Modified granulocyte colony-stimulating factor (G-CSF) and its binding chimeric cytokine receptor

Abstract

Described herein are methods and compositions for selective activation of cells with variant cytokine receptors and cytokine pairs, where the cytokine receptor comprises the extracellular domain (ECD) of the granulocyte colony-stimulating factor receptor (G-CSFR). In certain embodiments, the methods and compositions described herein are useful for exclusive activation of cells for adoptive cell transfer therapy. Accordingly, included herein are methods 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 of treating a subject in need thereof, comprising administering to the subject cells expressing a variant receptor comprising the extracellular domain of G-CSFR and co-administering a variant cytokine that activates the variant receptor.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application Nos. 63 / 171,933, filed April 7, 2021, 63 / 171,950, filed April 7, 2021, 63 / 171,980, filed April 7, 2021, and 63 / 172,025, filed April 7, 2021, each of which is incorporated herein by reference in its entirety for all purposes.

[0002] Sequence Listing Not applicable

[0003] Field In certain aspects, described herein are methods and compositions for selective activation of cells using a variant cytokine receptor and cytokine pair, wherein the cytokine receptor comprises a variant extracellular domain (ECD) of granulocyte colony-stimulating factor receptor (G-CSFR). Also disclosed herein are methods of treating a subject by adoptive cell transfer, comprising administering to the subject cells expressing the variant receptor and administering a variant cytokine to signal the cells expressing the variant receptor. Included in the disclosure are nucleic acids, expression vectors, and kits for producing cells expressing the variant cytokine and receptor, which kits also provide cytokines for binding to the variant receptor. In certain aspects, described herein are chimeric cytokine receptors comprising the extracellular domain of G-CSFR (granulocyte colony-stimulating factor receptor) and the intracellular domains of various cytokine receptors for selectively activating cytokine signaling in cells of interest. The present disclosure also includes methods, cells, and kits for use in adoptive cell transfer (ACT), comprising cells expressing a chimeric cytokine receptor, and / or an expression vector encoding the chimeric cytokine receptor, and / or a cytokine that binds to the chimeric cytokine receptor. In certain aspects, described herein are systems and methods for regulated paracrine signaling involving a chimeric cytokine receptor comprising the extracellular domain of G-CSFR (granulocyte colony-stimulating factor receptor) and the intracellular domains of various cytokine receptors to selectively activate cytokine signaling in cells of interest.

[0004] Also described herein are methods and compositions for the selective activation of cells using variant cytokine receptors and cytokine pairs, wherein the cytokine receptor comprises the extracellular domain (ECD) of interleukin-7 receptor alpha (IL-7Rα). [Background technology]

[0005] Over the past two decades, significant advances have been made in cancer treatment through adoptive cell transfer (ACT). ACT using endogenous tumor-infiltrating T cells (TILs) now reproducibly achieves objective clinical response rates exceeding 50% in advanced melanoma. ACT using T cells engineered to recognize B-cell leukemia (using CD19-directed chimeric antigen receptors, or CD19 CARs) has resulted in complete response rates of up to 90%, with the majority of patients achieving durable responses. ACT using T cells expressing engineered T cell receptors (TCRs) has shown promise against a variety of solid tumors. Successful ACT has also been reported using other effector cell types, such as natural killer (NK) cells, natural killer T cells (NKT cells), and macrophages, instead of T cells. Encouraged by these remarkable results, several companies have commercialized TIL, CAR, and engineered TCR ACT approaches.

[0006] The engraftment, proliferation, and persistence of T cells or other effector cells for CAR T cells are important determinants of clinical safety and efficacy. In the case of T cells, this is often addressed by systemic administration of IL-2 after CAR T cell transplantation and by expanding T cells ex vivo with IL-2 before transplantation. In addition to the intended immunostimulatory effects, systemic IL-2 treatment can also cause serious toxicities, such as vascular leak syndrome, which must be tightly controlled for patient safety. To manage these risks, patients typically require hospitalization for 2–3 weeks and intensive care unit (ICU) placement as a preventative measure. Furthermore, because IL-2 induces the proliferation of both effector T cells and regulatory (suppressor) T cells (5), administering IL-2 to patients is like stepping on the accelerator and brake pedal simultaneously. CAR T cells pose the opposite problem: T cell proliferation and persistence exceed safe levels in some patients and premature attenuation in others. Furthermore, they invariably eradicate normal B cells (which also express CD19), leaving patients partially immunocompromised. Ideally, it would be desirable to tightly control the number of tumor-reactive T cells following ACT, including the ability to safely enhance T cell proliferation, persistence, and efficacy, and to remove the transferred cells after eradicating the cancer. Other cell-based therapies, such as stem cell therapy, could also benefit from improved control over the proliferation, differentiation, and persistence of infused cells.

[0007] Human G-CSF (Neupogen®, filgrastim) and a PEGylated version of human G-CSF (Neulasta®, pegfilgrastim) are approved therapies used to treat neutropenia in cancer patients. G-CSF is a four-helix bundle (Hill, C. P. et al. Proc Natl Acad Sci US A. 1993 Jun 1;90(11):5167-71 (Non-Patent Document 1)), and the structure of G-CSF in complex with 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 (Non-Patent Document 2)). The G-CSF:G-CSFR complex is a 2:2 heterodimer. G-CSF has two binding interfaces with G-CSFR. One, termed site II, is the larger interface between G-CSF and the cytokine receptor homology (CRH) domain of G-CSFR, and the other, termed site III, is the smaller interface between G-CSF and the N-terminal Ig-like domain of G-CSFR.

[0008] Interleukin-7 (IL-7) is an example of a safe and well-tolerated cytokine, but its effectiveness in cancer immunotherapy is limited. IL-7 is a growth factor for T and B cells, is important for thymic development, and supports the survival and homeostasis of naive and memory T cells. Unlike IL-2, IL-7 induces minimal Treg proliferation due to low expression levels of IL-7Rα on this suppressor lymphocyte population. IL-7 is not produced by hematopoietic cells but is secreted by stromal cells. IL-7 has been used in cancer immunotherapy to increase the number, persistence, and activity of T cells (Barata JT et al. Nat Immunol. 2019 Dec;20(12):1584-1593 (Non-Patent Document 3)). It has been shown to be well tolerated but exhibits negligible anticancer efficacy as monotherapy (Rosenberg et al. J Immunother. 2006 May-Jun;29(3):313-9 (Non-Patent Document 4)), (Sportes C. et al., Clin Cancer Res. 2010 Jan 15;16(2):727-35 (Non-Patent Document 5)), and (Sportes C., et al., J Exp Med. 2008 Jul 7;205(7):1701-14 (Non-Patent Document 6)). Furthermore, recombinant IL-7 made in Escherichia coli (E. coli) was found to be highly immunogenic, but this problem was ameliorated by producing IL-7 in mammalian cells (Conlon KC, et al., J Interferon Cytokine Res. 2019 Jan;39(1):6-21 (Non-Patent Document 7)). For these reasons, clinical development of IL-7 in oncology has been limited compared to more therapeutic cytokines such as IL-2 and IL-15. However, IL-7 is attractive for use as a ligand for chimeric receptors that induce stronger intracellular signals than those achieved by the native IL-7 receptor.

[0009] To mitigate the toxicity associated with systemic cytokine delivery, several groups have engineered T cells or NK cells to produce and secrete cytokines in an autocrine / paracrine manner. The goal is for the engineered T / NK cells to produce enough cytokines to consume themselves and neighboring cells, but not enough to cause systemic toxicity. For example, this strategy has been applied to improve chimeric antigen receptor (CAR) T cell and CAR NK cell therapies using cytokines such as IL-2, IL-15, IL-12, and IL-18. A common approach is to use retroviruses or lentiviruses to stably introduce the CAR gene and cytokine gene into T cell or NK cell populations. This can be achieved by various approaches, such as co-transduction of two viral vectors or the use of bicistronic viral vectors carrying two transgenes. T cells and NK cells co-expressing the CAR and cytokine transgenes are commonly referred to as "armored CARs" or "trucks."

[0010] Armored CARs have been evaluated in mouse tumor models and, to a lesser extent, in human clinical trials. Several studies have demonstrated that armored CAR T / NK cells are safe and effective compared to standard CAR T / NK cells (i.e., cells bearing a CAR but without a cytokine transgene). However, toxicity has been observed in some mouse studies (Ataca Atilla P., et al., J Immunother Cancer. 2020 Sep;8(2):e001229 (Non-Patent Document 8)) and clinical trials (Zhang L., et al., Clin Cancer Res. 2015 May 15;21(10):2278-88 (Non-Patent Document 9)). Furthermore, theoretical concerns have always existed regarding the concept of endowing T / NK cells (or any cell type) with the ability to produce autocrine growth factors, which could potentially lead to uncontrolled T / NK cell proliferation and secondary lymphoproliferative disorders or malignancies. Some researchers have attempted to mitigate this risk by co-expressing "suicide genes" that can be used to kill T / NK cells in response to drugs if toxicity, uncontrolled proliferation, or other concerns arise. However, the introduction of suicide genes by definition terminates the cell therapy and may leave patients with residual tumor burden. Therefore, novel compositions and methods are needed to mitigate the toxicity and safety risks associated with armored CAR approaches so that therapeutically relevant cytokine signals can be delivered to immune cells in a safe and controlled manner. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Hill,CP et al.Proc Natl Acad Sci US A.1993 Jun 1;90(11):5167-71 [Non-patent document 2] Tamada,T et al.Proc Natl Acad Sci US A.2006 Feb 28;103(9):3135-40

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Summary of the Invention

[0012] Summary

[0003] Described 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, wherein the at least one mutation in the Site II interface region is selected from the group of mutations consisting of L108R, D112R, E122R E122K, E123K, and E123R, and combinations thereof, wherein the mutation in the Site II interface region is relative to the corresponding amino acid position in the sequence set forth in SEQ ID NO: 1, and the at least one mutation in the Site III interface region comprises the mutation E46R relative to the corresponding amino acid position in the sequence set forth in SEQ ID NO: 1, and wherein the variant G-CSF selectively binds to a receptor comprising a variant extracellular domain (ECD) of the granulocyte colony-stimulating factor receptor (G-CSFR).

[0013] In certain aspects, described herein is a system for selectively activating a receptor expressed on a cell surface, comprising: (a) a variant G-CSF corresponding to SEQ ID NO: 83 or 84; and (b) a receptor comprising a variant ECD of G-CSFR, wherein the variant G-CSF preferentially binds to the receptor comprising the variant ECD of G-CSFR compared to an otherwise identical wild-type G-CSFR ECD; and the receptor comprising the variant ECD of G-CSFR preferentially binds to the variant G-CSF compared to an otherwise identical wild-type G-CSF, and wherein the variant G-CSFR comprises G2R-3 or G12 / 2R-1.

[0014] In some embodiments, the variant G-CSF binds to a receptor comprising a variant ECD of G-CSFR expressed by a cell. In some embodiments, the cell expressing a receptor comprising a variant ECD of G-CSFR is an immune cell. In some embodiments, the immune cell expressing a receptor comprising a variant ECD of G-CSFR is a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, optionally the cell is a stem cell, optionally the cell is a primary cell, optionally the cell is a human cell. In some embodiments, the T cell is a CD8 + T cells, cytotoxic CD8 + T cells, naive CD8+ T cells, naive CD4 + The T cells are selected from the group consisting of T cells, helper T cells, regulatory T cells, memory T cells, and γδ T cells.

[0015] In some embodiments, selective binding of a variant G-CSF to a receptor comprising a variant ECD of G-CSFR results in a cellular response comprising at least one of proliferation, viability, persistence, cytotoxicity, cytokine secretion, memory, and enhanced activity of cells expressing the receptor. In some embodiments, the receptor comprising a 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 some embodiments, the receptor comprising a variant ECD of G-CSFR comprises at least one mutation in the site II interface region of the G-CSFR ECD comprising one or both of an R141E or R167D mutation, and the at least one mutation in the site III interface region of the G-CSFR ECD comprises an R41E mutation, and the mutation in the variant G-CSFR corresponds to the amino acid position of the sequence set forth in SEQ ID NO: 2. In some embodiments, the receptor comprising a variant ECD of G-CSFR is a chimeric receptor.

[0016] In certain aspects, the present disclosure describes one or more nucleic acid sequence(s) encoding the variant G-CSF described herein. In certain aspects, the present disclosure describes one or more expression vector(s) comprising the nucleic acid sequence. In certain aspects, the present disclosure describes cells engineered to express the variant G-CSF described herein. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, optionally the cell is a stem cell, optionally the cell is a primary cell, optionally the cell is a human cell. In some embodiments, the T cell is a CD8 + T cells, cytotoxic CD8 + T cells, naive CD8 + T cells, naive CD4 + The T cells are selected from the group consisting of T cells, helper T cells, regulatory T cells, memory T cells, and γδ T cells.

[0017] In certain aspects, the present disclosure provides a system for selectively activating a receptor expressed on a cell surface, comprising: (a) a variant G-CSF described in any one of claims 1 to 14; and (b) a receptor comprising a variant ECD of G-CSFR, wherein the variant G-CSF preferentially binds to the receptor comprising the variant ECD of G-CSFR compared to an otherwise identical wild-type G-CSFR ECD, and the receptor comprising the variant ECD of G-CSFR preferentially binds to the variant G-CSF compared to an otherwise identical wild-type G-CSF. In some embodiments, the variant G-CSF comprises a combination of mutations at the Site II and Site III interfaces of the G-CSF variants numbered in Table 4A, wherein the mutations in the variant G-CSF correspond to amino acid positions in the sequence set forth in SEQ ID NO: 1.

[0018] In some embodiments, the variant G-CSF comprises the mutations E46R, L108K, D112R, E122R, and E123R relative to the corresponding amino acid positions in the sequence set forth in SEQ ID NO: 1, and the receptor comprising a variant ECD of G-CSFR comprises the mutations R41E, R141E, and R167D relative to the corresponding amino acid positions in the sequence set forth in SEQ ID NO: 2. In some embodiments, the variant G-CSF comprises the mutations E46R, L108K, D112R, and E122K relative to the corresponding amino acid positions in the sequence set forth in SEQ ID NO: 1, and the receptor comprising a variant ECD of G-CSFR comprises the mutations R41E, R141E, and R167D relative to the corresponding amino acid positions in the sequence set forth in SEQ ID NO: 2. In some embodiments, the variant G-CSF comprises the mutations E46R, L108K, D112R, and E123K relative to the corresponding amino acid positions in the sequence set forth in SEQ ID NO: 1, and the receptor comprising a variant ECD of G-CSFR comprises the mutations R41E, R141E, and R167D relative to the corresponding amino acid positions in the sequence set forth in SEQ ID NO: 2. In some embodiments, the variant G-CSF comprises the mutations E46R, L108K, D112R, and E122R relative to the corresponding amino acid positions in the sequence set forth in SEQ ID NO: 1, and the receptor comprising a variant ECD of G-CSFR comprises the mutations R41E, R141E, and R167D relative to the corresponding amino acid positions in the sequence set forth in SEQ ID NO: 2. In some embodiments, the variant G-CSF comprises the mutations E46R, L108K, D112R, and E123R relative to the corresponding amino acid positions in the sequence set forth in SEQ ID NO: 1, and the receptor comprising a variant ECD of G-CSFR comprises the mutations R41E, R141E, and R167D relative to the corresponding amino acid positions in the sequence set forth in SEQ ID NO: 2. In some embodiments, the variant G-CSF comprises the mutations E46R, L108K, D112R, E122K, and E123K relative to the corresponding amino acid positions in the sequence set forth in SEQ ID NO: 1, and the receptor comprising a variant ECD of G-CSFR comprises the mutations R41E, R141E, and R167D relative to the corresponding amino acid positions in the sequence set forth in SEQ ID NO: 2.

[0019] In some embodiments, the system further comprises one or more additional agonist or antagonist signaling protein(s), optionally wherein the one or more additional agonist or antagonist signaling protein(s) comprise one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s). In some embodiments, the system further comprises an antigen-binding signaling receptor. In some embodiments, the antigen-binding signaling receptor comprises at least one receptor selected from the group consisting of a natural T cell receptor, an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a natural B cell receptor, an engineered B cell receptor (BCR), a stress ligand receptor, and a pattern recognition receptor. In some embodiments, the antigen-binding signaling receptor is a CAR. In some embodiments, the cytokine or chemokine is selected from the group consisting of IL-18, IL-21, interferon alpha, interferon beta, interferon gamma, IL-17, IL-21, TNF-alpha, CXCL13, CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CD40 ligand, B-cell activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, and CCL19, and the receptor NKG2D. In some embodiments, the cytokine is IL-18. In some embodiments, the cytokine is human.

[0020] In certain aspects, described herein are methods of selectively activating a receptor expressed on the surface of a cell, comprising contacting a receptor comprising a variant ECD of G-CSFR with a variant G-CSF described herein. In some embodiments, the receptor comprising a variant ECD of G-CSFR is expressed on an immune cell, optionally the immune cell is a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, optionally the cell is a stem cell, optionally the cell is a primary cell, optionally the cell is a human cell. In some embodiments, the T cell is a CD8 + T cells, cytotoxic CD8 + T cells, naive CD8 + T cells, naive CD4 + The immune cells are selected from the group consisting of T cells, helper T cells, regulatory T cells, memory T cells, and γδ T cells. In some embodiments, the selective activation of the immune cells results in a cellular response comprising at least one of proliferation, viability, persistence, cytotoxicity, cytokine secretion, memory, and enhanced activity of cells expressing the receptor.

[0021] In certain aspects, the present disclosure describes methods of increasing an immune response in a subject in need thereof, comprising administering cell(s) expressing a receptor comprising a variant ECD of G-CSFR and administering or providing a variant G-CSF described herein. In certain aspects, the present disclosure describes methods of treating a disease in a subject in need thereof, comprising administering cell(s) expressing a receptor comprising a variant ECD of G-CSFR and administering or providing a variant G-CSF described herein to the subject. In some embodiments, the method is used to treat cancer. In some embodiments, the method is used to treat an inflammatory condition. In some embodiments, the method is used to treat an autoimmune disease. In some embodiments, the method is used to treat a degenerative disease. In some embodiments, the method is used to generate natural or engineered cells, tissues, or organs for transplantation. In some embodiments, the method is used to prevent or treat transplant rejection. In some embodiments, the method is used to treat an infectious disease. In some embodiments, the method further comprises administering or providing one or more additional agonist or antagonist signaling protein(s), optionally the one or more additional agonist or antagonist signaling protein(s) comprise one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s).

