] Example 20: Expression of the chimeric receptor results in the expression of 32D-IL-2Rβ cells and primary murine T cells after stimulation with orthogonal G-CSF. Cell proliferation.

[0595] To determine whether cells expressing chimeric cytokine receptors can be selectively activated in response to orthogonal versions of G-CSF, 32D-IL-2Rβ cells or primary murine T cells were transduced with chimeric receptors G2R-1 and G2R-2 containing wild-type G-CSFR ECDs (G2R-1 WT ECD, G2R-2 WT ECD) and chimeric receptors G2R-1 and G2R-2 containing G-CSFR ECDs with amino acid substitutions R41E, R141E, and R167D (G2R-1 134 ECD, G2R-2 134 ECD). Cells were stimulated with IL-2, wild-type G-CSF, or an orthogonal G-CSF (130G-CSF), which binds to G2R-1 134 ECD and G2R-2 134 ECD but has significantly reduced binding to wild-type G-CSFR. BrdU incorporation assay was performed to assess the ability of cells to promote cell cycle progression under cytokine stimulation ( Figure 3 ). 32D-IL-2Rβ cells expressing G2R-2 134 ECD showed cell cycle progression after stimulation with 130G-CSF (containing amino acid substitutions E46R, L108K, and D112R; 30 ng / ml), but did not undergo cell cycle progression after stimulation with wild-type G-CSF (30 ng / ml). The orthogonal nature of the engineered cytokine:receptor ECD pairs was further demonstrated by stimulating primary murine T cells in a "criss-cross" proliferation assay, in which cells expressing G2R-3 with WT, 130, 134, 304, or 307 ECDs were co-cultured with WT cells ( Figure 23 ) were stimulated with WT, 130, 304 or 307 cytokines (100 ng / ml) ( Figure 36 ). 130 ECD has the amino acid substitutions R41E and R167D. 304 ECD has the amino acid substitutions R41E, E93K, and R167D; while 304 cytokine has the amino acid substitutions E46R, L108K, D112R, and R147E. 307 ECD has the amino acid substitutions R41E, D197K, D200K, and R288E; while 307 cytokine has the amino acid substitutions S12E, K16D, E19K, and E46R. Figure 36 Panels A and B represent replicate experiments.

[0596] These results demonstrate that cells expressing orthogonal chimeric cytokine receptors are capable of selective activation and cell cycle progression following stimulation with orthogonal G-CSF CSF.

[0597] Example 21: Activation in 32D-IL2Rβ Cells and Primary Human T Cells Expressing Orthogonal Chimeric Cytokine Receptors Intracellular signaling and stimulation with orthogonal G-CSF.

[0598] To determine whether cells expressing chimeric cytokine receptors can selectively activate intracellular cytokine signaling events in response to orthogonal versions of G-CSF, 32D-IL-2Rβ cells were transduced with chimeric receptors G2R-1 and G2R-2 containing wild-type G-CSFR ECDs (G2R-1 WT ECD and G2R-2 WTECD) and G-CSFR ECDs carrying amino acid substitutions R41E, R141E, and R167D (G2R-1 134 ECD, G2R-2 134 ECD). Cells were stimulated with IL-2 (300 IU / ml), wild-type G-CSF (30 ng / ml), or an orthogonal G-CSF (130G-CSF-E46R_L108K_D112R; 30 ng / ml), which binds to G2R-1 134 ECD, G2R-2 134 ECD, but has significantly reduced binding to wild-type G-CSFR. Cell lysates were subjected to Western blotting to assess the ability of cells to activate cytokine signaling after exposure to cytokines ( Figure 37 ). Cells expressing G2R-2 134 ECD showed evidence of cytokine signaling after stimulation with 130 G-CSF but not wild-type G-CSF. In addition, cells expressing G2R-2 WT ECD failed to activate cytokine signaling after stimulation with 130 G-CSF.

[0599] The orthogonal nature of the engineered cytokine:receptor pairs was further demonstrated by Western blot analysis of primary murine T cells, 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 signaling events were measured ( Figure 38 A). IL-2 (300 IU / ml) and IL-12 (10 ng / ml) were used as control cytokines. Cells expressing G2R-3 WT ECD showed evidence of cytokine signaling after stimulation with IL-2, IL-12, or WT G-CSF. Cells expressing G2R-3 134 ECD showed evidence of cytokine signaling after stimulation with IL-2, IL-12, or 130 G-CSF. Cells expressing G2R-3 304 ECD showed evidence of cytokine signaling after stimulation with IL-2, IL-12, or 304 G-CSF.

[0600] The cell surface expression of the three ECD variants of G2R-3 was confirmed by flow cytometry ( Figure 38 B).

[0601] These results demonstrate that cells expressing orthogonal chimeric cytokine receptors are able to selectively activate intracellular cytokine signaling events upon stimulation with orthogonal G-CSF.

[0602] Example 22: Expression of G2R-3 leads to expansion, cell cycle progression, and cytokine-related changes in primary human T cells Intracellular signaling and immune phenotype

[0603] To determine whether the G2R-3 chimeric receptor can promote cytokine signaling-related events after stimulation with G-CSF in primary human T cells, TALs were transduced with a lentiviral vector encoding G2R-3. T cell expansion assays were performed to test the proliferation of cells when stimulated with IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. Viable cells were counted every 3-4 days. In contrast to their non-transduced counterparts, primary TALs expressing G2R-3 expanded in culture in response to G-CSF ( Figure 40 A). To determine whether cytokine signaling events were activated after stimulation with G-CSF, cell lysates were subjected to Western blotting to assess intracellular signaling. Cells were harvested from the amplification assay, washed, and stimulated with IL-2 (300 IU / ml) or wild-type G-CSF (100 ng / ml). Primary TAL expressing G2R-3 exhibited IL-2-dependent signaling events in response to G-CSF, with the expected exception that G-CSF induced Jak2 phosphorylation, while IL-2 induced Jak3 phosphorylation ( Figure 40 B).

[0604] BrdU incorporation assays were performed to assess cell cycle progression following stimulation with G-CSF. Cells were harvested from the expansion assay, washed, and replated in IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. Primary TAL expressing G2R-3 showed cell cycle progression in response to G-CSF ( Figure 40 C).

[0605] G-CSF-induced expansion of G2R-3-expressing cells was also demonstrated using primary PBMC-derived human T cells ( Figure 41 ). Cells expressing G2R-3 WT ECD expanded in response to WT G-CSF but not medium alone ( Figure 41 A). To demonstrate the cells' continued dependence on exogenous cytokines, cells from the G-CSF expansion condition were washed and replated in WT G-CSF (100 ng / mL), IL-7 (20 ng / mL) + IL-15 (20 ng / mL), or medium alone on culture day 21. Cells replated in the presence of G-CSF alone or IL-7 + IL-15 remained viable over time.

[0606] As assessed by flow cytometry, G-CSFR ECD expression remained stable on both CD4+ and CD8+ T cells between days 21-42 of expansion ( Figure 41 B).

[0607] Western blotting confirmed that primary PBMC-derived T cells expressing G2R-3 displayed IL-2-related signaling events in response to G-CSF ( Figure 42 A). Flow cytometry-based immunophenotyping of primary PBMC-derived T cells expanded for 42 days in WT G-CSF versus IL-7 + IL-15. Cells expressing G2R-3 WT ECD and cultured in G-CSF retained a phenotype similar to that of untransduced cells cultured in IL-7 + IL-15, with a predominantly CD62L+, CD45RO+ phenotype, indicative of a stem cell-like memory T cell phenotype (T SCM )( Figure 42 B, 42C). Similarly, central memory (T CM ), effect memory (T EM ) and terminal differentiation (T TE )T cell fractions were also similar.

[0608] These results demonstrate that the G2R-3 chimeric cytokine receptor is able to activate cytokine signaling events and promote cell cycle progression and expansion in primary cells. The immunophenotype of T cells expressing G2R-3 and long-term expanded in G-CSF was similar to that of untransduced cells expanded in IL-7 + IL-15.