[0022] In some embodiments, a subject is administered two or more cell populations that each express one or both of: (i) distinct chimeric receptors comprising a G-CSFR ECD; and (ii) at least one distinct variant form of G-CSF, wherein at least one population(s) of cells further expresses at least one distinct antigen binding signaling receptor(s), and optionally, the at least one distinct antigen binding signaling receptor(s) comprises at least one CAR(s). In some embodiments, one or both of the first and second population(s) of immune cells further express one or both of: (a) at least one additional agonist or antagonist signaling protein(s), optionally wherein the at least one or more additional agonist or antagonist signaling protein(s) comprises one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s); and (b) at least one antigen-binding signaling receptor. In some embodiments, the method further comprises one or more additional immune cell populations, each additional immune cell population expressing at least one of: (i) a distinct receptor comprising a distinct variant ECD of G-CSFR; (ii) a distinct variant G-CSF; (iii) a distinct agonist or antagonist signaling protein; and (iv) a distinct antigen-binding signaling receptor.

[0023] In some embodiments of this method, the cells expressing a receptor comprising a variant ECD of G-CSFR further express at least one antigen-binding signaling receptor(s). In some embodiments, the antigen-binding signaling receptor(s) comprise at least one receptor selected from the group consisting of a natural T cell receptor, an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a natural B cell receptor, an engineered B cell receptor (BCR), a stress ligand receptor, a pattern recognition receptor, and combinations thereof. In some embodiments, the antigen-binding signaling receptor(s) comprise one or more CAR(s). In some embodiments, the cytokine or chemokine is selected from the group consisting of IL-18, IL-21, interferon alpha, interferon beta, interferon gamma, IL-17, IL-21, TNF-alpha, CXCL13, CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CD40 ligand, B-cell activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, and CCL19, and the receptor NKG2D, and combinations thereof. In some embodiments, the cytokine is IL-18. In some embodiments, the cytokine is human.

[0024] In certain aspects, described herein are methods of treating a subject in need thereof, the methods comprising: (i) isolating an immune cell-containing sample; (ii) transducing or transfecting the immune cell(s) with nucleic acid sequence(s) encoding at least one receptor(s) comprising a variant ECD of G-CSFR; (iii) administering the immune cell(s) of (ii) to a subject; and (iv) contacting the immune cell(s) with one or more variant G-CSFs described herein that selectively bind to the receptor(s). In some embodiments, the subject has undergone immunoablative therapy prior to administering the cells to the subject. In some embodiments, the immune cell-containing sample is isolated from a subject to whom the cells will be administered. In some embodiments, the immune cell-containing sample is generated from cells from the subject to whom the cells will be administered or from a subject different from the subject to whom the cells will be administered; optionally, the cells are stem cells, optionally pluripotent stem cells. In some embodiments, the immune cell(s) are contacted with at least one variant G-CSF in vitro before the cells are administered to the subject. In some embodiments, the immune cell(s) are contacted with at least one variant G-CSF that binds to the receptor(s) for a time sufficient to activate signaling from the receptor(s).

[0025] In certain aspects, described herein are kits comprising cells encoding at least one receptor(s) comprising a variant ECD of G-CSFR and instructions for use, wherein the kit comprises at least one variant G-CSF as defined in any one of claims 1 to 14, and optionally, the cells are immune cells. In certain aspects, described herein are kits comprising (a) one or more nucleic acid sequence(s) encoding one or more receptor(s) comprising a variant ECD of G-CSFR, (b) at least one variant G-CSF as described herein, the nucleic acid sequence(s) as described herein, or one or more expression vector(s) as described herein, and (c) instructions for use. In some embodiments, the kit further comprises one or more expression vector(s) encoding one or more cytokine(s) or chemokine(s) selected from the group consisting of IL-18, IL-21, interferon alpha, interferon beta, interferon gamma, IL-17, IL-21, TNF-alpha, CXCL13, CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CD40 ligand, B-cell activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, and CCL19, and the receptor NKG2D, and combinations thereof. In some embodiments, the kit further comprises one or more expression vector(s) encoding at least one antigen-binding receptor(s). In some embodiments, the at least one antigen binding receptor(s) is selected from the group consisting of a natural T cell receptor, an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a natural B cell receptor, an engineered B cell receptor (BCR), a stress ligand receptor, a pattern recognition receptor, and combinations thereof. In some embodiments, the kit further comprises one or more expression vector(s) encoding the chimeric antigen receptor.In some embodiments, the cells further comprise one or more expression vector(s) encoding at least one cytokine(s) or chemokine(s) selected from the group consisting of IL-18, IL-21, interferon alpha, interferon beta, interferon gamma, IL-17, IL-21, TNF-alpha, CXCL13, CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CD40 ligand, B-cell activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, and CCL19, and the receptor NKG2D, and combinations thereof. In some embodiments, the cells further comprise one or more expression vector(s) encoding at least one antigen-binding signaling receptor(s). In some embodiments, the at least one antigen-binding signaling receptor(s) is selected from the group consisting of a natural T cell receptor, an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a natural B cell receptor, an engineered B cell receptor (BCR), a stress ligand receptor, a pattern recognition receptor, and combinations thereof. In some embodiments, the cell further comprises one or more expression vector(s) encoding at least one CAR(s), optionally wherein the CAR is a mesothelin CAR.

[0026] In certain aspects, chimeric receptors are described herein, the chimeric receptors comprising: (a) an extracellular domain (ECD) operably linked to at least one second domain; wherein the second domain is selected from the group consisting of: (b) an intracellular domain (ICD) comprising at least one signaling molecule binding site derived from the intracellular domain of a cytokine receptor, the at least one signaling molecule binding site being selected from the group consisting of an SHC binding site of interleukin (IL)-2Rβ, a STAT5 binding site of IL-2Rβ, an IRS-1 or IRS-2 binding site of IL-4Rα, a STAT6 binding site of IL-4Rα, an SHP-2 binding site of gp130, a STAT3 binding site of gp130, an SHP-1 or SHP-2 binding site of EPOR, an SHP-2 binding site of erythropoietin receptor (EPOR), an SHP-3 binding site of erythropoietin receptor (EPOR), an SHP-4 binding site of erythropoietin receptor (EPOR), an SHP-5 binding site of erythropoietin receptor (EPOR), an SHP-6 binding site of erythropoietin receptor (EPOR), an SHP-8 binding site of erythropoietin receptor (EPOR), an SHP-9 binding site of erythropoietin receptor (EPOR), an SHP-1 binding site of erythropoietin receptor (EPOR), an SHP-1 binding site of erythropoietin receptor (EPOR), an SHP-2 ... ), the STAT1 or STAT2 binding site of interferon alpha and beta receptor subunit 2 (IFNAR2), and the STAT1 binding site of interferon gamma receptor 1 (IFNγR1), or a combination thereof, wherein the ICD further comprises at least one Box 1 region and at least one Box 2 region of at least one protein selected from the group consisting of G-CSFR, gp130, EPOR, and interferon gamma receptor 2 (IFNγR2), or a combination thereof; and (c) at least one third domain comprising a transmembrane domain (TMD), wherein the ECD is N-terminal to the TMD and the TMD is N-terminal to the ICD.

[0027] In certain aspects, a chimeric receptor comprises an ECD operably linked to a second domain, the second domain comprising an ICD, the ICD comprising: (i) (a) the Box 1 and Box 2 regions of G-CSFR; (b) at least one signaling molecule binding site of IL-4Rα; or (ii) (a) Box1 and Box2 regions of gp130; (b) at least one signaling molecule binding site of gp130; or (iii) (a) Box 1 and Box 2 regions of the erythropoietin receptor (EPOR), (b) at least one signaling molecule binding site of the EPOR; or (iv) (a) the Box 1 and Box 2 regions of G-CSFR; (b) at least one signaling molecule binding site for interferon alpha and beta receptor subunit 2 (IFNAR2); or (v) (a) Box 1 and Box 2 regions of interferon gamma receptor 2 (IFNγR2); (b) at least one signaling molecule binding site for interferon gamma receptor 1 (IFNγR1); wherein the ECD is N-terminal to the TMD, and the TMD is N-terminal to the ICD.

[0028] In certain embodiments, the ECD of the chimeric receptor is the ECD of G-CSFR (granulocyte colony-stimulating factor receptor). In certain embodiments, the TMD of the chimeric receptor is the TMD of G-CSFR, and optionally, the TMD is a wild-type TMD. In certain embodiments, the active form of the chimeric receptor forms a homodimer, and optionally, activation of the chimeric receptor causes a cellular response comprising at least one of proliferation, viability, persistence, cytotoxicity, cytokine secretion, memory, and enhanced activity of cells expressing the 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.

[0029] In certain embodiments, the chimeric receptor is expressed in a cell, optionally an immune cell, optionally an immune cell, wherein the immune cell is a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, optionally the cell is a stem cell, optionally the cell is a primary cell, optionally the cell is a human cell.+ T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, naive CD8 + The T cells are selected from the group consisting of T cells, helper T cells, regulatory T cells, memory T cells, and γδ T cells.

[0030] In certain embodiments, the ICD comprises: (a) the amino acid sequence of one or both of SEQ ID NOs: 90 or 91; or (b) the amino acid sequence of one or both of SEQ ID NOs: 90 or 92; or (c) the amino acid sequence of SEQ ID NO: 93, or (d) the amino acid sequence of SEQ ID NO: 94, or (e) the amino acid sequence of one or both of SEQ ID NOs: 95 or 96; or (f) the amino acid sequence of SEQ ID NO: 97 or 98, or (g) the amino acid sequence of SEQ ID NO: 99 or 100 Includes:

[0031] In certain embodiments, the transmembrane domain comprises the sequence set forth in SEQ ID NO:88.

[0032] In certain aspects, the present disclosure describes one or more nucleic acid sequence(s) encoding the chimeric receptors described herein. In certain embodiments, the nucleic acid sequence(s) of the ECD of G-CSFR are encoded by the nucleic acid sequence(s) set forth in any one of SEQ ID NOs: 85, 86, or 87. In certain embodiments, the present disclosure describes one or more expression vector(s) comprising the nucleic acid sequence(s). In certain embodiments, the expression vector(s) are selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and a plasmid.

[0033] In certain aspects, the present disclosure describes one or more nucleic acid sequence(s) encoding a chimeric receptor, wherein the chimeric receptor comprises an ECD operably linked to a second domain; the second domain comprises: (i) (a) the Box 1 and Box 2 regions of G-CSFR; (b) at least one signaling molecule binding site of IL-4Rα; or (ii) (a) Box1 and Box2 regions of gp130; (b) at least one signaling molecule binding site of gp130; or (iii) (a) Box 1 and Box 2 regions of the erythropoietin receptor (EPOR), (b) at least one signaling molecule binding site of the EPOR; or (iv) (a) the Box 1 and Box 2 regions of G-CSFR; (b) at least one signaling molecule binding site for interferon alpha and beta receptor subunit 2 (IFNAR2); or (v) (a) Box 1 and Box 2 regions of interferon gamma receptor 2 (IFNγR2); (b) at least one signaling molecule binding site for interferon gamma receptor 1 (IFNγR1); Includes:

[0034] In certain embodiments of the nucleic acid(s) described herein, the ECD is the ECD of G-CSFR (granulocyte colony-stimulating factor receptor). In certain embodiments of the nucleic acid(s) described herein, the TMD is the TMD of G-CSFR, and optionally, the TMD is a wild-type TMD. In certain embodiments of the nucleic acid(s) described herein, the ECD of G-CSFR is encoded by the nucleic acid sequence set forth in any one of SEQ ID NOs: 85, 86, or 87. In certain embodiments, the nucleic acid sequence(s) comprise (a) a sequence encoding an ICD comprising the amino acid sequence of one or both of SEQ ID NOs: 90 or 91, or (b) a sequence encoding an ICD comprising the amino acid sequence of one or both of SEQ ID NOs: 90 or 92, or (c) a sequence encoding an ICD comprising the amino acid sequence of SEQ ID NO: 93, or (d) a sequence encoding an ICD comprising the amino acid sequence of SEQ ID NO: 94, or (e) a sequence encoding an ICD comprising the amino acid sequence of one or both of SEQ ID NOs: 95 or 96, or (f) a sequence encoding an ICD comprising the amino acid sequence of SEQ ID NO: 97 or 98, or (g) a sequence encoding an ICD comprising the amino acid sequence of SEQ ID NO: 99 or 100.

[0035] In certain embodiments, one or more expression vector(s) comprise the nucleic acid sequence(s) described herein, hi certain embodiments, the expression vector(s) are selected from the group consisting of retroviral vectors, lentiviral vectors, adenoviral vectors, and plasmids.

[0036] In certain aspects, described herein are cells comprising a nucleic acid sequence(s) encoding the chimeric receptor described herein. In certain embodiments, the cells are immune cells, optionally the immune cells are T cells, optionally NK cells, optionally NKT cells, optionally B cells, optionally plasma cells, optionally macrophages, optionally dendritic cells, optionally the cells are stem cells, optionally the cells are primary cells, and optionally the cells are human cells. In certain embodiments, the T cells are CD8 + T cells, cytotoxic CD8+ T cells, naive CD4 + T cells, naive CD8 + In certain embodiments, the cell is selected from the group consisting of a T cell, a helper T cell, a regulatory T cell, a memory T cell, and a γδ T cell. In certain embodiments, the cell comprises a nucleic acid sequence(s) described herein. In certain embodiments, the cell comprises an expression vector(s) described herein.

[0037] In certain aspects, described herein are methods for selectively activating a chimeric receptor expressed on the surface of a cell, comprising contacting the chimeric receptor with a cytokine that selectively binds to the chimeric receptor. In certain embodiments, the active form of the chimeric receptor forms a homodimer, and optionally, activation of the chimeric receptor elicits a cellular response comprising at least one of proliferation, viability, persistence, cytotoxicity, cytokine secretion, memory, and enhanced activity of cells expressing the receptor, and optionally, the chimeric receptor is activated upon contact with the cytokine.

[0038] In certain embodiments, the cytokine that selectively binds to the chimeric receptor is G-CSF, optionally the chimeric receptor is activated upon contact with G-CSF, optionally the G-CSF is wild-type G-CSF, and optionally the extracellular domain of G-CSFR is a wild-type extracellular domain. In certain embodiments, the chimeric receptor is expressed in a cell, optionally an immune cell, optionally the immune cell is a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, optionally the cell is a stem cell, optionally the cell is a primary cell, optionally the cell is a human cell.

[0039] In some embodiments, a first population of immune cells expresses the chimeric receptor and a second population of immune cells expresses a cytokine that binds to the chimeric receptor, and optionally, one or both of the first and second population(s) of immune cells further express at least one distinct antigen binding signaling receptor(s), and optionally, the at least one distinct antigen binding signaling receptor(s) comprises at least one CAR. In some embodiments, one or both of the first and second population(s) of immune cells further express one or both of: (a) at least one additional agonist or antagonist signaling protein(s), optionally wherein the at least one or more additional agonist or antagonist signaling protein(s) comprises one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s), and (b) at least one antigen-binding signaling receptor. In some embodiments, each of the first and second populations of immune cells expresses a distinct chimeric receptor comprising a distinct variant ECD of G-CSFR and a distinct variant G-CSF. In some embodiments, the method further comprises one or more additional immune cell populations, each additional immune cell population expressing at least one of: (i) a distinct receptor comprising a distinct variant ECD of G-CSFR; (ii) a distinct variant G-CSF; (iii) a distinct agonist or antagonist signaling protein; and (iv) a distinct antigen-binding signaling receptor.

[0040] In certain aspects, described herein are methods of producing a chimeric receptor in a cell, the methods comprising introducing into a cell one or more nucleic acid sequence(s) described herein, or one or more expression vector(s) described herein, optionally the method comprises gene editing, and optionally the cell is an immune cell, optionally the immune cell is a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, optionally the cell is a stem cell, optionally the cell is a primary cell, and optionally the cell is a human cell.

[0041] In certain aspects, described herein are methods of treating a subject in need thereof, the methods comprising administering to the subject cells expressing a chimeric receptor described herein and providing to the subject a cytokine that specifically binds to the chimeric receptor. In certain embodiments, an active form of the chimeric receptor forms a homodimer, and optionally, activation of the chimeric receptor elicits a cellular response comprising at least one of proliferation, viability, persistence, cytotoxicity, cytokine secretion, memory, and enhanced activity of cells expressing the receptor, and optionally, the chimeric receptor is activated upon contact with the cytokine. In certain embodiments, the cytokine is G-CSF, optionally, the G-CSF is wild-type G-CSF, and optionally, the extracellular domain of G-CSFR is a wild-type extracellular domain.

[0042] In certain embodiments of the methods described herein, the chimeric receptor is expressed in a cell, optionally an immune cell, optionally an immune cell, wherein the immune cell is a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, optionally the cell is a stem cell, optionally the cell is a primary cell, optionally the cell is a human cell.

[0043] In certain embodiments, the method further comprises administering or providing at least one additional agonist or antagonist signaling protein(s), optionally wherein the one or more additional agonist or antagonist signaling protein(s) comprise one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s). In certain embodiments, the subject is administered two or more cell populations, each expressing a distinct chimeric receptor comprising the G-CSFR ECD and each expressing a distinct variant form of G-CSF. In certain embodiments, the cells expressing the chimeric receptor further express at least one antigen-binding signaling receptor. In certain embodiments, the antigen-binding signaling receptor comprises at least one receptor(s) selected from the group consisting of a natural T cell receptor, an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a natural B cell receptor, an engineered B cell receptor (BCR), a stress ligand receptor, a pattern recognition receptor, and combinations thereof. In certain embodiments, the antigen-binding signaling receptor is a CAR. In certain embodiments, the at least one cytokine(s) or chemokine(s) is selected from the group consisting of IL-18, IL-21, interferon α, interferon β, interferon γ, IL-17, IL-21, TNF-α, CXCL13, CCL3 (MIP-1α), CCL4 (MIP-1β), CD40 ligand, B cell activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, or CCL19, or the receptor NKG2D, and combinations thereof. In certain embodiments, the cytokine is IL-18. In certain embodiments, the cytokines are human.

[0044] In certain embodiments, the method is used to treat cancer, such as, but not limited to, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, ovarian cancer, colon cancer, endometrial cancer, hematological malignancies, kidney cancer (renal cell), leukemia, lymphoma, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, sarcoma, and thyroid cancer. In certain embodiments, the method is used to treat an autoimmune disease. In certain embodiments, the method is used to treat an inflammatory condition. In certain embodiments, the method is used to treat a degenerative disease. In certain embodiments, the method is used to generate natural or engineered cells, tissues, or organs for transplantation. In certain embodiments, the method is used to treat or prevent allograft rejection.

[0045] In certain embodiments, the method further comprises administering or providing at least one additional agonist or antagonist signaling protein(s), optionally the one or more additional agonist or antagonist signaling protein(s) comprise one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s). In some embodiments, the subject is administered two or more cell populations, each expressing a distinct chimeric receptor and each expressing a distinct variant form of a cytokine.