[0609] Example 23: Orthogonal G-CSF induces expansion and proliferation in primary human T cells expressing G2R-3 with an orthogonal ECD proliferation

[0610] Whether the chimeric cytokine receptor G2R-3 with 304 (R41E_E93K_R167D) or 307 (R41E_D197K_D200K_R288E) ECD can induce proliferation and expansion in response to stimulation with orthogonal ligands 130, 304 or 307 G-CSF was assessed, and primary PBMC-derived human T cells were transduced with lentiviral vectors encoding G R-3 304 ECD or G2R-3 307 (R41E_D197K_D200K_R288E) ECD. T cell growth assays were performed to assess the expansion fold of cells when cultured with IL-2 (300 IU / ml), 304 G-CSF (100 ng / ml), 307 G-CSF (100 ng / ml) or no cytokines. Viable cells were counted every 3-4 days. T cells expressing G2R-3 304 ECD expanded in culture in response to IL-2 or 304G-CSF ( Figure 43 A). T cells expressing G2R-3 307 ECD expand in culture in response to IL-2 or 307 G-CSF ( Figure 43 B). Untransduced T cells expand only in response to IL-2 ( Figure 43 C).

[0611] BrdU incorporation assays were performed to assess cell cycle progression following stimulation with 130, 304, and 307G-CSF in a cross design. Cells were harvested from the expansion assay, washed, and re-plated in IL-2 (300 IU / ml), 130G-CSF (100 ng / ml), 304G-CSF (100 ng / ml), 307G-CSF (100 ng / ml), or no cytokine. Primary human T cells expressing G2R-3 304ECD exhibited cell cycle progression in response to 130 or 304G-CSF, but not in response to 307G-CSF. Figure 44 T cells expressing G2R-3 307 ECD showed cell cycle progression in response to 307 G-CSF, but not in response to 130 or 304 G-CSF. All T cells showed cell cycle progression in response to IL-2.

[0612] Results showed that the chimeric receptors G2R-3 304 ECD and G2R-3 307 ECD were able to induce selective cell cycle progression and expansion of primary human CD4+ and CD8+ T cells after stimulation with orthogonal 304 or 307 G-CSF, respectively. Furthermore, 130 G-CSF stimulated the proliferation of cells expressing G2R-3 304 ECD, but not G2R-3 307 ECD.

[0613] Example 24: G-CSFR ECD in transduction with G21R-1, G21R-2, G12R-1 and G2R-3 chimeric receptor constructs Expression on the surface of primary human tumor-associated lymphocytes (TAL)

[0614] To assess whether chimeric cytokine receptor constructs could be expressed on the surface of primary human tumor-associated lymphocytes (TALs), TALs were transduced with lentiviral vectors encoding the G21R-1, G21R-2, G12R-1, and G2R-3 chimeric receptors, and the cells were tested for G-CSFR ECD expression on the cell surface by flow cytometry ( Figure 39 ). For all four chimeric cytokine receptor designs, G-CSFR ECD-positive cells were detected.

[0615] 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 G-CSFR ECD chimeric receptor design is expressed on the surface of primary cells.

[0616] Example 25: G-CSFR ECD in primary murine T cells transduced with G12R-1 and G21R-1 chimeric receptor constructs On the surface

[0617] To determine whether G12R-1 and G21R-1 chimeric receptors can be expressed on the surface of primary T cells, primary murine T cells were transduced with retroviral vectors encoding G12R-1 and G21R-1 chimeric receptors and analyzed by flow cytometry ( Figure 45 ).

[0618] The results showed that G-CSFR ECD was expressed on the surface of primary murine CD4+ and CD8+ T cells transduced with retroviral vectors encoding G12R-1 and G21R-1.