[0046] In certain embodiments, the method includes: (i) isolating an immune cell-containing sample; (ii) introducing a nucleic acid sequence encoding a chimeric cytokine receptor into the immune cells; (iii) administering the immune cells of (ii) to a subject; and (iv) contacting the immune cells with a cytokine that binds to the chimeric receptor. In certain embodiments, the subject has undergone immunoablative therapy before administering or injecting the cells into the subject. In certain embodiments, the immune cell-containing sample is isolated from the subject to whom the cells will be administered. In certain embodiments, the immune cell-containing sample is isolated from a subject different from the subject to whom the cells will be administered. In some embodiments, the immune cell-containing sample is generated from cells derived from the subject to whom the cells will be administered or a subject different from the subject to whom the cells will be administered, optionally wherein the cells are stem cells, optionally pluripotent stem cells. In certain embodiments, the immune cells are contacted with the cytokine in vitro before the cells are administered or infused into the subject. In certain embodiments, the immune cells are contacted with the cytokine for a time sufficient to activate signaling from the chimeric receptor. In certain embodiments, the cytokine is G-CSF, optionally the G-CSF is wild-type G-CSF, and optionally the extracellular domain of G-CSFR is a wild-type extracellular domain.

[0047] In certain aspects, described herein are kits comprising at least one expression vector(s) encoding one or more chimeric receptor(s) described herein and instructions for use, optionally the kits comprising at least one cytokine(s) that bind to the chimeric receptor(s). In some embodiments, the kits further comprise one or more expression vector(s) encoding at least one additional agonist or antagonist signaling protein(s), optionally the one or more additional agonist or antagonist signaling protein(s) comprising one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s). In some embodiments, the kit further comprises one or more expression vector(s) encoding one or more cytokine(s) or chemokine(s) selected from the group consisting of IL-18, IL-21, interferon alpha, interferon beta, interferon gamma, IL-17, IL-21, TNF-alpha, CXCL13, CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CD40 ligand, B-cell activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, or CCL19, or the receptor NKG2D, and combinations thereof. In some embodiments, the kit further comprises one or more expression vector(s) encoding at least one antigen-binding receptor(s). In some embodiments, the at least one antigen-binding receptor(s) is selected from the group consisting of a natural T cell receptor, an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a natural B cell receptor, an engineered B cell receptor (BCR), a stress ligand receptor, a pattern recognition receptor, and combinations thereof. In some embodiments, the kit further comprises one or more expression vector(s) encoding the one or more CAR(s), optionally wherein the CAR(s) are mesothelin CARs. In some embodiments, the kit further comprises one or more expression vector(s) encoding one or more separate chimeric receptor(s) described herein.

[0048] In certain aspects, described herein are kits comprising cells encoding one or more chimeric receptor(s) described herein, where optionally the cells are immune cells, and instructions for use, and optionally the kits comprise at least one cytokine(s) that bind to the chimeric receptor(s). In some embodiments, the cells further comprise one or more expression vector(s) encoding at least one additional agonist or antagonist signaling protein(s), where optionally the one or more additional agonist or antagonist signaling protein(s) comprise one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s). In some embodiments, the cells further comprise one or more expression vector(s) encoding at least one cytokine(s) or chemokine(s) selected from the group consisting of IL-18, IL-21, interferon alpha, interferon beta, interferon gamma, IL-17, IL-21, TNF-alpha, CXCL13, CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CD40 ligand, B-cell activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, or CCL19, or the receptor NKG2D, and combinations thereof. In some embodiments, the cells further comprise one or more expression vector(s) encoding at least one cytokine(s) or chemokine(s) selected from the group consisting of IL-18, IL-21, interferon alpha, interferon beta, interferon gamma, IL-17, IL-21, TNF-alpha, CXCL13, CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CD40 ligand, B-cell activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, or CCL19, or the receptor NKG2D, and combinations thereof. In some embodiments, the cells further comprise one or more expression vector(s) encoding at least one antigen-binding signaling receptor(s).In some embodiments, the at least one antigen-binding signaling receptor(s) is selected from the group consisting of a natural T cell receptor, an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a natural B cell receptor, an engineered B cell receptor (BCR), a stress ligand receptor, a pattern recognition receptor, and combinations thereof. In some embodiments, the cell further comprises one or more expression vector(s) encoding at least one CAR(s), optionally wherein the CAR(s) is a mesothelin CAR. In some embodiments, the cell further comprises one or more expression vector(s) encoding at least one distinct chimeric receptor described herein.

[0049] In certain aspects, described herein are systems for selectively activating cells, the systems comprising: (i) a receptor comprising a variant extracellular domain (ECD) of granulocyte colony-stimulating factor receptor (G-CSFR); (ii) a variant G-CSF that selectively binds to the receptor of (i); and one or both of: (a) at least one additional agonist or antagonist signaling protein(s), optionally wherein the one or more additional agonist or antagonist signaling protein(s) comprise one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s); and (b) at least one antigen-binding signaling receptor(s). In some embodiments, the at least one additional cytokine(s) or chemokine(s) comprises at least one of interleukin (IL)-18, IL-21, interferon α, interferon β, interferon γ, IL-17, IL-21, TNF-α, CXCL13, CCL3 (MIP-1α), CCL4 (MIP-1β), CD40 ligand, B-cell activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, CCL19, NKG2D receptor, and combinations thereof. In some embodiments, the at least one additional cytokine(s) comprises IL-18. In some embodiments, the antigen binding signaling receptor(s) comprises at least one of a natural T cell receptor, an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a natural B cell receptor, an engineered B cell receptor (BCR), a stress ligand receptor, a pattern recognition receptor, and combinations thereof. In some embodiments, the antigen-binding signaling receptor comprises a CAR, and optionally, the CAR is a mesothelin CAR. In some embodiments, 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 some embodiments, the 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 the sequence set forth in SEQ ID NO: 2. In some embodiments, the at least one mutation in the Site II interface region of the G-CSFR ECD is selected from the group consisting of R141E, R167D, K168D, K168E, L171E, L172E, Y173K, Q174E, D197K, D197R, M199D, D200K, D200R, V202D, R288D, and R288E of the sequence set forth in SEQ ID NO: 2.

[0050] In some embodiments, the at least one mutation in the Site III interface region of the G-CSFR ECD is selected from the group consisting of amino acid positions 30, 41, 73, 75, 79, 86, 87, 88, 89, 91, and 93 of amino acids 2-308 of the sequence set forth in SEQ ID NO: 2. In some embodiments, the at least one mutation in the Site III interface region of the G-CSFR ECD is selected from the group consisting of S30D, R41E, Q73W, F75KF, S79D, L86D, Q87D, I88E, L89A, Q91D, Q91K, and E93K of the sequence set forth in SEQ ID NO: 2. In some embodiments, the G-CSFR ECD comprises a combination of mutations with design numbers set forth in Tables 4, 22, and 23, wherein the mutations correspond to amino acid positions of the sequence set forth in SEQ ID NO: 2.

[0051] In some embodiments, the G-CSFR ECD comprises the following mutations of the sequence set forth in SEQ ID NO: 2: R41E, R141E, and R167D. In some embodiments, the receptor comprising a variant ECD of G-CSFR is a chimeric receptor.

[0052] In some embodiments, the chimeric receptor is operably linked to at least one second domain, wherein the second domain comprises at least one signaling molecule binding site derived from the intracellular domain (ICD) of one or more cytokine receptor(s), the at least one signaling molecule binding site being selected from the group consisting of a STAT3 binding site of G-CSFR, a STAT3 binding site of glycoprotein 130 (gp130), a SHP-2 binding site of gp130, a SHC binding site of IL-2Rβ, an IGF-1 binding site, an IGF-2 binding site, an IGF-3 binding site, an IGF-4 binding site, an IGF-5 binding site, an IGF-6 binding site, an IGF-8 binding site, an IGF-9 binding site, an IGF-10 binding site, an IGF-11 binding site, an IGF-12 binding site, an IGF-13 binding site, an IGF-14 binding site, an IGF-15 binding site, an IGF-16 binding site, an IGF-17 binding site, an IGF-18 binding site, an IGF-19 binding site, an IGF-2 binding site, an IGF-2 binding site, an IGF-19 ... STAT5 binding site in IL-2Rβ, STAT3 binding site in IL-2Rβ, STAT1 binding site in IL-2Rβ, STAT5 binding site in IL-7Rα, phosphatidylinositol 3-kinase (PI3K) binding site in IL-7Rα, STAT4 binding site in IL-12Rβ2, STAT5 binding site in IL-12Rβ2, STAT3 binding site in IL-12Rβ2, STAT5 binding site in IL-21R, STAT3 binding site in IL-21R, STAT1 binding site in IL-21R, the ICD comprises the Box 1 and Box 2 regions of a protein selected from the group consisting of G-CSFR, gp130 (glycoprotein 130), and IL-2Rβ, and optionally the TMD is a wild-type TMD.

[0053] In some embodiments, the chimeric receptor is operably linked to at least one second domain, the second domain comprising: (i) (a) the Box1 and Box2 regions of gp130, and (b) the C-terminal region of IL-2Rβ, or (ii) (a) the Box 1 and Box 2 regions of the G-CSFR, and (b) the C-terminal region of IL-2Rβ, or (iii) (a) the Box 1 and Box 2 regions of the G-CSFR, and (b) the C-terminal region of IL-12Rβ2, or (iv) (a) the Box 1 and Box 2 regions of the G-CSFR, and (b) the C-terminal region of IL-21R, or (v) (a) the Box1 and Box2 regions of IL-2Rβ, and (b) the C-terminal region of IL-2Rβ, or (vi) (a) the Box 1 and Box 2 regions of the G-CSFR, and (b) the C-terminal region of IL-7Rα, or (vii) (a) the Box 1 and Box 2 regions of G-CSFR; (b) the C-terminal region of IL-4Rα, or (viii) (a) Box1 and Box2 regions of gp130; (b) the C-terminal region of gp130, or (ix) (a) Box 1 and Box 2 regions of the erythropoietin receptor (EPOR), (b) the C-terminal region of EPOR, or (x) (a) the Box 1 and Box 2 regions of G-CSFR; (b) the C-terminal region of interferon alpha and beta receptor subunit 2 (IFNAR2), or (xi) (a) Box 1 and Box 2 regions of interferon gamma receptor 2 (IFNγR2); (b) C-terminal region of interferon gamma receptor 1 (IFNγR1) Includes:

[0054] In some embodiments, the ECD is N-terminal to the TMD, and the TMD is N-terminal to the ICD. In some embodiments, a receptor comprising a variant ECD of G-CSFR is expressed on a cell. In some embodiments, the cell is an immune cell, optionally the immune cell is a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, optionally the cell is a stem cell, optionally the cell is a primary cell, optionally the cell is a human cell. In some embodiments, the T cell is a CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, naive CD8 +The variant G-CSF is selected from the group consisting of T cells, helper T cells, regulatory T cells, memory T cells, and γδ T cells. In some embodiments, activation of a receptor comprising a variant ECD of G-CSFR by a variant G-CSF results in a cellular response comprising at least one of proliferation, viability, persistence, cytotoxicity, cytokine secretion, memory, enhanced activity, and combinations thereof, of cells expressing the receptor. In some embodiments, 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 some embodiments, 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 the sequence set forth in SEQ ID NO: 1. In some embodiments, at least one mutation in the Site II interface region of the variant G-CSF is selected from the group of mutations selected from the group consisting of S12E, S12K, S12R, K16D, L18F, E19K, Q20E, D104K, D104R, L108K, L108R, D109R, D112R, D112K, T115E, T115K, T116D, Q119E, Q119R, E122K, E122R, and E123R. In some embodiments, at least one mutation in the Site III interface region of the variant G-CSF is selected from the group of mutations selected from the group consisting of 38, 39, 40, 41, 46, 47, 48, 49, and 147 of the sequence set forth in SEQ ID NO: 1.

[0055] In some embodiments, at least one mutation in the site III interface region of the variant G-CSF is selected from the group of mutations selected from the group consisting of T38R, Y39E, K40D, K40F, L41D, L41E, L41K, E46R, L47D, V48K, V48R, L49K, and R147E. In some embodiments, the cells express both a receptor comprising a variant ECD of G-CSFR and at least one additional cytokine(s) or chemokine(s). In some embodiments, two or more cell populations each express one or more distinct chimeric receptor(s) comprising the G-CSFR ECD and each express one or more distinct variant forms of G-CSF. In some embodiments, the first population of immune cells further express one or both of: (a) at least one additional agonist or antagonist signaling protein(s), optionally the one or more additional agonist or antagonist signaling protein(s) comprising one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s); and (b) at least one antigen-binding signaling receptor(s). In some embodiments, the cells are immune cells, and the immune cells further express an antigen-binding signaling receptor, which selectively binds to an antigen expressed on the second cells. In some embodiments, the antigen-binding signaling receptor(s) comprises a chimeric antigen receptor (CAR), optionally a mesothelin CAR. In some embodiments, the additional cytokine comprises IL-18. In some embodiments, the second cell is a cancer cell.

[0056] In certain aspects, the present disclosure describes one or more nucleic acid sequence(s) encoding the systems described herein. In certain aspects, the present disclosure describes one or more expression vector(s) comprising the nucleic acid sequence(s) described herein. In certain aspects, the present disclosure describes one or more cell(s) engineered to express the systems described herein. In some embodiments, the cell(s) are immune cells, optionally the immune cells are T cells, optionally NK cells, optionally NKT cells, optionally B cells, optionally plasma cells, optionally macrophages, optionally dendritic cells, optionally the cells are stem cells, optionally the cells are primary cells, optionally the cells are human cells. In some embodiments, the T cells are CD8 + T cells, cytotoxic CD8 + T cells, naive CD8+ T cells, naive CD4 + The T cells are selected from the group consisting of T cells, helper T cells, regulatory T cells, memory T cells, and γδ T cells.

[0057] In certain aspects, a method for selectively activating a receptor comprising a variant ECD of G-CSFR expressed on the surface of a cell is provided, comprising administering to the cell one or more nucleic acid sequence(s) encoding one or more receptor(s) comprising a variant ECD of G-CSFR of a system described herein, and (i) at least one additional agonist or antagonist signaling protein(s), optionally wherein the one or more additional agonist or antagonist signaling protein(s) are selected from the group consisting of one or more cytokine(s), chemokines, and the like of a system described herein. and (ii) at least one additional agonist or antagonist signaling protein(s) comprising a nucleotide(s), a hormone(s), an antibody(ies), or derivative(s) thereof, or other affinity reagent(s), and one or both of at least one antigen-binding signaling receptor(s) of the system described herein, and contacting the receptor(s) comprising a variant ECD of G-CSFR with one or more variant G-CSF, or a variant G-CSF of the system described herein. In some embodiments, the receptor(s) are expressed on immune cells, optionally the immune cells are T cells, optionally NK cells, optionally NKT cells, optionally B cells, optionally plasma cells, optionally macrophages, optionally dendritic cells, optionally the cells are stem cells, optionally the cells are primary cells, and optionally the cells are human cells. In some embodiments, the T cells are CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, naive CD8 +The immune cells are selected from the group consisting of T cells, helper T cells, regulatory T cells, memory T cells, and γδ T cells. In some embodiments, selective activation of the receptor(s) expressed on the immune cells results in a cellular response comprising at least one of immune cell proliferation, viability, persistence, cytotoxicity, cytokine secretion, memory, enhanced activity, and combinations thereof. In some embodiments, a first population of immune cells expresses a receptor comprising a variant ECD of G-CSFR and a second population of immune cells expresses a variant G-CSF, and optionally, one or both of the first and second population(s) of immune cells further express at least one distinct antigen binding signaling receptor(s), and optionally, the at least one distinct antigen binding signaling receptor(s) comprises at least one CAR. In some embodiments, one or both of the first and second population(s) of immune cells further express one or both of: (a) at least one additional agonist or antagonist signaling protein(s), optionally wherein the at least one or more additional agonist or antagonist signaling protein(s) comprises one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s); and (b) at least one antigen-binding signaling receptor(s).

[0058] In some embodiments, the first and second populations of immune cells each express at least one distinct receptor(s) comprising a distinct variant ECD of G-CSFR and at least one distinct variant G-CSF. In some embodiments, the method further includes one or more additional immune cell populations, each additional immune cell population expressing at least one of: (i) a distinct receptor comprising a distinct variant ECD of G-CSFR, (ii) a distinct variant G-CSF, (iii) a distinct agonist or antagonist signaling protein, and (iv) a distinct antigen-binding signaling receptor.

[0059] In certain aspects, the present disclosure describes methods of producing a cell expressing a variant ECD of a G-CSFR of a system described herein and (i) at least one additional agonist or antagonist signaling protein(s), optionally wherein the at least one or more additional agonist or antagonist signaling protein(s) comprises one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s) of a system described herein, and (ii) one or more receptor(s) comprising one or both of at least one antigen-binding signaling receptor(s) of a system described herein, the method comprising introducing into the cell one or more nucleic acid(s) or expression vector(s) encoding the receptor(s), and one or both of (i) and (ii). In some embodiments, a first population of immune cells expresses a receptor comprising a variant ECD of G-CSFR, and a second population of immune cells expresses a variant G-CSF. In some embodiments, one or both of the first and second population(s) of immune cells further express one or both of: (a) at least one additional agonist or antagonist signaling protein(s), optionally wherein the at least one or more additional agonist or antagonist signaling protein(s) comprises one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s), and (b) at least one antigen-binding signaling receptor(s).

[0060] In certain aspects, described herein are methods of increasing an immune response in a subject in need thereof, comprising administering to the subject an immune cell(s) described herein. In certain aspects, described herein are methods of treating a disease in a subject in need thereof, comprising administering to the subject an immune cell(s) described herein. In some embodiments, the method further comprises administering or providing to the subject a variant G-CSF. In some embodiments, the method is used to treat cancer. In some embodiments, the method is used to treat an inflammatory condition. In some embodiments, the method is used to treat an autoimmune disease or condition. In some embodiments, the method is used to treat a degenerative disease. In some embodiments, the method is used to generate natural or engineered cells, tissues, or organs for transplantation. In some embodiments, the method is used to prevent or treat transplant rejection. In some embodiments, the method is used to treat an infectious disease. In some embodiments, the method further comprises administering or providing at least one additional active agent, optionally the at least one additional active agent comprises at least one additional agonist or antagonist signaling protein(s), optionally the one or more additional agonist or antagonist signaling protein(s) comprise one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s).