[0619] Example 26: G-CSF induces cytokines in primary PBMC-derived human T cells expressing G21R-1 or G21R-2 sub-signaling events

[0620] To determine whether G21R-1 and G21R-2 constructs were able to 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 determine the extent of STAT3 phosphorylation, a measure of STAT3 activation ( Figure 46 ). After stimulation with G-CSF (100 ng / ml), the number of cells expressing phosphorylated STAT3 increased in the G-CSFR-positive cell subpopulation transduced with G21R-1 or G21R-2. In contrast, G-CSFR-negative (i.e., non-expressing) cells did not show an increase in phosphorylated STAT3 after stimulation with G-CSF, but did show an increase after stimulation with IL-21.

[0621] These results demonstrate that G21R-1 and G21R-2 chimeric receptors are able to activate IL-21-related cytokine signaling events in primary human T cells following stimulation with G-CSF.

[0622] Example 27: G-CSF induces intracellular signaling in primary murine T cells expressing G21R-1 or G-12R-1 event

[0623] To determine whether the chimeric cytokine receptor G21R-1 is able to 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 after stimulation with G-CSF. Live cells were gated for CD8 or CD4, and the percentage of cells that stained positive for phosphorylated STAT3 in the CD8 and CD4 cell populations was determined after stimulation with either IL-21 (1 ng / ml) or G-CSF (100 ng / ml) without cytokine stimulation. After stimulation with G-CSF, cells expressing G21R-1 (but not transduced cells) showed an increase in the amount of phosphorylated STAT3 ( Figure 47 A and 47B). Western blot was performed to assess intracellular cytokine signaling in cells expressing G21R-1 or G12R-1 after G-CSF stimulation. As expected, cells expressing G21R-1 and stimulated with G-CSF showed increased phosphorylation of STAT3, with slight increases in phospho-STAT4 and phospho-STAT5 ( Figure 47 C). As expected, robust STAT4 phosphorylation in response to G-CSF was observed in cells expressing G12R-1. G-CSF did not induce any signaling events in mock-transduced cells. (Note that in the G12R-1 group, the positive control (hIL-12 10 ng / ml) did not appear to induce any signaling events; this is likely due to poor binding of human IL-12 to murine IL-12R.)

[0624] The results showed that G21R-1 and G12R-1 were able to induce cytokine signaling events in primary murine T cells after stimulation with G-CSF.

[0625] Example 28: G-CSF induces expression of G2R-2, G2R-3, G7R-1, G21 / 7R-1, G27 / 2R-1, G21 / 2R-1, Proliferation and intracellular signaling events in primary murine T cells expressing G12 / 2R-1 or G21 / 12 / 2R-1

[0626] To assess cytokine signaling events and cell proliferation mediated by chimeric cytokine receptors, primary murine T cells were transduced with retroviral vectors encoding G2R-2, G2R-3, G7R-1, G21 / 7R-1, G27 / 2R-1, G21 / 2R-1, G12 / 2R-1, or G21 / 12 / 2R-1. BrdU incorporation assays were performed to assess cell cycle progression after stimulation with G-CSF. Cells were harvested, washed, and replated in IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. Figure 48 A. Figure 48 B) or G21 / 2R-1, G12 / 2R-1 or G21 / 12 / 2R-1( Figure 49 A. Figure 49 G-CSF-induced cell cycle progression was observed in primary mouse T cells (B). G-CSF RECD expression was also detected by flow cytometry ( Figure 48 C, Figure 49 C).

[0627] By Western blotting, multiple cytokine signaling events were observed in response to G-CSF (100 ng / ml) in cells expressing the indicated chimeric cytokine receptors, but not in mock-transduced cells ( Figure 48 D. Figure 49 D). In general, the observed cytokine signaling events were expected based on the signaling domains incorporated into various ICD designs ( Figure 23 and Figure 24 As an example, the G7R-1 chimeric receptor induces phosphorylation of STAT5 ( Figure 48 D), which is expected to be due to the incorporation of STAT5 binding sites from IL-7Rα ( Figure 23 As a second example, the G21 / 2R-1 chimeric receptor induces phosphorylation of STAT3 ( Figure 49 D), which is expected to be due to the incorporation of STAT3 binding sites from G-CSFR ( Figure 24 As a third example, the G12 / 2R-1 chimeric receptor induces phosphorylation of STAT4, which is expected to be due to the incorporation of the STAT4 binding site from IL-12Rβ2 ( Figure 24 Other chimeric cytokine receptors display other distinct patterns of intracellular signaling events.