[0061] In certain aspects, described herein are methods of treating a subject in need thereof, the method comprising: (i) isolating an immune cell-containing sample; and (ii) administering to the immune cells one or more nucleic acid sequence(s) encoding one or more receptor(s) comprising a variant ECD of G-CSFR of the system of any one of claims 48 to 54, and (a) at least one additional active agent, optionally comprising at least one additional agonist or antagonist signaling protein(s), and optionally, one or more additional agonist or antagonist signaling proteins. The protein(s) comprise at least one additional active agent, including one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s) of the system described herein, and optionally, (b) one or both of the antigen-binding signaling receptors of the system described herein; (iii) administering the immune cell(s) of (ii) to a subject; and (iv) contacting the immune cell(s) with a variant G-CSF that specifically binds to a receptor(s) comprising a variant ECD of G-CSFR. In some embodiments, the subject has undergone immunoablative therapy prior to administering or infusing the immune cell(s) to the subject. In some embodiments, the immune cell-containing sample is isolated from a subject to which the cell(s) were administered. In some embodiments, the immune cell-containing sample is isolated from a subject different from the subject to which the cell(s) were administered. In some embodiments, the immune cell-containing sample is generated from source cells derived from a subject to whom the source cells will be administered, and optionally the cells are stem cells, optionally pluripotent stem cells. In some embodiments, the immune cell-containing sample is generated from source cells derived from a subject different from the subject to whom the source cells will be administered, and optionally the cells are stem cells, optionally pluripotent stem cells.In some embodiments, the immune cell(s) are contacted with the variant G-CSF or additional cytokine(s) or chemokine(s) in vitro before the immune cell(s) are administered to a subject. In some embodiments, the immune cell(s) are contacted with the variant G-CSF for a time sufficient to activate signaling from a receptor(s) comprising a variant ECD of G-CSFR of a system described herein.

[0062] In certain aspects, described herein are kits comprising one or more receptor(s) comprising a variant ECD of G-CSFR of the systems described herein, and (a) one or more additional cytokine(s) and chemokine(s) of the systems described herein, and (b) cells encoding one or both of one or more antigen-binding signaling receptor(s) of the systems described herein, and instructions for use, optionally wherein the cells are immune cells. In certain aspects, kits comprise (i) receptor(s) comprising a variant ECD of G-CSFR of the systems described herein, and (a) at least one additional active agent(s), optionally wherein the at least one additional active agent(s) comprises at least one additional agonist or antagonist signaling protein(s), optionally wherein the one or more additional agonist or antagonist signaling protein(s) is one or more cytokine(s), chemokine(s), hormone(s) of the systems described herein. ), at least one additional active agent(s) including a G-CSF, antibody(ies), or derivative(s) thereof, or other affinity reagent(s), and (b) one or more nucleic acid sequence(s) or expression vector(s) encoding one or both of one or more antigen-binding signaling receptors of the systems described herein; (ii) one or more variant G-CSF; and (iii) instructions for use, wherein the receptor and one or both of (a) and (b) are located on the same or separate nucleic acid sequences or expression vectors.

[0063] In certain aspects, a system is provided comprising: (i) a receptor(s) comprising a variant ECD of G-CSFR of the system described herein; and (a) at least one additional active agent(s), optionally wherein the at least one additional active agent(s) comprises at least one additional agonist or antagonist signaling protein(s), and optionally wherein the one or more additional agonist or antagonist signaling protein(s) are one or more cytokine(s), chemokine(s), hormone(s) of the system described herein. , at least one additional active agent(s) comprising an antibody(ies), or derivative(s) thereof, or other affinity reagent(s); and (b) cells comprising one or more nucleic acid sequence(s) or expression vector(s) encoding one or both of at least one antigen-binding signaling receptor(s) of the systems described herein; and (ii) instructions for use, optionally wherein the kit comprises one or more variant G-CSFs that specifically bind to receptor(s) comprising a variant ECD of G-CSFR.

[0064] In certain aspects, disclosed herein is a chimeric receptor comprising: (i) the extracellular domain (ECD) of interleukin receptor alpha (IL-7Rα); (ii) a transmembrane domain (TMD); and (iii) an intracellular domain (ICD) of a cytokine receptor that differs from the wild-type human IL-7Rα intracellular signaling domain set forth in SEQ ID NO: 109, wherein the ECD and TMD are each operably linked to the ICD. In some embodiments, the carboxy-terminus (C-terminus) of the ECD is linked to the amino-terminus (N-terminus) of the TMD, and the C-terminus of the TMD is linked to the N-terminus of the ICD. In some embodiments, the ECD is the ECD of native human IL-7Rα. In some embodiments, the TMD is the TMD of IL-7Rα. In some embodiments, the TMD is the TMD of native human IL-7Rα. In some embodiments, the ICD comprises at least one signaling molecule binding site derived from the intracellular domain of a cytokine receptor, and optionally the at least one signaling molecule binding site comprises: (a) a JAK1 binding site (Box 1 and 2 regions) of IL-2Rβ, IL-4Rα, IL-7Rα, IL-21R, or gp130; (b) an SHC binding site of IL-2Rβ; (c) a STAT5 binding site of IL-2Rβ or IL-7Rα; (d) a STAT3 binding site of IL-21R or gp130; (e) a STAT4 binding site of IL-12Rβ2; (f) a STAT6 binding site of IL-4Rα; (g) an IRS-1 or IRS-2 binding site of IL-4Rα; and (h) an SHP-2 binding site of gp130; (i) a PI3K binding site of IL-7Rα; or a combination thereof. In some embodiments, the ICD comprises at least the intracellular signaling domain of a receptor that is activated by homodimerization or heterodimerization with the common gamma chain (gc).In some embodiments, the ICD comprises at least the intracellular signaling domain of a cytokine receptor selected from the group consisting of IL-2Rβ (interleukin-2 receptor beta), IL-4Rα (interleukin-4 receptor alpha), IL-9Rα (interleukin-9 receptor alpha), IL-12R β2 (interleukin-12 receptor), IL-21R (interleukin-21 receptor), and glycoprotein 130 (gp130), and combinations thereof.

[0065] In a particular aspect, a chimeric receptor comprises an ECD and a TMD of IL-7Rα operably linked to an ICD, wherein the ICD comprises: (i) (a) Box1 and Box2 regions of IL-2Rβ; (b) the SHC binding site of IL-2Rβ, and (c) the STAT5 binding site of IL-2Rβ, or (ii) (a) Box1 and Box2 regions of IL-7Rα; (b) the SHC binding site of IL-2Rβ, and (c) the STAT5 binding site of IL-2Rβ, or (iii) (a) Box1 and Box2 regions of IL-2Rβ; (b) SHC binding site of IL-2Rβ; (c) the STAT5 binding site of IL-2Rβ, and (d) the STAT4 binding site of IL-12Rβ2, or (iv) (a) Box1 and Box2 regions of IL-7Rα; (b) SHC binding site of IL-2Rβ; (c) the STAT5 binding site of IL-2Rβ, and (d) the STAT4 binding site of IL-12Rβ2, or (v) (a) the Box1 and Box2 regions of IL-21R, and (b) the STAT3 binding site of IL-21R, or (vi) (a) the Box1 and Box2 regions of IL-7Rα, and (b) the STAT3 binding site of IL-21R, or (vii) (a) Box1 and Box2 regions of IL-21R; (b) the STAT3 binding site of IL-21R, and (c) STAT5 and PI3 kinase binding sites of IL-7Rα; (viii) (a) Box1 and Box2 regions of IL-7Rα; (b) the STAT3 binding site of IL-21R, and (c) STAT5 and PI3 kinase binding sites of IL-7Rα; (ix) (a) Box1 and Box2 regions of IL-2Rβ; (b) SHC binding site of IL-2Rβ; (c) the STAT5 binding site of IL-2Rβ, and (d) the STAT3 binding site of IL-21R, or (x) (a) Box1 and Box2 regions of IL-7Rα; (b) SHC binding site of IL-2Rβ; (c) the STAT5 binding site of IL-2Rβ, and (d) the STAT3 binding site of IL-21R, or (xi) (a) Box1 and Box2 regions of IL-2Rβ; (b) SHC binding site of IL-2Rβ; (c) STAT5 binding site of IL-2Rβ; (d) STAT4 binding site of IL12Rβ2, and (e) the STAT3 binding site of IL-21R, or (xii) (a) Box1 and Box2 regions of IL-7Rα; (b) SHC binding site of IL-2Rβ; (c) STAT5 binding site of IL-2Rβ; (d) STAT4 binding site of IL12Rβ2, and (e) the STAT3 binding site of IL-21R, or (xiii) (a) Box1 and Box2 regions of IL-4Rα; (b) the IRS-1 or IRS-2 binding site of IL-4Rα, and (c) the STAT6 binding site of IL-4Rα, or (xiv) (a) Box1 and Box2 regions of IL-7Rα; (b) the IRS-1 or IRS-2 binding site of IL-4α, and (c) the STAT6 binding site of IL-4α, or (xv) (a) Box1 and Box2 regions of gp130; (b) the SHP-2 binding site of gp130, and (c) the STAT3 binding site of gp130, or (xvi) (a) Box1 and Box2 regions of IL-7Rα; (b) the SHP-2 binding site of gp130, and (c) STAT3 binding site of gp130 In some embodiments, (b) is N-terminal to (c), or (c) is N-terminal to (b), or (c) is N-terminal to (d), or (d) is N-terminal to (c), or (d) is N-terminal to (e), or (e) is N-terminal to (d).

[0066] In some embodiments, the ICD comprises a sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 192-214.

[0067] In some embodiments, the activated form of the chimeric receptor forms a heterodimer, and optionally, activation of the chimeric receptor results in a cellular response comprising at least one of proliferation, viability, persistence, cytotoxicity, cytokine secretion, memory, and enhanced activity of cells expressing the chimeric receptor, and optionally, the chimeric receptor is activated upon contact with interleukin (IL)-7. In some embodiments, the IL-7 is wild-type human IL-7. In some embodiments, the IL-7 comprises one, two, three, four, or five mutations compared to wild-type IL-7. In some embodiments, the IL-7 comprises one or more chemical modifications.

[0068] In some embodiments, the chimeric receptor is expressed on a cell, and the cell is an immune cell, optionally the immune cell is a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, optionally the cell is a stem cell, optionally the cell is a primary cell, and optionally the cell is a human cell. In some embodiments, the T cell is a CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, naive CD8 + The receptor is selected from the group consisting of T cells, helper T cells, regulatory T cells, memory T cells, and γδ T cells. In some embodiments, activation of the receptor by IL-7 results in a cellular response that includes at least one of proliferation, viability, persistence, cytotoxicity, cytokine secretion, memory, and enhanced activity of cells expressing the receptor.

[0069] In certain aspects, the present disclosure describes one or more nucleic acid sequence(s) encoding the receptors described herein. In certain aspects, the present disclosure describes one or more expression vector(s) comprising the nucleic acid sequence(s) described herein. In certain aspects, the present disclosure describes cells comprising the nucleic acid sequence(s) or expression vector(s) described herein. In some embodiments, the cell is an immune cell, optionally the immune cell is a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, optionally the cell is a stem cell, optionally the cell is a primary cell, optionally the cell is a human cell. In some embodiments, the T cell is a CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, naive CD8 + The T cells are selected from the group consisting of T cells, helper T cells, regulatory T cells, memory T cells, and γδ T cells.

[0070] In certain aspects, the present disclosure describes a system for activating a receptor expressed on a cell surface, the system comprising: (a) a chimeric receptor described herein; and (b) IL-7.

[0071] In certain aspects, the present disclosure describes a system for activating immune cells, the system comprising: (a) a chimeric receptor described herein; (b) IL-7; and (c) an antigen-binding signaling receptor.

[0072] In certain aspects, the present disclosure describes a system for activating immune cells, comprising: (a) a chimeric receptor described herein; (b) IL-7; and (c) at least one or more additional agonist or antagonist signaling protein(s), optionally wherein the one or more additional agonist or antagonist signaling protein(s) comprise one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent.

[0073] In some embodiments, the system further comprises at least one antigen-binding signaling receptor. In some embodiments, the at least one antigen-binding signaling receptor comprises at least one receptor selected from the group consisting of a natural T cell receptor, an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a natural B cell receptor, an engineered B cell receptor (BCR), a stress ligand receptor, a pattern recognition receptor, and combinations thereof. In some embodiments, the at least one antigen-binding signaling receptor is a CAR. In some embodiments, the cytokine or chemokine is selected from the group consisting of IL-18, IL-21, interferon alpha, interferon beta, interferon gamma, IL-17, IL-21, TNF-alpha, CXCL13, CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CD40 ligand, B cell-activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, CCL19, NKG2D receptor, and combinations thereof. In some embodiments, the cytokine is IL-18. In some embodiments, the cytokines are human.

[0074] In certain aspects, described herein are methods of activating a chimeric receptor expressed on the surface of a cell, comprising contacting the chimeric receptor with IL-7 to activate the chimeric receptor, wherein the chimeric receptor comprises (i) an extracellular domain (ECD) of IL-7Rα, (ii) a transmembrane domain (TMD), and (iii) an intracellular domain (ICD) of a cytokine receptor that differs from the wild-type human IL-7Rα intracellular signaling domain set forth in SEQ ID NO: 109, wherein the ECD and TMD are each operably linked to the ICD. In some embodiments, the chimeric receptor is a chimeric receptor described herein.

[0075] In certain aspects, described herein are methods of producing a chimeric receptor in a cell, the methods comprising introducing into the cell a nucleic acid sequence(s) described herein or an expression vector(s) described herein. In some embodiments, the methods further comprise editing the sequence(s) of the sequence(s) or vector(s) into the genome of the cell.

[0076] In some embodiments of the methods described herein, the cell is an immune cell, optionally the immune cell is a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, optionally the cell is a stem cell, optionally the cell is a primary cell, optionally the cell is a human cell. In some embodiments, the T cell is a CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, naive CD8 + The T cells are selected from the group consisting of T cells, helper T cells, regulatory T cells, memory T cells, and γδ T cells.

[0077] In certain aspects, described herein are methods of increasing an immune response in a subject in need thereof, comprising administering to the subject a cell(s) that express a chimeric receptor described herein, and administering or providing to the subject IL-7.

[0078] In certain aspects, described herein are methods of treating a subject in need thereof, comprising administering to the subject a cell(s) that express a chimeric receptor described herein and administering or providing IL-7 to the subject. In some embodiments, the method is used to treat cancer. In some embodiments, the method is used to treat an autoimmune disease. In some embodiments, the method is used to treat an inflammatory condition. In some embodiments, the method is used to treat a degenerative disease. In some embodiments, the method is used to generate natural or engineered cells, tissues, or organs for transplantation. In some embodiments, the method is used to prevent or treat transplant rejection. In some embodiments, the method is used to treat an infectious disease.

[0079] In certain embodiments, the method is used to treat a degenerative disease or condition, including, but not limited to, age-related neurodegenerative diseases and conditions.

[0080] In certain embodiments, the methods are used to generate natural or engineered cells, tissues, or organs for transplantation.

[0081] In some embodiments, the method further comprises administering or providing at least one or more additional agonist or antagonist signaling protein(s), optionally wherein the one or more additional agonist or antagonist signaling protein(s) comprise one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s). In some embodiments, the subject is administered cells expressing at least one additional distinct chimeric receptor. In some embodiments, the at least one additional distinct chimeric receptor is a chimeric receptor comprising a variant ECD of granulocyte colony-stimulating factor receptor (G-CSFR). In some embodiments, the cells expressing at least one additional distinct chimeric receptor comprising a variant ECD of G-CSFR are contacted with one or more variant G-CSFs, and optionally the subject is administered the one or more variant G-CSFs. In some embodiments, the method includes: i) isolating an immune cell-containing sample; (ii) transducing or transfecting the immune cell(s) with nucleic acid sequence(s) encoding chimeric cytokine receptor(s); (iii) administering the immune cell(s) of (ii) to a subject; and (iv) contacting the immune cell(s) with IL-7.

[0082] In some embodiments, the method further comprises introducing into the immune cell(s) a nucleic acid sequence(s) encoding at least one additional agonist or antagonist signaling protein(s), optionally the one or more additional agonist or antagonist signaling protein(s) comprise one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s). In some embodiments, the at least one cytokine or chemokine is selected from the group consisting of IL-18, IL-21, interferon alpha, interferon beta, interferon gamma, IL-17, IL-21, TNF-alpha, CXCL13, CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CD40 ligand, B-cell activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, CCL19, NKG2D receptor, and combinations thereof. In some embodiments, the method further comprises introducing into the immune cell(s) a nucleic acid sequence(s) encoding at least one antigen-binding signaling receptor. In some embodiments, the at least one antigen-binding signaling receptor is selected from the group consisting of a natural T cell receptor, an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a natural B cell receptor, an engineered B cell receptor (BCR), a stress ligand receptor, a pattern recognition receptor, and combinations thereof. In some embodiments, the subject has undergone immunoablative therapy prior to administering the cells to the subject. In some embodiments, the immune cell-containing sample is isolated from the subject to which the cells are administered. In some embodiments, the immune cell-containing sample is isolated from a subject different from the subject to which the cells will be administered. In some embodiments, the immune cell-containing sample is generated from cells derived from a subject different from the subject to which the cells will be administered, optionally wherein the cells are stem cells, optionally pluripotent stem cells. In some embodiments, the immune cells are contacted in vitro with one or both of IL-7 or variant G-CSF prior to administering the cells to the subject.In some embodiments, immune cells are contacted with one or both of IL-7 or variant G-CSF for a time sufficient to activate signaling from a chimeric receptor described herein.

[0083] In some embodiments, the cells administered to the subject further express at least one antigen-binding signaling receptor selected from the group consisting of a natural T cell receptor, an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a natural B cell receptor, an engineered B cell receptor (BCR), a stress ligand receptor, a pattern recognition receptor, and combinations thereof. In some embodiments, the cells administered to the subject further express a receptor comprising a variant ECD of G-CSFR. In some embodiments, 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 some embodiments, the cells administered to the subject further express IL-18.

[0084] In certain aspects, described herein are kits comprising cells encoding a chimeric receptor described herein, where the cells are optionally immune cells, and instructions for use, optionally comprising IL-7, and optionally comprising a variant G-CSF described herein. In certain aspects, described herein are kits comprising one or more expression vector(s) comprising nucleic acid sequence(s) encoding a chimeric receptor described herein, and instructions for use, optionally comprising IL-7, and optionally comprising a variant G-CSF described herein. In some embodiments, the kits further comprise one or more expression vector(s) encoding at least one additional agonist or antagonist signaling protein(s), optionally comprising one or more additional agonist or antagonist signaling protein(s), including one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent. In some embodiments, the kit further comprises one or more expression vector(s) encoding a cytokine or chemokine selected from the group consisting of IL-18, IL-21, interferon α, interferon β, interferon γ, IL-17, IL-21, TNF-α, CXCL13, CCL3 (MIP-1α), CCL4 (MIP-1β), CD40 ligand, B-cell activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, CCL19, NKG2D receptor, and combinations thereof. In some embodiments, the kit further comprises one or more expression vector(s) encoding at least one receptor selected from the group consisting of a natural T cell receptor, an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a natural B cell receptor, an engineered B cell receptor (BCR), a stress ligand receptor, a pattern recognition receptor, and combinations thereof. In some embodiments, the kit further comprises an expression vector encoding a chimeric antigen receptor.In some embodiments, the cells further comprise one or more expression vector(s) encoding at least one additional agonist or antagonist signaling protein(s), optionally the one or more additional agonist or antagonist signaling protein(s) comprise one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s). In some embodiments, the cells further comprise one or more expression vector(s) encoding at least one cytokine or chemokine selected from the group consisting of IL-18, IL-21, interferon alpha, interferon beta, interferon gamma, IL-17, IL-21, TNF-alpha, CXCL13, CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CD40 ligand, B-cell activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, CCL19, NKG2D receptor, and combinations thereof. In some embodiments, the cells further comprise one or more expression vector(s) encoding at least one antigen-binding signaling receptor(s). In some embodiments, the at least one antigen-binding signaling receptor(s) is selected from the group consisting of a natural T cell receptor, an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a natural B cell receptor, an engineered B cell receptor (BCR), a stress ligand receptor, a pattern recognition receptor, and combinations thereof. In some embodiments, the cell further comprises one or more expression vector(s) encoding the at least one CAR(s). [Brief explanation of the drawings]

[0085] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description and accompanying drawings.

[0086] [Figure 1] FIG. 1 depicts a diagram showing the structure of the Site II and III interface of the 2:2 G-CSF:G-CSFR heterodimer complex.

[0087] [Figure 2] FIG. 1 depicts a diagram outlining the strategy used for G-CSF:G-CSFR interface design.

[0088] [Figure 3] 1 depicts a graph showing the energy components of Site II interface interactions.

[0089] [Figure 4] FIG. 1 depicts the structure of the Site II interface and a diagram showing the interactions of Arg167 (left) and Arg141 (right) of G-CSFR(CRH) with G-CSF residues at the Site II interface.

[0090] [Figure 5] Schematic showing wild-type G-CSF at Site II (left) and an overlay of the same region of a triple ZymeCAD™ mean field pack for Site II design #35 (right).

[0091] [Figure 6] 1 depicts an image of SDS-PAGE showing the results of G-CSF pull-down assays of G-CSF Design #s: 6, 7, 8, 9, 15, 17, 30, 34, 35, and 36 (upper panel), and co-expressed and purified Site II Design Complex #s: 6, 7, 8, 9, 15, 17, 30, 34, 35, and 36 (lower panel) (1st lane: WT G-CSF control, 2nd lane: WT G-CSF:G-CSFR(CRH) control).

[0092] [Figure 7] 1 depicts an image of an SDS-PAGE showing the results of a G-CSF pull-down assay of G-CSF design #s: 6, 7, 8, 9, 15, 17, 30, 34, 35, and 36 co-expressed with WT-G-CSFR (upper panel), and a G-CSF pull-down assay of WT G-CSF co-expressed with G-CSFR design #s: 6, 7, 8, 9, 15, 17, 30, 34, 35, and 36 (lower panel, lane 1 WT:WT G-CSF:G-CSFR pull-down control).

[0093] [Figure 8] 1 depicts a graph showing the energy components of Site III interface interactions.

[0094] [Figure 9] FIG. 1 depicts the structure of the Site III interface and a diagram showing the interactions of R41 (left) and E93 (right) of G-CSFR(Ig) with G-CSF residues at the Site III interface.

[0095] [Figure 10] Figure 1 shows the results of a G-CSF pull-down assay of designs #401 and 402 (top panel) and of co-expressed and purified site II / III design complexes #401 and 402 (pull-down of WT G-CSFR and WT G-CSF co-expressed with design #401 and 402 G-CSFR) (bottom panel). Images of SDS-PAGE are shown, with the first lane being the WT G-CSF control and the second lane being the WT G-CSF:G-CSFR(Ig-CRH) control.

[0096] [Figure 11] 1 depicts a graph showing the size-exclusion chromatography profiles of WT (SX75) G-CSF and design 130 (SX75), 303 (SX200), and 401 (SX200) G-CSF variants after TEV cleavage.

[0097] [Figure 12] 1 depicts a graph showing the size exclusion chromatography profiles of WT (SX75) and purified design 401 (SX200) and 402 (SX200) G-CSFR(Ig-CRH)E variants after TEV cleavage.

[0098] [Figure 13]Graphs depicting binding SPR sensorgrams of WT, Designs #130, #401, and #402 G-CSFs binding to their cognate or mispaired G-CSFRs (Ig-CRH). Each G-CSF-to-G-CSFR pair is labeled below each panel. Representative steady-state fits used to derive KDs for the cognate pairs of Designs #401 and #402 are shown below each sensorgram.

[0099] [Figure 14] Graphs are presented showing: upper left panel: DSC thermograms of WT G-CSF, designs #130 and #134 G-CSFE; upper right panel: DSC thermograms of WT G-CSFR(Ig-CRH), designs #130 and #134 G-CSFR(Ig-CRH)E; lower left panel: DSC thermograms of designs #401 and #402 G-CSFE; lower right panel: DSC thermograms of designs #300, #303, #304 and #307 G-CSFE.

[0100] [Figure 15] Graphs depicting the results of a bromo-deoxyuridine (BrdU) assay showing proliferating 32D-IL-2RβIL2Rb cells expressing A) G-CSFRWT-ICDIL-2Rb (homodimer) or B) G-CSFRWT-ICDIL-2Rb + G-CSFRWT-ICDgc (heterodimer). Cells were stimulated with no cytokine, IL-2 (300 IU / ml), or G-CSFRWT (100 ng / ml in A, 30 ng / ml in B).

[0101] [Figure 16] Graph showing the results of a BrdU assay demonstrating proliferation of 32D-IL-2Rβ cells expressing A) G-CSFR137-ICDgp130-IL-2Rb (homodimer) or B) G-CSFR137-ICDIL-2Rb + G-CSFR137-ICDgc (heterodimer). Cells were stimulated with no cytokine, IL-2 (300 IU / ml), G-CSFRWT (30 ng / ml), or G-CSFR137 (30 ng / ml).

[0102] [Figure 17] Figure 1 shows the results of a BrdU assay demonstrating proliferation of 32D-IL-2Rβ cells expressing A) G-CSFRWT-ICDgp130-IL-2Rb (homodimer) or B) G-CSFRWT-ICDIL-2Rb + G-CSFRWT-ICDgc (heterodimer). Cells were stimulated with no cytokine, IL-2 (300 IU / ml), G-CSFRWT (30 ng / ml), or G-CSFR137 (30 ng / ml).

[0103] [Figure 18] Western blots showing signal transduction in 32D-IL-2Rβ cells expressing G-CSFRWT-ICDgp130-IL-2Rb (homodimer), G-CSFR137-ICDgp130-IL-2Rb (homodimer), G-CSFRWT-ICDIL-2Rb + G-CSFRWT-ICDgc (heterodimer), or G-CSFR137-ICDIL-2Rb + G-CSFR137-ICDgc (heterodimer). Cells were stimulated with no cytokine, IL-2 (300 IU / ml), G-CSFRWT (30 ng / ml), or G-CSFR137 (30 ng / ml).

[0104] [Figure 19] Graphs depicting the results of BrdU incorporation assays assessing cell cycle progression of primary murine T cells (or untransduced cells) expressing the indicated chimeric receptors in response to stimulation with no cytokine, IL-2, or WT, 130, 304, or 307 cytokines. A and B represent repeat experiments.

[0105] [Figure 20] Schematic representation of the native IL-2Rβ, IL-2Rγc, and G-CSFR subunits, and the G2R-1 receptor subunit design.

[0106] [Figure 21]This graph shows the proliferation (fold change in cell number) of 32D-IL-2Rβ cells (a 32D cell line stably expressing the human IL-2Rβ subunit) expressing the indicated G-CSFR chimeric receptor subunits and stimulated with WT G-CSF, IL-2, or no cytokine. G / γc is tagged with a Myc epitope at its N-terminus (Myc / G / γc), and G / IL-2Rβ is tagged with a Flag epitope at its N-terminus (Flag / G / IL-2Rβ); these epitope tags aid in detection by flow cytometry and do not affect receptor function. Additionally, the lower panels in B–D show the percentage of cells expressing the G-CSFR ECD (%G-CSFR+) under each culture condition. Squares represent cells stimulated with IL-2. Triangles represent cells stimulated with G-CSF. Circles represent cells not stimulated with cytokine.

[0107] [Figure 22] Graphs depicting proliferation (fold change in cell number) of human T cells expressing Flag-tagged G / IL-2R β subunit alone, Myc-tagged G / γ subunit alone, 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.

[0108] [Figure 23] 20 depicts a schematic diagram of native and chimeric receptors showing JAK, STAT, Shc, SHP-2, and PI3K binding sites. The shaded scheme includes receptors from FIG. 20.

[0109] [Figure 24] 2A-2C depict schematic diagrams of chimeric receptors showing JAK, STAT, Shc, SHP-2, and PI3K binding sites. The shaded scheme includes receptors from FIGS. 20 and 23.

[0110] [Figure 25]FIG. 1 shows a diagram of the lentiviral plasmid containing the G2R-2 cDNA insert.

[0111] [Figure 26] Graphs depicting G-CSFR ECD expression assessed by flow cytometry in cells transduced with G2R-2: A) 32D-IL-2Rβ cell line; B) PBMC-derived human T cells and human tumor-associated lymphocytes (TAL).

[0112] [Figure 27] Graphs showing proliferation (fold change in cell number) of cells expressing G2R-2 compared to non-transduced cells. A) Human PBMC-derived T cells; B, C) Human tumor-associated lymphocytes (TAL) from two independent experiments. Squares represent cells stimulated with IL-2. Triangles represent cells stimulated with G-CSF. Circles represent cells not stimulated with cytokine.

[0113] [Figure 28] Figure 1 shows graphs showing proliferation (fold change in cell number) of CD4- or CD8-selected human tumor-associated lymphocytes expressing G2R-2 compared to non-transduced cells. A) Non-transduced CD4-selected cells; B) Non-transduced CD8-selected cells; C) G2R-2-transduced CD4-selected cells; D) G2R-2-transduced CD8-selected cells. The dotted gray line represents cells stimulated with IL-2. The solid black line represents cells stimulated with G-CSF. The dashed gray line represents cells not stimulated with cytokine.

[0114] [Figure 29]Figure 1 depicts a graph showing the proliferation (fold change in cell number) of CD4+ or CD8+ tumor-associated lymphocytes expressing G2R-2. Cells were first grown in G-CSF or IL-2 as indicated. Cells were then plated with either IL-2, G-CSF, or medium alone. The solid gray line represents cells stimulated with IL-2. The solid gray line represents cells stimulated with IL-2. The solid black line represents cells stimulated with G-CSF. The dashed light gray line represents cells grown with IL-2 and stimulated with medium alone. The dashed dark gray line represents cells grown with G-CSF and stimulated with medium alone.

[0115] [Figure 30] Graphs depicting the immunophenotype (by flow cytometry) of CD4- or CD8-selected tumor-associated lymphocytes (TALs) expressing G2R-2 chimeric receptor constructs versus untransduced cells after expansion with G-CSF or IL-2. A) Percentage of viable cells displaying CD4+, CD8+, or CD3-CD56+ cell surface phenotypes. B) Percentage of viable cells displaying the indicated cell surface phenotypes based on CD45RA and CCR7 expression.

[0116] [Figure 31] Graphs depicting the results of a BrdU incorporation assay to assess proliferation of primary human T cells expressing G2R-2 versus non-transduced cells. T cells were selected by culture in IL-2 or G-CSF, as indicated, prior to the assay. A) Tumor-associated lymphocytes; B) PBMC-derived T cells.

[0117] [Figure 32]Graphs depict the results of a BrdU incorporation assay to assess proliferation of primary murine T cells expressing G2R-2 or single-chain G / IL-2Rβ (a component of G2R-1) versus mock-transduced cells. A) Transduction efficiency as reflected by the percentage of cells expressing the G-CSFR ECD (by flow cytometry) after culture with the indicated cytokines; B) Percentage of BrdU incorporation among all live cells in response to the indicated cytokines; C) Percentage of BrdU incorporation by cells expressing the G-CSFR ECD (G-CSFR+ cells). All cells were grown with IL-2 for 3 days before the assay. Squares represent cells stimulated with IL-2. Triangles represent cells stimulated with G-CSF. Circles represent cells not stimulated with cytokines.

[0118] [Figure 33] Western blots for detecting the indicated cytokine signaling events in human primary T cells expressing G2R-2 versus non-transduced cells. β-actin, total Akt, and histone H3 serve as protein loading controls. A, B) Tumor-associated lymphocytes (TAL); C) PBMC-derived T cells.

[0119] [Figure 34] Western blots for detecting the indicated cytokine signaling events in primary murine T cells expressing G2R-2 or single-chain G / IL-2Rβ (derived from G2R-1) versus mock-transduced cells. The arrow indicates the specific phospho-JAK2 band; other larger bands are presumably the result of cross-reactivity of the primary anti-phospho-JAK2 antibody with phospho-JAK1. β-Actin and histone H3 served as protein loading controls.

[0120] [Figure 35]Figure 1 depicts a graph showing the results of a BrdU incorporation assay assessing cell cycle progression of 32D-IL-2Rβ cells (or non-transduced cells) expressing the indicated chimeric receptors in response to stimulation with no cytokine, IL-2 (300 IU / mL), or WT G-CSF (30 ng / mL), or WT G-CSF (30 ng / mL).

[0121] [Figure 36] Graphs depicting the results of BrdU incorporation assays assessing cell cycle progression of primary murine T cells (or untransduced cells) expressing the indicated chimeric receptors in response to stimulation with no cytokine, IL-2, WT G-CSF, or G-CSF variants 130, 304, or 307. A and B represent repeat experiments.

[0122] [Figure 37] Western blots are shown to detect the indicated cytokine signaling events in 32D-IL-2Rβ cells (or non-transduced cells) expressing the indicated chimeric receptor subunits in response to stimulation with no cytokine, IL-2, WT G-CSF, or 130 G-CSF. β-Actin and histone H3 serve as protein loading controls.

[0123] [Figure 38] A) Western blots for detecting the indicated cytokine signaling events in primary murine T cells expressing the indicated chimeric receptor subunits in response to stimulation with no cytokine, IL-2, WT G-CSF, 130 G-CSF, or 304 G-CSF. β-Actin and histone H3 serve as protein loading controls. B) Transduction efficiency of the cells used in panel A, assessed by flow cytometry using an antibody specific for the extracellular domain of the human G-CSF receptor.

[0124] [Figure 39]Figure 1 shows plots showing G-CSFR ECD expression by flow cytometry in primary human tumor-associated lymphocytes (TALs) transduced with the indicated chimeric receptor constructs. Live CD3+, CD56- cells were gated on CD8 or CD4, and G-CSFR ECD expression is shown for each population.

[0125] [Figure 40] Graphs and images depict the expansion, proliferation, and signaling of primary human tumor-associated lymphocytes (TALs) expressing G2R-3 versus untransduced cells. A) Graph showing the results of a T cell proliferation assay in which cells were transduced with a lentivirus encoding G2R-3, washed, and then replated with IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. Viable cells were counted every 3–4 days. Squares represent cells stimulated with IL-2. Triangles represent cells stimulated with G-CSF. Circles represent cells not stimulated with cytokine. B) Western blot to assess intracellular signaling events. Cells were harvested from the proliferation assay and stimulated with IL-2 (300 IU / ml) or wild-type G-CSF (100 ng / ml). The arrow indicates the specific phospho-JAK2 band at 125 kDa; the larger band is presumably the result of cross-reactivity of the primary anti-phospho-JAK2 antibody with phospho-JAK1. β-Actin and histone H3 served as protein loading controls. C) Graph showing the results of a BrdU incorporation assay to assess T cell proliferation. Cells were harvested from the proliferation assay, washed, and then replated in IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine.

[0126] [Figure 41]Graphs showing fold expansion and G-CSFR ECD expression of primary human PBMC-derived T cells expressing G2R-3 with WT ECD versus untransduced cells. A) Graph showing the results of a T cell proliferation assay in which cells were transduced with a lentivirus encoding G2R-3. On day 1, WT G-CSF (100 ng / ml) or no cytokine (media only) was added to the culture medium. Cells were then supplemented with media containing WT G-CSF or no cytokines until day 21. On day 21 of expansion, cells were washed and replated with WT G-CSF (100 ng / mL), IL-7 (20 ng / mL), and IL-15 (20 ng / mL), or no cytokines. Viable cells were counted every 2–4 days. Squares represent cells stimulated with G-CSF. Triangles represent cells stimulated with G-CSF and replated with IL-7 and IL-15 on day 21. Circles represent cells not stimulated with cytokines. Diamonds represent cells stimulated with G-CSF and replated with medium alone on day 21. B) Graph showing expression of G-CSFR ECD measured by flow cytometry on days 21 or 42 of expansion.

[0127] [Figure 42] Figures 1A-C show graphs depicting the intracellular signaling and immunophenotype of primary human PBMC-derived T cells expressing G2R-3 versus non-transduced cells. A) Western blot to assess intracellular signaling events. Cells were harvested from proliferation assays and stimulated with IL-2 (300 IU / ml) or wild-type G-CSF (100 ng / ml). β-actin serves as a protein loading control. B, C) Representative flow cytometry plots and graphs showing the immunophenotype, as assessed by flow cytometry, of cells expressing G2R-3 versus non-transduced cells at day 42 of expansion.

[0128] [Figure 43]Graphs showing the fold expansion of primary human PBMC-derived T cells expressing G2R-3 with either the 304 or 307 ECD versus untransduced cells. A) Graph showing the results of a T cell proliferation assay in which cells were transduced with a lentivirus encoding the G2R-3 304 ECD. B) Graph showing the results of a T cell proliferation assay in which cells were transduced with a lentivirus encoding the G2R-3 307 ECD. C) Graph showing the results of a T cell proliferation assay using untransduced cells. On day 2, IL-2 (300 IU / mL), 304 G-CSF (100 ng / mL), 307 G-CSF (100 ng / mL), or no cytokine (media only) was added to the cultures as indicated and replenished every 2 days thereafter. Viable cells were counted every 3–4 days. Diamonds represent cells stimulated with 304 G-CSF. Squares represent cells stimulated with 307 G-CSF. Triangles represent cells stimulated with IL-2. Inverted triangles represent cells not stimulated with cytokines.