[0628] Results indicate 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 capable of inducing cytokine signaling events and proliferation in primary murine T cells following stimulation with G-CSF. Furthermore, distinct patterns of intracellular signaling events can be generated by incorporating different signaling domains into the ICD of the chimeric receptor.

[0629] Example 29: Orthogonal G-CSF-induced expansion and proliferation of primary human T cells expressing G12 / 2R-1 with an orthogonal ECD proliferation, cytokine-related intracellular signaling, and immune phenotype

[0630] 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 130G-CSF, primary PBMC-derived human T cells were transduced with a lentiviral vector encoding G12 / 2R-1 134 ECD. T cell growth assays were performed to assess the fold expansion of cells when cultured with IL-2 (300 IU / ml), 130G-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 expanded in culture in response to IL-2 or 130G-CSF ( Figure 50 A), but showed limited transient expansion in culture medium alone.

[0631] On day 19 of the experiment, T cells that had been expanded in 130G-CSF or IL-2 were washed three times and re-plated in IL-2, 130G-CSF, or culture medium alone. In culture medium alone, T cells showed reduced viability and decreased numbers ( Figure 50 B) In contrast, T cells replated in IL-2 or G-CSF 130 showed sustained viability and stable numbers.

[0632] The expression of G12 / 2R-1134 ECD detected by flow cytometry using an antibody against the G-CSF receptor increased between days 4 and 16 on CD4+ and CD8+ T cells expanded by stimulation with 130G-CSF ( Figure 50 C) BrdU incorporation assay was performed to assess cell cycle progression after stimulation with 130G-CSF.

[0633] To assess cell cycle progression by BrdU assay, cells were harvested from the expansion assay, washed, and replated in IL-2 (300 IU / ml), IL-2 and IL-12 (10 ng / mL), 130G-CSF (300 ng / ml), or no cytokines. Primary human T cells expressing G12 / 2R-1 134 ECD showed cell cycle progression in response to 130G-CSF, IL-2, or IL-2 + IL-12, whereas untransduced cells responded to IL-2 alone or IL-2 + IL-12 ( Figure 51 A).

[0634] After a 16-day culture period, immunophenotyping was performed by flow cytometry using antibodies against CD62L and CD45RO to compare T cells expressing G12 / 2R-1 134 ECD expanded in 130G-CSF with untransduced cells expanded in IL-2. Both T cell populations showed similar proportions of stem cell-like memory (T SCM), central memory (T CM ), effect memory (T EM ) and terminal differentiation (T TE ) phenotype( Figure 51 B. Figure 51 C).

[0635] Similar experiments were performed with the chimeric cytokine receptor G12 / 2R-1 with 304 ECD (as opposed to 134 ECD). Primary PBMC-derived human T cells were transduced with a lentiviral vector encoding the G12 / 2R-1 304 ECD. T cell growth assays were performed to assess the expansion fold of cells when cultured with IL-2 (300 IU / ml), 130G-CSF (100 ng / ml), 304G-CSF (100 ng / mL), or culture medium alone. Viable cells were counted every 4-5 days. T cells expressing G12 / 2R-1 with 304 ECD could expand in the presence of IL-2, 130G-CSF, or 304G-CSF, but not in culture medium alone, whereas untransduced cells could expand only in response to IL-2 ( Figure 52 A).

[0636] To assess cell cycle progression by BrdU assay, T cells expressing G12 / 2R-1 304 ECD that had been previously expanded in 130G-CSF or 304G-CSF were harvested from the expansion assay, washed, and replated in IL-2 (300 IU / ml), 130G-CSF (100 ng / ml), 304G-CSF (100 ng / mL), 307G-CSF (100 ng / mL), or medium alone. T cells expressing G12 / 2R-1 304 ECD exhibited cell cycle progression in response to 130 or 304G-CSF, but not in response to 307G-CSF or medium alone ( Figure 52 B).