[0129] [Figure 44]

[0033] Figure 1 depicts a graph showing the results of a BrdU incorporation assay to assess proliferation of primary human PBMC-derived T cells expressing G2R-3 with 304 or 307 ECD versus untransduced cells. Cells were transduced with lentivirus encoding the G2R-3 304 ECD or 307 ECD and grown in 304 or 307 G-CSF (100 ng / mL). Untransduced cells were grown in IL-2 (300 IU / mL). On day 12 of growth, cells were washed and replated with IL-2 (300 IU / mL), 130 G-CSF (100 ng / mL), 304 G-CSF (100 ng / mL), 307 G-CSF (100 ng / mL), or no cytokine.

[0130] [Figure 45] 1 depicts a graph showing G-CSFR ECD expression by flow cytometry in primary mouse T cells transduced with the indicated chimeric receptor constructs.

[0131] [Figure 46] G-CSF-induced phosphorylation of STAT3 (detected by flow cytometry) in primary PBMC-derived human T cells expressing G21R-1 or G21R-2. Cells were subdivided (i.e., gated) into G-CSFR-positive (upper panel) or G-CSFR-negative (lower panel) populations.

[0132] [Figure 47] Graphs and images depict G-CSF-induced biochemical signaling events in primary mouse T cells expressing G21R-1 or G12R-1. A) Graph showing STAT3 phosphorylation (detected by flow cytometry) in CD4+ or CD8+ cells transduced with G21R-1 and stimulated with no cytokine, IL-21, or G-CSF. B) Graph showing the percentage of cells staining positive for phospho-STAT3 after stimulation with no cytokine (filled circles), IL-21 (squares), or WT G-CSF (gray circles). Live cells were gated by CD8 or CD4, and the percentage of phospho-STAT3-positive cells is shown for each population. C) Western blot to assess the indicated cytokine signaling events in cells expressing G21R-1 or G12R-1 and stimulated with IL-21, IL-12, or WT G-CSF. β-Actin and histone H3 served as protein loading controls.

[0133] [Figure 48-1]Figure 48 presents graphs and images showing proliferation, G-CSFR ECD expression, and WT G-CSF-induced intracellular signaling events in primary murine T cells expressing G2R-2, G2R-3, G7R-1, G21 / 7R-1, and G27 / 2R-1, or mock-transduced T cells. A, B) Graphs showing the results of a BrdU incorporation assay to assess T cell proliferation. Cells were harvested, washed, and then replated in IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. Panels A and B represent replicate experiments. C) Graph showing G-CSFR ECD expression by flow cytometry in primary murine T cells transduced with the indicated chimeric receptor constructs. D) Western blot to assess the indicated cytokine signaling events in cells expressing 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.

[0134] [Figure 48-2] See description of Figure 48-1.

[0135] [Figure 49]Figure 1 shows graphs and images depicting 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 G21 / 12 / 2R-1, or mock-transduced T cells. A, B) Graphs depicting the results of a BrdU incorporation assay to assess T cell proliferation. Cells were harvested, washed, and then replated in IL-2 (300 IU / ml), wild-type G-CSF (100 ng / ml), or no cytokine. Panels A and B represent replicate experiments. C) Graph showing G-CSFR ECD expression by flow cytometry in primary murine T cells transduced with the indicated chimeric receptor constructs. D) Western blot to assess the indicated cytokine signaling events in cells expressing G21 / 2R-1, G12 / 2R-1, and G21 / 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.

[0136] [Figure 50]Graphs showing fold expansion and G-CSFR ECD expression of primary human PBMC-derived T cells expressing G12 / 2R-1 with the 134 ECD versus untransduced cells. A) Graph showing the results of a T cell proliferation assay in which cells were transduced with lentivirus encoding the G12 / 2R-1 134 ECD and grown in IL-2 (300 IU / ml), G-CSF (100 ng / ml), or medium. Viable cells were counted every 4–5 days. Squares represent cells unstimulated with cytokines. Triangles represent cells stimulated with G-CSF. Diamonds represent cells stimulated with IL-2. B) Graph showing the results of a T cell proliferation assay in which cells were transduced as in panel A. On day 19 of expansion, cells were washed and replated with IL-2, G-CSF, or medium alone. Viable cells were counted every 4–5 days. Squares represent cells not stimulated with cytokines. Light gray diamonds represent cells stimulated with 130 G-CSF. Dark gray diamonds represent cells stimulated with IL-2. Light gray inverted triangles represent cells first stimulated with IL-2 and then re-plated on day 19 with medium alone. Dark gray triangles represent cells first stimulated with 130 G-CSF and then re-plated on day 19 with medium alone. C) Graph showing G-CSFR ECD expression measured by flow cytometry on days 4 or 16 of expansion.

[0137] [Figure 51] Graphs depicting the proliferation and immunophenotype of primary human PBMC-derived T cells expressing the G12 / 2R-1 134 ECD versus untransduced cells. A) Graph showing the results of a BrdU incorporation assay to assess T cell proliferation. Cells were harvested, washed, and then replated with IL-2 (300 IU / ml), IL-2 + IL-12 (300 IU / ml and 10 ng / ml, respectively), 130G-CSF (300 ng / ml), or medium alone. B, C) Representative flow cytometry plots and graphs showing the immunophenotype, as assessed by flow cytometry, of cells expressing G12 / 2R-1 with the 134 ECD versus untransduced cells on day 16 of expansion.

[0138] [Figure 52] Figure 1 shows graphs depicting the fold expansion and proliferation of primary human PBMC-derived T cells expressing G12 / 2R-1 with the 304 ECD versus untransduced cells. A) Graph showing the results of a T cell proliferation assay in which cells were transduced with lentivirus encoding the G12 / 2R-1 134 ECD and grown in IL-2 (300 IU / mL), 130 G-CSF (100 ng / ml), 304 G-CSF (100 ng / ml), or media alone. Untransduced cells were cultured in IL-2, 130 G-CSF, 304 G-CSF, or media alone. Viable cells were counted every 3–4 days. Inverted triangles represent cells not stimulated with cytokines. Triangles represent cells stimulated with IL-2. Circles represent cells stimulated with 130 G-CSF. Diamonds represent cells stimulated with 304 G-CSF. B) On day 12, cells were harvested from the proliferation assay, washed, and then replated with IL-2 (300 IU / ml), 130 G-CSF (300 ng / ml), 304 G-CSF (100 ng / ml), 307 G-CSF (100 ng / ml), or medium alone.

[0139] [Figure 53] Western blots are shown 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 proliferation assays and stimulated with 304 G-CSF (100 ng / mL), IL-2 (300 IU / mL), IL-2 and IL-12 (10 ng / mL), or medium alone, as indicated. The black arrows and small protruding panels on the right indicate molecular weight markers at 115 kDa and 140 kDa on the protein ladder. β-Actin and histone H3 served as protein loading controls.

[0140] [Figure 54]A size-exclusion UV trace of refolded 130a1 G-CSF (labeled as GCSF_130a1) and the corresponding SDS PAGE purity gel stained with Coomassie blue are shown. Refolded 130a1 G-CSF eluted in the expected amount compared to standards of known molecular weight.

[0141] [Figure 55] Shown are the results of BrdU incorporation assays to assess proliferation of (A, B, C) OCI-AML1 cells that naturally express wild-type (WT) human G-CSFR or (D, E) 32D clone 3 cells that naturally express WT murine G-CSFR. Cells were stimulated with the following cytokines: (A) WT G-CSF or G-CSF variants 130, 130a1, 130b1, 130a2, or 130b2; (B) WT G-CSF or G-CSF variants 130, 130a1, or 130b1, or medium alone (no cytokines added); (C) WT G-CSF or G-CSF variants 130, 130a1, or 130a1b1, or medium alone; (D) WT G-CSF or G-CSF variants 130, 130a1, or 130b1, or medium alone; and (E) WT G-CSF or G-CSF variants 130, 130a1, or 130a1b1, or medium alone. Filled squares represent WT G-CSF, filled triangles represent 130 G-CSF, filled inverted triangles represent 130a1 G-CSF, open circles represent 130a2 G-CSF, filled diamonds represent 130b1 G-CSF, open squares represent 130bd G-CSF, and open diamonds represent 130a1b1 G-CSF.

[0142] [Figure 56]

[0033] Figure 1 depicts the results of a BrdU incorporation assay to assess proliferation of PBMC-derived human T cells expressing G12 / 2R-1 134 ECD. Cells were stimulated with (A) medium alone, medium and IL-2, IL-2 plus IL-12, or G-CSF variants 130, 130a1, 130b1, 130a2, or 130b2, or (B) medium alone, medium and IL-2, IL-2 plus IL-12, or G-CSF variants 130 or 130a1b1. Results are shown for T cells expressing G12 / 2R-1 134 ECD (i.e., T cells that were G-CSFR+ by flow cytometry).

[0143] [Figure 57] These figures show the results of an in vivo experiment to determine the safety and efficacy of adoptive cell therapy using tumor-specific CD8+ T cells (Thy1.1+OT-I T cells) retrovirally transduced to express the G2R-3 134 ECD and then injected into syngeneic, immunocompetent mice bearing established mammary tumors (NOP23 tumor line). Starting on the day of T cell infusion (day 0), mice were randomized and treated with vehicle (n = 4 animals) or 130a1 G-CSF (10 μg / dose; n = 4 animals) daily for 14 days, followed by every other day for 14 days, for a total of 21 doses. (A) Tumor area (length × width) from day 2 to day 80. Each line represents the results from an individual animal. (B) High-resolution view of tumor area from day 2 to day 20. (C) Kaplan-Meier plot showing the percentage of animals surviving from day 0 to day 80. (D) Proliferation of OT-I T cells in peripheral blood, shown as the mean percentage of Thy1.1+ (OT-I) cells to all CD8+ T cells (mean + / - SEM). (E) Percentage of neutrophils to all CD45+ cells in peripheral blood (mean + / - SEM). (F) Percentage of eosinophils to all CD45+ cells in peripheral blood (mean + / - SEM). (G) Percentage of monocytes to all CD45+ cells in peripheral blood (mean + / - SEM). Gray circles represent vehicle-treated animals, and black triangles represent animals treated with 130a1 G-CSF.

[0144] [Figure 58] These figures show the results of an in vivo experiment to determine the safety and efficacy of adoptive cell therapy using tumor-specific CD8+ T cells (Thy1.1+OT-I T cells) retrovirally transduced to express the G12 / 2R-1 134 ECD and then injected into syngeneic, immunocompetent mice bearing established mammary tumors (NOP23 tumor line). Starting on the day of T cell infusion (day 0), mice were randomized and treated with vehicle (n = 4 animals) or 130a1 G-CSF (10 μg / dose; n = 3 animals) daily for 14 days, followed by every other day for 14 days, for a total of 21 doses. (A) Tumor area (length × width) from day 2 to day 80. Each line represents the results from an individual animal. (B) High-resolution view of tumor area from day 2 to day 20. (C) Kaplan-Meier plot showing the percentage of animals surviving from day 0 to day 80. (D) Proliferation of OT-I T cells in peripheral blood, shown as the mean percentage of Thy1.1+ (OT-I) cells to all CD8+ T cells (mean + / - SEM). (E) Percentage of neutrophils to all CD45+ cells in peripheral blood (mean + / - SEM). (F) Percentage of eosinophils to all CD45+ cells in peripheral blood (mean + / - SEM). (G) Percentage of monocytes to all CD45+ cells in peripheral blood (mean + / - SEM). Gray circles represent vehicle-treated animals, and black triangles represent animals treated with 130a1 G-CSF.

[0145] [Figure 59]Figures 4 and 5 show results from three control groups from the experiment. Tumor-specific CD8 T cells (Thy1.1+OT-I T cells) underwent a mock retroviral transduction procedure and then injected into syngeneic, immunocompetent mice bearing established mammary tumors (NOP23 tumor line). Starting on the day of T cell infusion (day 0), mice were randomized and treated with vehicle (n = 4 animals), IL-2 (30,000 IU / dose; n = 5 animals), or 130a1 (10 μg / dose; n = 5 animals) daily for 14 days, followed by every other day for 14 days, for a total of 21 doses. (A) Tumor area (length × width) from day 2 to day 80. Each line represents the results from an individual animal. (B) High-resolution view of tumor area from day 2 to day 20. (C) Kaplan-Meier plot showing the percentage of animals surviving from day 0 to day 80. (D) Proliferation of OT-I T cells in peripheral blood, shown as the mean percentage of Thy1.1+ (OT-I) cells to all CD8+ T cells (mean + / - SEM). (E) Percentage of neutrophils to all CD45+ cells in peripheral blood (mean + / - SEM). (F) Percentage of eosinophils to all CD45+ cells in peripheral blood (mean + / - SEM). (G) Percentage of monocytes to all CD45+ cells in peripheral blood (mean + / - SEM). Gray circles represent vehicle-treated animals, gray squares represent IL-2-treated animals, and black triangles represent 130a1 G-CSF-treated animals.

[0146] [Figure 60-1] Figure 60 depicts a schematic of wild-type cytokine receptor subunits and additional chimeric receptor designs.

[0147] [Figure 60-2] See description of Figure 60-1.

[0148] [Figure 61] 1 depicts flow cytometry data showing cell surface expression of G-CSFR 134 ECD on human PBMC-derived CD3+ T cells transduced with G4R 134 ECD. Non-transduced T cells served as a negative control.

[0149] [Figure 62] (A) Graph showing proliferation (fold change in cell number) of human PBMC-derived T cells expressing G4R 134 ECD cultured in medium alone, medium and IL-2, 130a1 G-CSF, or 130a1 G-CSF plus IL-2. (B) Results for untransduced T cells cultured in medium alone, medium and IL-2, or 130a1 G-CSF plus 307 G-CSF. The 307 G-CSF variant was included in this latter condition to serve as a control for another arm of this experiment (not shown). We previously demonstrated, and confirm here, that neither 130a1 nor 307 G-CSF induces proliferation of untransduced T cells. Diamonds represent cells cultured in 130a1 G-CSF (panel A) or 130a1 G-CSF plus 307 G-CSF (panel B). Triangles represent cells cultured with IL-2. Light grey circles represent cells cultured with 130a1 G-CSF and IL-2. Black circles represent cells cultured in medium alone (no cytokines).

[0150] [Figure 63] (B) Graph showing the results of a BrdU incorporation assay to assess proliferation of primary human T cells expressing (A) G4R 134 ECD compared to non-transduced T cells. Cells were stimulated with medium alone, IL-2, IL-4, IL2 + IL-4, 130a1 G-CSF, or 130a1 G-CSF + IL-2. Data are presented separately for CD4+ and CD8+ T cell subsets. Data in panel A are gated on T cells expressing G4R 134 ECD (detected using an antibody against human G-CSFR).

[0151] [Figure 64]Western blots show the detection of indicated biochemical signaling events in human PBMC-derived T cells expressing G4R 134 ECD versus non-transduced cells. Cells were stimulated with IL-2, IL-4, or 130a1 G-CSF. β-actin and histone H3 served as protein loading controls.

[0152] [Figure 65] 1 depicts flow cytometry data showing cell surface expression of G-CSFR 134 ECD on human PBMC-derived CD3+ T cells transduced with lentivirus encoding G6R 134 ECD. Non-transduced T cells served as a negative control.

[0153] [Figure 66] Figure 1 depicts a graph showing proliferation (fold change in cell number) of (A) human PBMC-derived T cells expressing G6R 134 ECD or (B) untransduced T cells cultured in medium alone or medium containing IL-2, 130a1 G-CSF, or 130a1 G-CSF + IL-2. Diamonds represent cells stimulated with 130a1 G-CSF. Triangles represent cells stimulated with IL-2. Light gray circles represent cells stimulated with 130a1 G-CSF + IL-2. Black circles represent cells stimulated with medium alone (no cytokines).

[0154] [Figure 67] (B) Graph showing the results of a BrdU incorporation assay to assess proliferation of PBMC-derived human T cells expressing (A) G6R 134 ECD compared to non-transduced T cells. Cells were stimulated with medium alone, medium and IL-2, IL-6, IL2 + IL-6, 130a1 G-CSF, or 130a1 G-CSF + IL-2. Data are shown separately for CD4+ and CD8+ T cell subsets. Data in panel A were gated on T cells expressing G4R 134 ECD (detected using an antibody against human G-CSFR).

[0155] [Figure 68] Western blots are shown to detect the indicated biochemical signaling events in human PBMC-derived T cells expressing G6R 134 ECD versus untransduced cells. Cells were stimulated with IL-2, IL-6, or 130a1 G-CSF. Histone H3 served as a protein loading control. Note that in the P-STAT3 image, a dark high-molecular-weight band appears under IL-2 stimulation conditions (particularly in untransduced T cells). This is residual signal from previous probing of this membrane with a phospho-STAT5 antibody. The lower-molecular-weight band (arrow) represents phospho-STAT3.

[0156] [Figure 69] 1 depicts flow cytometry data showing cell surface expression of G-CSFR 134 ECD on human PBMC-derived CD3+ T cells transduced with GEPOR 134 ECD. Non-transduced T cells served as a negative control.

[0157] [Figure 70] Figure 1 depicts graphs showing the proliferation (fold change in cell number) of (A) primary human PBMC-derived T cells expressing GEPOR 134 ECD or (B) untransduced T cells cultured in medium alone or medium containing IL-2, 130a1 G-CSF, or 130a1 G-CSF + IL-2. For untransduced T cells, 307 G-CSF was added to the 130a1 G-CSF condition to serve as a control for another arm of this experiment (not shown). We have previously demonstrated, and this is confirmed herein, that neither 130a1 nor 307 G-CSF induces proliferation of untransduced T cells. Diamonds represent cells cultured in 130a1 G-CSF + / - 307 G-CSF. Triangles represent cells cultured in IL-2. Light gray circles represent cells cultured in 130a1 G-CSF + IL-2. The black circles represent cells cultured in medium alone.

[0158] [Figure 71]Western blots show the detection of indicated biochemical signaling events in human PBMC-derived T cells expressing GEPOR 134 ECD versus untransduced cells. Cells were stimulated with medium alone, medium plus IL-2, or 130a1 G-CSF. β-Actin and histone H3 served as protein loading controls.

[0159] [Figure 72] 1 depicts flow cytometry data showing cell surface expression of G-CSFR 134 ECD on human PBMC-derived CD3+ T cells transduced with GIFNAR 134 ECD. Non-transduced T cells served as a negative control.

[0160] [Figure 73] Figure 1 depicts graphs showing proliferation (fold change in cell number) of primary human PBMC-derived T cells that were (A) untransduced or (B) transduced to express GIFNAR 134 ECD and cultured in medium alone or medium containing IL-2, 130a1 G-CSF, or 130a1 G-CSF + IL-2. Diamonds represent cells cultured in 130a1 G-CSF. Triangles represent cells cultured in IL-2. Light gray circles represent cells cultured in 130a1 G-CSF + IL-2. Black circles represent cells cultured in medium alone.

[0161] [Figure 74] Western blots are shown to detect the indicated biochemical signaling events in human PBMC-derived T cells expressing GIFNAR 134 ECD versus untransduced cells. Cells were stimulated with medium alone, medium plus IL-2, IFNα, or 130a1 G-CSF. β-Actin and histone H3 served as protein loading controls.

[0162] [Figure 75]Flow cytometry data showing the expression of Myc and Flag tags on the surface of human PBMC-derived T cells transduced with (A) GIFNGR-1 307 ECD, (B) G2R3 134 ECD, (C) GIFNGR-1 307 ECD and G2R3 134 ECD, or (D) untransduced T cells. G2R3 134 ECD is tagged with a Myc epitope at its N-terminus (EQKLISEEDL), and GIFNGR-1 307 ECD is tagged with a Flag epitope at its N-terminus (DYKDDDDK). These epitope tags aid in flow cytometric detection and do not affect receptor function. Plots were gated on CD3+ cells.

[0163] [Figure 76] Flow cytometry data showing the expression of Myc and Flag tags on the surface of human PBMC-derived T cells transduced with (A) GIFNGR-2 307 ECD, (B) G2R3 134 ECD, (C) GIFNGR-2 307 ECD and G2R3 134 ECD, or (D) untransduced T cells. G2R3 134 ECD is tagged with a Myc epitope at its N-terminus (EQKLISEEDL), and GIFNGR-2 307 ECD is tagged with a Flag epitope at its N-terminus (DYKDDDDK). These epitope tags aid in flow cytometric detection and do not affect receptor function. Plots were gated on CD3+ cells.

[0164] [Figure 77]Figure 1 depicts a graph showing the proliferation (fold change in cell number) of primary human PBMC-derived T cells that were (A) untransduced, (B) transduced to express GIFNGR-1 307 ECD, or (C) cotransduced to express GIFNGR-1 307 ECD and G2R-3 134 ECD. T cells were cultured in medium alone or medium containing the indicated combinations of IL-2, 130a1 G-CSF, and 307 G-CSF. Diamonds and light gray circles represent cells cultured with the indicated combinations of 130a1 G-CSF, 307 G-CSF, and IL-2. Triangles represent cells cultured in IL-2. Black circles represent cells cultured in medium alone.

[0165] [Figure 78] Figure 1 depicts graphs showing proliferation (fold change in cell number) of primary human PBMC-derived T cells that were (A) untransduced, (B) transduced to express GIFNGR-2 307 ECD, or (C) cotransduced to express GIFNGR-2 307 ECD and G2R-3 134 ECD. T cells were cultured in medium alone or medium containing the indicated combinations of IL-2, 130a1 G-CSF, and 307 G-CSF. Diamonds and light gray circles represent cells cultured with the indicated combinations of 130a1 G-CSF, 307 G-CSF, and IL-2. Triangles represent cells cultured in IL-2. Black circles represent cells cultured in medium alone.

[0166] [Figure 79]

[0033] Figure 1 depicts graphs showing the results of a BrdU incorporation assay to assess proliferation of human PBMC-derived T cells expressing G2R-3 134 ECD and / or GIFNGR-1 307 ECD. Data are shown for (A) untransduced T cells or (B) CD4+ and (C) CD8+ T cells expressing the indicated chimeric receptors (see X-axis). Cells were stimulated with the indicated combinations of IL-2, IFNg, 130a1 G-CSF, 307 G-CSF, or medium alone.

[0167] [Figure 80]

[0033] Figure 1 depicts graphs showing the results of a BrdU incorporation assay to assess proliferation of human PBMC-derived T cells expressing G2R-3 134 ECD and / or GIFNGR-2 307 ECD. Data are shown for (A) untransduced T cells or (B) CD4+ and (C) CD8+ T cells expressing the indicated chimeric receptors (see X-axis). Cells were stimulated with the indicated combinations of IL-2, IFNg, 130a1 G-CSF, 307 G-CSF, or medium alone.

[0168] [Figure 81] Figure 1 shows Western blots to detect the indicated biochemical signaling events in human PBMC-derived T cells expressing GIFNGR-1 307 ECD or GIFNGR-2 307 ECD versus untransduced cells. Cells were stimulated with medium alone, medium and IL-2, IFNγ, or G-CSF. β-actin and histone H3 served as protein loading controls.

[0169] [Figure 82] Figure 1 shows flow cytometry data showing cell surface expression of G2R-3 134 ECD and mesothelin-specific CAR on human PBMC-derived CD3+ T cells transduced with the following monocistronic or bicistronic lentiviral constructs: (A) untransduced T cells (negative control), (B) mesothelin CAR only, (C) G2R-3 134 ECD only, (D) CAR_T2A_G2R-3-134 ECD (with the gene fragments mesothelin CAR, T2A site, and G2R-3 134 ECD in this order), and (E) G2R-3-134 ECD_T2A_CAR (with the gene fragments mesothelin CAR, T2A site, and G2R-3 134 ECD in this order).

[0170] [Figure 83]Figure 1 shows flow cytometry data showing cell surface expression of G12 / 2R-1 134 ECD and mesothelin-specific CAR on human PBMC-derived CD3+ T cells transduced with the following monocistronic or bicistronic lentiviral constructs: (A) untransduced T cells (negative control), (B) mesothelin CAR only, (C) G2R-3 134 ECD only, (D) CAR_T2A_G12 / 2R-1-134 ECD (with the gene fragments mesothelin CAR, T2A site, and G12 / 2R-1 134 ECD in this order), and (E) G12 / 2R-1-134 ECD_T2A_CAR (with the gene fragments mesothelin CAR, T2A site, and G12 / 2R-1 134 ECD in this order).

[0171] [Figure 84]

[0039] Figure 1 depicts the results of a BrdU incorporation assay to assess proliferation of PBMC-derived human T cells modified as follows: (A) untransduced or expressing only the mesothelin CAR, or (B) expressing the G2R3 134 ECD, or the bicistronic CAR_T2A_G2R3-134 ECD, or the bicistronic CAR_T2A_G12 / 2R-1-134 ECD, or the bicistronic G12 / 2R-1-134ECD_T2A_CAR construct. Cells were stimulated with medium alone, or medium and IL-2 or 130a1 G-CSF. Results in panel B are shown for T cells positive for G-CSFR ECD expression by flow cytometry.

[0172] [Figure 85-1]Figure 85 shows the results of an in vitro coculture assay to evaluate the functional properties of the mesothelin CAR in PBMC-derived human CD4+ T cells expressing: (A) mesothelin CAR alone, (B) G12 / 2R-1 134 ECD alone, or the following bicistronic constructs containing the mesothelin CAR: (C) CAR_T2A_G2R3-134 ECD, (D) G2R3-134 ECD_T2A_CAR, (E) CAR_T2A_G12 / 2R-1-134 ECD, or (F) G12 / 2R-1-134ECD_T2A_CAR. Cells were left unstimulated or stimulated with OVCAR3 cells for 13 hours, after which intracellular flow cytometry detected the expression of cytokines IFNγ, TNFα, and IL-2, and surface molecules CD69 and CD137. Results in panels A, C, D, E, and F are gated on cells expressing the mesothelin CAR.

[0173] [Figure 85-2] See description of Figure 85-1.

[0174] [Figure 86] Schematic diagram of natural cytokine receptors including IL-7Rα, IL-2Rβ, gp130, IL-21R, IL-12Rβ2 and IL-4Rα.

[0175] [Figure 87-1] Figure 87 is a schematic diagram of the 7 / 2R-1, 7 / 2R-2, 7 / 2 / 12R-1, 7 / 2 / 12R-2, 7 / 21R-1, 7 / 21R-2, 7 / 7 / 21R-1, 7 / 7 / 21R-2, 7 / 2 / 21R-1, 7 / 2 / 21R-2, 7 / 2 / 12 / 21R-1, 7 / 2 / 12 / 21R-2, 7 / 2 / 12 / 21R-3, 7 / 2 / 12 / 21R-4, 7 / 4R-1, 7 / 4R-2, 7 / 6R-1, and 7 / 6R-2 receptor subunit designs.

[0176] [Figure 87-2] See description of Figure 87-1.

[0177] [Figure 88] Graphs depicting human CD127 (IL-7Rα) and Flag-tag expression assessed by flow cytometry in (A) primary human PBMC-derived T cells transduced with 7 / 2R-1, (B) untransduced T cells, or (C) T cells transduced with 7 / 2R-1 but stained as a fluorescence minus one (FMO) control (i.e., antibodies against CD127 were omitted from the antibody staining panel). 7 / 2R-1 is tagged with a Flag epitope at its N-terminus; this epitope tag aids detection by flow cytometry and does not affect receptor function. Receptor expression was analyzed 12 days after lentiviral transduction.

[0178] [Figure 89] (B) Graph showing proliferation (fold change in cell number) of primary human PBMC-derived T cells expressing (A) 7 / 2R-1 and cultured with IL-7, IL-7 + IL-15, or no cytokines compared to non-transduced T cells. Diamonds represent cells stimulated with IL-7. Triangles represent cells stimulated with IL-7 and IL-15. Circles represent cells cultured in medium alone (no cytokines).

[0179] [Figure 90] (B) Graph showing the results of a BrdU incorporation assay to assess proliferation of primary human T cells expressing (A) 7 / 2R-1 compared to non-transduced T cells. Cells were stimulated with IL-7, IL-2, IL-2 + IL-7, or medium alone (no cytokines). Results are shown separately for CD4+ and CD8+ T cell subsets.

[0180] [Figure 91] Western blots are shown to detect the indicated cytokine signaling events in human primary T cells expressing 7 / 2R-1 versus non-transduced T cells. β-Actin and Histone H3 serve as protein loading controls. Cells were stimulated with IL-7, IL-2, IL-2 + IL-7, or medium alone (no cytokines).

[0181] [Figure 92] 1 shows an SDS-PAGE gel for detecting unmodified 130a1 G-CSF and the PEGylated forms of 130a1 G-CSF (PEG20k_G-CSF_130a1) and 307 G-CSF (PEG20k_G-CSF_307). The gel was stained with Coomassie brilliant blue.

[0182] [Figure 93] These figures show the results of an in vivo experiment to determine the safety and efficacy of adoptive cell therapy using tumor-specific CD8 T cells (Thy1.1+ OT-I T cells) retrovirally transduced to express G4R 134 ECD and then injected into syngeneic, immunocompetent mice bearing established mammary tumors (NOP23 tumor line). Starting on the day of T cell infusion (day 0), mice were randomized and treated with vehicle (n = 3 animals) or 130a1 G-CSF (10 μg / dose; n = 3 animals) daily for 14 days, followed by every other day for 14 days, for a total of 21 doses. (A) Tumor area (length × width) from day 0 to day 46. Each line represents results from an individual animal. (B) Proliferation of OT-I T cells in peripheral blood, shown as the mean percentage of Thy1.1+ (OT-I) cells relative to all CD8+ T cells (mean + / - standard deviation [SD]). (C) Percentage of OT-I T cells (Thy1.1+) exhibiting a T effector memory (Tem; CD44+CD62L-) phenotype (mean + / - SD). (D) Percentage of host (Thy1.1-) CD8+ T cells exhibiting a Tem phenotype (mean + / - SD). (E) Percentage of OT-I T cells (Thy1.1+) expressing programmed death-1 (PD-1) (mean + / - SD). (F) Percentage of host (Thy1.1-) CD8+ T cells expressing PD-1 (mean + / - SD). (G) Percentage of host (Thy1.1-) CD3+ T cells relative to total CD45+ cells in peripheral blood (mean + / - SD). (H) Percentage of host (Thy1.1-) CD19+ B cells relative to total CD45+ cells in peripheral blood (mean + / - SD). Grey circles represent vehicle-treated animals, black triangles represent 130a1 G-CSF-treated animals.

[0183] [Figure 94] These figures show the results of an in vitro experiment comparing PEGylated and non-PEGylated versions of G-CSF (wild-type, 130a1, and 307) for their ability to stimulate proliferation of cells expressing G2R-3 134 ECD or the native G-CSF receptor. Data are presented as the mean and standard deviation of duplicate wells. (A) Human PBMC-derived CD4 and CD8 T cells expressing G2R-3 134 ECD were stimulated with the indicated cytokines: human IL-2 (Proleukin, 300 IU / ml, positive control), medium alone (negative control), or the indicated concentrations of wild-type G-CSF, PEGylated wild-type G-CSF, 130a1 G-CSF, or PEGylated 130a1 G-CSF. Cells were cultured for 48 hours and assessed by BrdU incorporation assay. Results are shown for T cells that were positive for human G-CSFR. (B) A similar experiment was performed using the human myeloid cell line OCI-AML-1, which naturally expresses the wild-type human G-CSF receptor. Cells were stimulated with the indicated cytokines: human GM-CSF (20 ng / ml, positive control), medium alone (negative control), or the indicated concentrations of wild-type G-CSF, PEGylated wild-type G-CSF, 130a1 G-CSF, PEGylated 130a1 G-CSF, 307 G-CSF, or PEGylated 307 G-CSF. Cells were cultured for 48 hours and assessed by BrdU incorporation assay.

[0184] [Figure 95]

[0033] Figure 1 shows the results of an in vitro Western blot experiment comparing the ability of PEGylated versus non-PEGylated versions of 130a1 G-CSF to induce the indicated biochemical signaling events in human PBMC-derived T cells expressing the G2R-3 134 ECD. T cells were stimulated for 20 minutes with human IL-2 (Proleukin, 300 IU / ml) or the indicated concentrations (ng / ml) of the non-PEGylated or PEGylated (PEG) versions of 130a1 G-CSF. Histone H3 and total S6 served as gel loading controls.

[0185] [Figure 96] These figures show the results of an in vivo experiment comparing the efficacy of PEGylated (PEG) versus non-PEGylated versions of 130a1 G-CSF given daily, every 3 days, or weekly. Tumor-specific CD8+ T cells (Thy1.1+OT-I T cells) were retrovirally transduced to express the G2R-3 134 ECD and then injected into syngeneic, immunocompetent mice bearing established mammary tumors (NOP23 tumor line). Tumor size (length x width) is plotted over 46 days. Each line represents results from an individual animal. The right panel is an expanded version of the left panel. Starting on the day of T cell infusion (day 0), mice were randomized and treated with vehicle, 130a1 G-CSF, or PEG-130a1 G-CSF as indicated. (A, B) "Daily" treatment cohorts received the indicated cytokine (or vehicle) daily for 14 days, followed by every other day for 14 days, for a total of 21 doses. (C, D) The "every 3 day" dosing cohort received a total of 9 doses of the indicated cytokine every 3 days. (E, F) The "weekly" dosing cohort received a total of 4 doses of the indicated cytokine every 7 days.

[0186] [Figure 97] Figure 96 shows the proliferation and effector memory (Tem) phenotype of OT-I T cells in peripheral blood at the indicated time points from the experiment shown in Figure 96. The left panel shows the percentage of Thy1.1+ (OT-I) T cells relative to total CD8+ T cells (mean + / - SD). The right panel shows the percentage of Thy1.1+ (OT-I) T cells with the Tem (CD44+CD62L-) phenotype ((mean + / - SD)). (A, D) "Daily" dosing cohort. (B, E) "Every 3 days" dosing cohort. (C, F) "Weekly" dosing cohort.

[0187] [Figure 98]Figures 102 and 103 show the percentages of the indicated immune cell subsets (relative to total CD45+ cells) in peripheral blood at the indicated time points from the "every 3 day" cohorts from the experiments shown in Figures 102 and 103. Means and standard deviations are plotted. (A) Neutrophils (CD3-, CD19-, NK1.1-, CD11b+, CD11c-, Ly6G+). (B) Monocytes (CD3-, CD19-, NK1.1-, CD11b+, CD11c-, Ly6G-, SSC-low (Ly6C+ or Ly6C-)). (C) Eosinophils (CD3-, CD19-, NK1.1-, CD11b+, CD11c-, Ly6G-, SSC-high). (D) CD3+ T cells. (E) CD19+ B cells. (F) NK1.1+ natural killer cells.

[0188] [Figure 99] In vitro data from an IL-18-regulated paracrine signaling (CPS) experiment are shown. Human PBMC-derived T cells were transduced with either a bicistronic vector encoding a mesothelin-specific CAR, a T2A site, and a G12 / 2R-1 134 ECD (Meso CAR_G12 / 2R-1) or a tricistronic vector encoding a mesothelin-specific CAR, a T2A site, a G12 / 2R-1 134 ECD, and human IL-18 (Meso CAR_G12 / 2R-1+h18). (A) T cells were assessed by flow cytometry for expression of the mesothelin CAR (X-axis) and G12 / 2R-1 134 ECD (Y-axis). (B) In vitro BrdU assay was used to assess T cell proliferation in response to medium alone, human IL-2 (Proleukin, 300 IU / ml), human IL-18 (100 ng / ml), human IL-2 + human IL-12 (20 ng / ml), IL-2 + IL-12 + IL-18, or 130a1 G-CSF (100 ng / ml). (C) ELISA was used to assess human IL-18 levels in culture supernatants from T cells stimulated for 48 hours with medium alone, human IL-2 (Proleukin, 300 IU / ml) + human IL-12 (20 ng / ml), or 130a1 G-CSF (100 ng / ml).

[0189] [Figure 100] Results of in vitro IL-18 CPS experiments using mouse T cells are shown. Mouse CD4 and CD8 T cells were transduced with either a monocistronic vector encoding the G12 / 2R-1 134 ECD or a bicistronic vector encoding the G12 / 2R 134 ECD, a T2A site, and mouse IL-18 (G12 / 2R-1 134 ECD + m18). (A) T cells were assessed for expression of the G12 / 2R-1 134 ECD by flow cytometry. (B) An in vitro BrdU assay was used to assess T cell proliferation in response to medium alone, human IL-2 (Proleukin, 300 IU / ml), mouse IL-18 (100 ng / ml), human IL-2 + mouse IL-12 (10 ng / ml), IL-2 + IL-12 + IL-18, or 130a1 G-CSF (100 ng / ml). (C) ELISA was used to assess the levels of mouse IL-18 in culture supernatants from T cells stimulated for 48 h with medium alone, human IL-2 (Proleukin, 300 IU / ml) plus mouse IL-12 (10 ng / ml), or 130a1 G-CSF (100 ng / ml).