[0637] Results demonstrate that G12 / 2R-1 134 ECD induces cell cycle progression and expansion of primary human CD4+ and CD8+ T cells following stimulation with the orthogonal 130G-CSF. Cells expressing G12 / 2R-1 134 ECD and expanded with 130G-CSF exhibit a T cell memory phenotype similar to that of untransduced cells expanded with IL-2. Furthermore, G12 / 2R-1 304 ECD induces selective cell cycle progression and expansion of T cells following stimulation with 130 or 304G-CSF, but not in response to 307G-CSF.

[0638] Example 30: Orthogonal G-CSF induces proliferation in primary human T cells expressing G2R-3 or G12 / 2R-1 with orthogonal ECDs. induce different intracellular signaling events

[0639] To assess intracellular signaling events, primary PBMC-derived human T cells were transduced with lentiviral vectors encoding G2R-3 304 ECD or G12 / 2R-1 304 ECD. Western blotting was performed to assess intracellular cytokine signaling in cells expressing G2R-3 304 ECD or G12 / 2R-1 304 ECD or in untransduced cells following stimulation with 304G-CSF (100 ng / mL), IL-2 (300 IU / mL), IL-2 and IL-12 (10 ng / mL), or medium alone. In transduced and untransduced T cells, strong phosphorylation of STAT5 was detected in response to stimulation with IL-2 + IL-12 or IL-2 alone. Figure 53 ). Strong phosphorylation of STAT4 was detected in response to stimulation with both IL-2 and IL-12, but only weak phosphorylation of STAT4 was detected in response to stimulation with IL-2 alone. In cells expressing G2R-3 304 ECD, weak phosphorylation of STAT4 and strong phosphorylation of STAT5 were detected in response to stimulation with 304G-CSF, similar to the pattern observed in response to IL-2 alone. In cells expressing G12 / 2R-1 304 ECD, strong phosphorylation of STAT4 and STAT5 was detected in response to stimulation with 304G-CSF, similar to the pattern observed in response to IL-2 and IL-12. Untransduced T cells showed no response to 304G-CSF.

[0640] The results showed that G12 / 2R-1 with the 304 ECD was able to induce cytokine signaling events, including robust phosphorylation of STAT4 and STAT5 in response to stimulation with 304G-CSF. Following stimulation with 304G-CSF, a different pattern of signaling events was observed in cells expressing the G2R-3 304 ECD, including robust phosphorylation of STAT5 but not STAT4.

[0641] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

[0642] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

[0643] Table 12: G-CSFR WT -ICD gp130-IL-2Rβ sequence

[0644]

[0645]

[0646] Table 13: G-CSFR WT -ICD IL-2Rβ sequence

[0647]

[0648]

[0649] Table 14: G-CSFR WT -ICD γc sequence

[0650]

[0651]

[0652] Table 15A: Chimeric Cytokine Receptors

[0653]

[0654] Table 15B: Chimeric Cytokine Receptors

[0655]

[0656] Table 16

[0657]

[0658]

[0659] Table 17: Signal peptides. The signal peptide consists of one of the following:

[0660]

[0661] Table 18: Wild-type G-CSFR extracellular domain (ECD): G-CSFR ECD consists of one of the following:

[0662]

[0663]

[0664]

[0665]

[0666]

[0667] Table 19: Transmembrane domain (TM). TM consists of one of the following:

[0668]

[0669] Table 20: Intracellular domain (ICD). The ICD consists of one of the following:

[0670]

[0671]

[0672]

[0673]

[0674]

[0675]

[0676]

[0677]

[0678]

[0679]

[0680]

[0681]

[0682]

Claims

1. A system for selectively activating a receptor expressed on a cell surface, the system comprising: (a) a receptor comprising a variant extracellular domain (ECD) of granulocyte colony stimulating factor receptor (G-CSFR); and (b) variant G-CSF, in: (i) the mutations in the variant G-CSF are E46R, L108K and D112R mutations corresponding to amino acid positions of SEQ ID NO. 1; and the mutations in the receptor are R41E and R167D mutations, which correspond to residues numbered from 2 to 308 of SEQ ID NO. 2; or (ii) the mutations in the variant G-CSF are E46R, L108K, D112R and R147E mutations corresponding to amino acid positions of SEQ ID NO. 1; and the mutations in the receptor are R41E, E93K and R167D mutations corresponding to residues numbered from 2 to 308 of SEQ ID NO. 2; or (iii) the mutations in the variant G-CSF are S12E, K16D, E19K and E46R mutations corresponding to amino acid positions of SEQ ID NO. 1; and the mutations in the receptor are R41E, D197K, D200K and R288E mutations, which correspond to residues numbered from 2 to 308 of SEQ ID NO. 2; or (iv) the mutations in the variant G-CSF are E46R, L108K, D112R, E122R and E123R mutations corresponding to amino acid positions of SEQ ID NO. 1; and the mutations in the receptor are R41E, R141E and R167D mutations, which correspond to residues numbered from 2 to 308 of SEQ ID NO.

2.

2. The system of claim 1, wherein the receptor is a chimeric receptor.

3. The system of any one of claims 1-2, wherein the receptor is expressed on a cell.

4. The system of claim 3, wherein the cells are immune cells.

5. The system of claim 4, wherein the immune cells are selected from the group consisting of T cells, NK cells, NKT cells, B cells, plasma cells, macrophages, and dendritic cells.

6. The system of claim 5, wherein the cells are stem cells or primary cells.

7. The system of claim 5, wherein the cells are human cells.

8. The system of any one of claims 1-7, wherein the ECD comprises at least 90% identity to SEQ ID NO:

2.

9. The system of any one of claims 1-8, wherein the variant G-CSF comprises at least 90% identity to SEQ ID NO:

1.

10. A nucleic acid encoding the receptor and / or the variant G-CSF in the system of any one of claims 1 to 9. An expression vector comprising the nucleic acid according to claim 10 .

12. Use of the system of any one of claims 1 to 9 for the preparation of a kit for selectively activating a receptor on a cell surface.

13. The use according to claim 12, wherein the cell is an immune cell.

14. The use according to claim 13, wherein the immune cells are selected from the group consisting of T cells, NK cells, NKT cells, B cells, plasma cells, macrophages and dendritic cells.

15. The use according to claim 12, wherein the cells are stem cells or primary cells.

16. The use according to claim 12, wherein the cells are human cells.

17. The use of any one of claims 12-16, wherein the selective activation of the immune cells results in a cellular response selected from the group consisting of: increased proliferation, viability, and activity of the immune cells.

18. A cell comprising the receptor of any one of the systems of claims 1-9 or the variant G-CSF of any one of the systems of claims 1-9.

19. A kit for treating a subject in need thereof, comprising: A cell encoding the receptor of any one of claims 1-9 and instructions for use.

20. The kit of claim 19, wherein the kit further comprises a variant G-CSF according to any one of claims 1 to 9 that binds to the receptor.

21. The kit of any one of claims 19-20, wherein the cell is an immune cell.

22. A kit for producing a system for selectively activating a receptor expressed on a cell surface, the kit comprising: (a) the nucleic acid according to claim 10 or the expression vector according to claim 11; (b) the variant G-CSF of any one of claims 1 to 9, the nucleic acid of claim 10, or the expression vector of claim 11; and (c) Instructions for use.

23. A kit for producing a chimeric receptor expressed on a cell, comprising: A cell and instructions for use, wherein the cell comprises an expression vector encoding the receptor in the system of any one of claims 1-9.

24. The kit of claim 23, wherein the cells are bacterial cells.

25. The kit of any one of claims 23-24, wherein the kit comprises the variant G-CSF in the system of any one of claims 1-9 bound to the receptor.

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