[0190] [Figure 101]This figure shows the results of an in vitro IL-18 CPS experiment using mouse OT-I T cells. OT-I T cells were transduced with retroviral vectors encoding G2R-2 134 ECD, G2R-3 134 ECD, G12 / 2R-1 134 ECD, or G12 / 2R-1 134 ECD + m18 (abbreviated as G12 / 2R / 18C in the figures). To detect secreted cytokines, T cells were washed 5 days later and cultured for 48 hours in medium alone (negative control) or medium containing human IL-2 (Proleukin 300 IU / ml), human IL-2 + mouse IL-12 (10 ng / ml), or PEGylated (Peg)130a1 (100 ng / ml). A multiplex assay was then performed to detect 32 cytokines. (A) Mouse IL-18 and (B) mouse IFN-gamma are shown. The results for other cytokines are shown in Table 35.

[0191] [Figure 102]These figures show the results of an in vivo experiment comparing the characteristics of G7R-1, G2R-2, G12 / 2R-1, and G12 / 2R-1+m18 (abbreviated as G12 / 2R / 18C), all containing the 134 ECD, in OT-I T cells from a NOP23 mammary tumor model. Tumor-specific CD8+ T cells (Thy1.1+ OT-I T cells) were retrovirally transduced to express G7R-1, G2R-2, G12 / 2R-1, or G12 / 2R-1+m18 (all containing the 134 ECD) and subsequently injected into syngeneic, immunocompetent mice bearing established NOP23 mammary tumors. Starting on the day of T cell infusion (day 0), mice were randomized to receive either vehicle or PEGylated (PEG) 130a1 G-CSF (10 μg / dose) once weekly for a total of four treatments. (A-D) Flow cytometry results showing receptor (G-CSFR) expression on transduced OT-I T cells on the day of T cell infusion (day 0). (E-H) Tumor size (length x width) over 50 days. Each line represents an individual animal. (I-L) Expansion and persistence of Thy1.1+ OT-I cells in serial blood samples. (M-P) Percentage of Thy1.1+ OT-I T cells exhibiting a T effector memory (Tem) phenotype (CD44+CD62L-) in the blood.

[0192] [Figure 103]Figure 1 shows the results of an in vivo experiment in the NOP23 mammary tumor model comparing various doses of PEGylated (PEG) 130a1 G-CSF or wild-type IL-2. Thy1.1+OT-I T cells expressing G2R-3 134 ECD were injected into syngeneic, immunocompetent mice bearing established tumors. Starting on the day of T cell injection (day 0), mice were randomized into six cytokine treatment groups: vehicle only (shown in all panels); PEG-130a1 G-CSF 2 μg / dose, four doses per week (panels A, F, K, P); (3) PEG-130a1 G-CSF 0.4 μg / dose, four doses per week (panels B, G, L, Q); (4) PEG-130a1 G-CSF 0.08 μg / dose, four doses per week (panels C, H, M, R); (5) PEG-130a1 G-CSF (6) human IL-2 (Proleukin) 30,000 IU / day for 14 days followed by 30,000 IU every 2 days for 14 days (Panels E, J, O, T). Serial blood samples were analyzed by flow cytometry for the following parameters: (A-E) Percentage of Thy1.1+ OT-I cells relative to total CD8+ T cells (mean + / - SD). (F-J) Percentage of Thy1.1+ OT-I cells with a T effector memory (Tem) phenotype (CD44+CD62L-) (mean + / - SD). (K-O) Percentage of neutrophils (CD3-, CD19-, NK1.1-, CD11b+, CD11c-, Ly6G+) relative to total CD45+ cells (mean + / - SD). (P-T) Percentage of eosinophils (CD3-, CD19-, NK1.1-, CD11b+, CD11c-, Ly6G-, SSC-high) relative to all CD45+ cells (mean + / - SD). DETAILED DESCRIPTION OF THE INVENTION

[0193] Detailed Description Briefly, as described in more detail below, described herein are methods and compositions for selective activation of cells using a variant cytokine receptor and cytokine pair, wherein the cytokine receptor comprises a variant extracellular domain (ECD) of granulocyte-colony stimulating factor receptor (G-CSFR). In certain embodiments, the methods and compositions described herein are useful for exclusive activation of cells for adoptive cell transfer (ACT) therapy. Accordingly, included herein are methods for producing cells expressing a variant receptor that is selectively activated by a cytokine that does not bind to its native receptor. Also disclosed herein are methods of treating a subject in need thereof, comprising administering to the subject cells expressing a receptor comprising a variant ECD of G-CSFR and co-administering a variant of G-CSF that binds to the variant ECD of G-CSFR. In certain aspects, the compositions and methods described herein address the unmet need for selective activation of cells for adoptive cell transfer procedures, and may reduce or eliminate the need to immunodeplete the subject prior to adoptive cell transfer or to administer broad-acting stimulatory cytokines such as IL-2.

[0194] definition Terms used in the claims and specification, unless otherwise specified, are defined as set forth below.

[0195] The term "treatment" means any therapeutically beneficial result in the treatment of a disease state, for example, a cancer disease state, such as reducing its severity or progression, remission, or cure.

[0196] The term "in vivo" refers to a process that occurs within a living organism.

[0197] As used herein, the term "mammal" includes both humans and non-humans, including, but not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0198] The term "sufficient amount" means an amount sufficient to produce a desired effect, for example, an amount sufficient to selectively activate a receptor expressed on a cell.

[0199] The term "therapeutically effective amount" is an amount effective to ameliorate symptoms of the disease.

[0200] 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.

[0201] As used herein, the term "extracellular domain" (ECD) refers to the domain of a receptor (e.g., G-CSFR) that is external to the plasma membrane when expressed on the surface of a cell. In certain embodiments, the ECD of G-CSFR comprises at least a portion of SEQ ID NO:2 or SEQ ID NO:7.

[0202] As used herein, the term "intracellular domain" (ICD) refers to the domain of a receptor that is located intracellularly when the receptor is expressed on the cell surface.

[0203] 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.

[0204] The term "cytokine" refers to a small protein (approximately 5-20 kDa) that can bind to a cytokine receptor and induce cell signaling upon 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.

[0205] The term "cytokine receptor" refers to a receptor that binds to a cytokine, including cytokine receptors type 1 and type 2. Cytokine receptors include, but are not limited to, G-CSFR, IL-2R (interleukin-2 receptor), IL-7R (interleukin-7 receptor), IL-12R (interleukin-12 receptor), and IL-21R (interleukin-21 receptor).

[0206] As used herein, the term "chimeric receptor" refers to a transmembrane receptor that has been engineered to have at least a portion of at least one domain (e.g., the ECD, ICD, TMD, or C-terminal region) derived from the sequence of one or more different transmembrane proteins or receptors.

[0207] 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 between G-CSF and G-CSFR, located at the interface between the cytokine receptor homolog (CRH) domains of G-CSF and G-CSFR.

[0208] 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 interface between G-CSF and G-CSFR, located at the interface between the N-terminal Ig-binding domains of G-CSF and G-CSFR.

[0209] As used herein, the term "at least a portion" or "portion" refers, in certain embodiments, to greater than 75%, 80%, 90%, 95%, or 99% of the length of consecutive nucleic acid bases or amino acids of a SEQ ID NO: described herein. In certain embodiments, at least a portion of a domain or binding site described herein (e.g., an ECD, ICD, transmembrane, C-terminal region, or signaling molecule binding site) can be greater than 75%, 80%, 90%, 95%, or 99% identical to a SEQ ID NO: described herein.

[0210] The term "wild-type" refers to the naturally occurring amino acid sequence of a polypeptide or the native nucleic acid sequence of a gene encoding a polypeptide described herein. The wild-type sequence of a protein or gene is the most common sequence of the polypeptide or gene in that species of protein or gene.

[0211] The terms "variant cytokine-receptor pair," "variant cytokine and receptor pair," "variant cytokine and receptor design(s)," "variant cytokine-receptor switch," or "orthogonal cytokine-receptor pair" refer to a pair of engineered proteins that (a) do not bind to the native cytokine or cognate receptor, and (b) have been modified by changing amino acids so that they specifically bind to a corresponding engineered (variant) ligand or receptor.

[0212] As used herein, the term "variant receptor" or "orthogonal receptor" refers to a genetically engineered receptor of a variant cytokine-receptor pair, including chimeric receptors.

[0213] As used herein, the term "variant ECD" refers to an engineered extracellular domain of a receptor (eg, G-CSFR) of a variant cytokine-receptor pair.

[0214] 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.

[0215] As used herein, "does not bind" or "cannot bind" refers to no detectable binding or little binding, i.e., having a binding affinity much lower than that of the natural ligand.

[0216] As used herein, the term "selectively activate" or "selective activation," when referring to a cytokine and a variant receptor, refers to the cytokine preferentially binding to the variant receptor, and the receptor being activated by the cytokine binding to the variant receptor. In certain aspects, the cytokine selectively activates a chimeric receptor that has co-evolved to specifically bind to the cytokine. In certain aspects, the cytokine is a wild-type cytokine and selectively activates a chimeric receptor expressed on a cell, but a native wild-type receptor for the cytokine is not expressed in the cell.

[0217] As used herein, the term "enhanced activity" refers to an increase in the activity of a variant receptor expressed on a cell upon stimulation with a variant cytokine, which activity is the activity observed for the native receptor upon stimulation with the native cytokine.

[0218] The term "antigen-binding signaling receptor" refers to any cell surface protein or protein complex that can bind to an antigen and generate an intracellular signal upon antigen binding.

[0219] As used herein, the term "agonist signaling protein" refers to a protein that binds to a target binding molecule (e.g., a protein receptor or antigen), such that the binding induces one or more signaling events in a target cell bearing the target binding molecule. Conversely, the term "antagonist signaling protein" refers to a protein that binds to a target binding molecule (e.g., a protein receptor or antigen), such that the binding inhibits one or more signaling events in a target cell bearing the target binding molecule (e.g., by interfering with other agonist proteins binding to the same target molecule).

[0220] As used herein, the term "affinity reagent" refers to any molecule (eg, protein, nucleic acid, etc.) that has the ability to bind to any target molecule.

[0221] The term "immune cell" refers to any cell known to function to support an organism's immune system (including innate and adaptive immune responses), including, but not limited to, lymphocytes (e.g., B cells, plasma cells, and T cells), natural killer cells (NK cells), macrophages, monocytes, dendritic cells, neutrophils, and granulocytes. Immune cells include stem cells, immature immune cells, and differentiated cells. Immune cells also include any cell subpopulation, whether rare or abundant in the body. In certain embodiments, immune cells are identified as such by possessing known markers (e.g., cell surface markers) of immune cell types and subpopulations.

[0222] The term "T cell" refers to a mammalian immune effector cell that may be characterized by expression of CD3 and / or a T cell antigen receptor, and these cells may be engineered to express orthologous cytokine receptors. In some embodiments, T cells are naive CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, helper T cells, e.g., T H 2. T H 9. T H 11. T H 22, T FH ;regulatory T cells, e.g. R 1, Natural T Reg , inducible T Reg memory T cells, e.g., selected from central memory T cells, effector memory T cells, NKT cells, and γδT cells;

[0223] The term "G-CSFR" refers to granulocyte colony-stimulating factor receptor. G-CSFR may also be referred to as GCSFR, G-CSF receptor, colony-stimulating factor 3 receptor, CSF3R, CD114 antigen, or SCN7. Human G-CSFR is encoded by a gene having Ensembl identification number: ENSG00000119535. Human G-CSFR is encoded by a cDNA sequence corresponding to GeneBank accession number NM_156039.3.

[0224] The term "G-CSF" refers to granulocyte colony-stimulating factor. G-CSF may also be called colony-stimulating factor 3 and CSF3. Human G-CSF is encoded by the gene having Ensembl identification number: ENSG00000108342. Human G-CSF is encoded by the cDNA sequence corresponding to GeneBank accession number KP271008.1.

[0225] "JAK" may also be referred to as Janus kinase. JAK is a family of intracellular non-receptor tyrosine kinases that transmit cytokine-mediated signals via the JAK-STAT pathway, and includes JAK1, JAK2, JAK3, and TYK2. Human JAK1 is encoded by the gene having Ensembl identification number: ENSG00000162434. Human JAK1 is encoded by the cDNA sequence corresponding to GeneBank accession number NM_002227. Human JAK2 is encoded by the gene having Ensembl identification number: ENSG00000096968. Human JAK2 is encoded by the cDNA sequence corresponding to GeneBank accession number NM_001322194. Human JAK3 is encoded by the gene having Ensembl identification number: ENSG00000105639. Human JAK3 is encoded by the cDNA sequence corresponding to GeneBank accession number NM_000215. Human TYK2 is encoded by the gene having Ensembl identification number: ENSG00000105397. Human TYK2 is encoded by the cDNA sequence corresponding to GeneBank accession number NM_001385197.

[0226] STATs may also be referred to as signal transducers and activators of transcription. STATs are a family of seven STAT proteins: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. Human STAT1 is encoded by the gene having Ensembl identification number: ENSG00000115415. Human STAT1 is encoded by the cDNA sequence corresponding to GeneBank accession number: NM_007315. Human STAT2 is encoded by the gene having Ensembl identification number: ENSG00000170581. Human STAT2 is encoded by the cDNA sequence corresponding to GeneBank accession number: NM_005419. Human STAT3 is encoded by the gene having Ensembl identification number: ENSG00000168610. Human STAT3 is encoded by the cDNA sequence corresponding to GeneBank accession number: NM_139276. Human STAT4 is encoded by the gene having Ensembl identification number: ENSG00000138378. Human STAT4 is encoded by the cDNA sequence corresponding to GeneBank accession number: NM_003151. Human STAT5A is encoded by the gene having Ensembl identification number: ENSG00000126561. Human STAT5A is encoded by the cDNA sequence corresponding to GeneBank accession number: NM_003152. Human STAT5B is encoded by the gene having Ensembl identification number: ENSG00000173757. Human STAT5B is encoded by the cDNA sequence corresponding to GeneBank accession number: NM_012448. Human STAT6 is encoded by the gene having Ensembl identification number: ENSG00000166888. Human STAT6 is encoded by a cDNA sequence corresponding to GeneBank accession number: NM_003153.

[0227] SHC may also be referred to as Src homology 2 domain-containing transforming protein. Shc is a family of three isoforms, including p66Shc, p52Shc, and p46Shc, SHC1, SHC2, and SHC3. Human SHC1 is encoded by the gene having Ensembl identification number: ENSG00000160691. Human SHC1 is encoded by the cDNA sequence corresponding to GeneBank accession number: NM_183001. Human SHC2 is encoded by the gene having Ensembl identification number: ENSG00000129946. Human SHC2 is encoded by the cDNA sequence corresponding to GeneBank accession number: NM_012435. Human SHC3 is encoded by the gene having Ensembl identification number: ENSG00000148082. Human SHC3 is encoded by a cDNA sequence corresponding to GeneBank accession number: NM_016848.

[0228] SHP-2 may also be referred to as non-receptor protein tyrosine phosphatase 11 (PTPN11) and protein tyrosine phosphatase 1D (PTP-1D). Human SHP-2 is encoded by a gene having Ensembl identification number: ENSG00000179295. Human SHP-2 is encoded by a cDNA sequence corresponding to GeneBank accession number: NM_001330437.

[0229] PI3K may also be referred to as phosphatidylinositol-4,5-bisphosphate 3-kinase. The catalytic subunit of PI3K may be referred to as PIK3CA. Human PIK3CA is encoded by a gene having Ensembl identification number: ENSG00000121879. Human PIK3CA is encoded by a cDNA sequence corresponding to GeneBank accession number: NM_006218.

[0230] EPOR may also be referred to as erythropoietin receptor. Human EPOR is encoded by a gene having Ensembl identification number: ENSG00000187266. Human EPOR is encoded by a cDNA sequence corresponding to GeneBank accession number: NM_000121.

[0231] IFNγR1 may also be referred to as interferon gamma receptor 1. Human IFNγR1 is encoded by a gene having Ensembl identification number: ENSG00000027697. Human IFNγR1 is encoded by a cDNA sequence corresponding to GeneBank accession number: NM_000416.

[0232] IFNγR2 may also be referred to as interferon gamma receptor 2. Human IFNγR2 is encoded by a gene having Ensembl identification number: ENSG00000159128. Human IFNγR2 is encoded by a cDNA sequence corresponding to GeneBank accession number: NM_001329128.

[0233] IFNAR2 may also be referred to as interferon alpha and beta receptor subunit 2. Human IFNAR2 is encoded by the gene having Ensembl identification number: ENSG00000159110. Human IFNAR2 is encoded by a cDNA sequence corresponding to GeneBank accession number: NM_000874.

[0234] Abbreviations used in this application include the following: ECD (extracellular domain), ICD (intracellular domain), TMD (transmembrane domain), G-CSFR (granulocyte colony-stimulating factor receptor), G-CSF (granulocyte colony-stimulating factor), IL (interleukin), IL-2R (interleukin-2 receptor), IL-12R (interleukin-12 receptor), IL-21R (interleukin-21 receptor) and IL-7R or IL-7Rα (interleukin-7 receptor), IL-18 (interleukin-18), IL-21 (interleukin-21), IL-17 (interleukin-17), TNF-α (tumor necrosis factor alpha), CXCL13 (C-X-C motif chemokine ligand 13), CCL3 (C-C motif chemokine ligand 3 or MIP-1α), CCL4 (C-C motif chemokine ligand 4) or MIP-1β), CD40 ligand, B-cell activating factor (BAFF), Flt3 ligand, CCL21 (C-C motif chemokine ligand 21), CCL5 (C-C motif chemokine ligand 5), XCL1 (XC motif chemokine ligand 1), CCL19 (C-C motif chemokine ligand 19), and NKG2D (killer cell lectin-like receptor K1). IL-2R may also be referred to herein as IL-2RG, IL-2Rgc, γc, or IL-2Rγ. For selected chimeric cytokine receptor designs: "G-CSFRwt-ICDIL-2Rb" is also referred to herein as "G / IL-2Rb"; "G-CSFRwt-ICDgc" is also referred to herein as "G / gc"; "G-CSFR137-ICDgp130-IL-2Rb" is also referred to herein as "G2R-2 with 137ECD"; "G-CSFR137-ICDIL-2Rb+GCSFR137-ICDgc" is also referred to herein as "G2R-1 with 137ECD".

[0235] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0236] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, is also specifically disclosed, unless the context clearly dictates otherwise. Each smaller range from any stated value or intervening value in a stated range to any other stated value or intervening value in that stated range is included within the invention. The upper and lower limits of these smaller ranges may or may not independently be included within the range, and each range within these smaller ranges, including either one, neither, or both of those limits, is included within the invention, subject to any specifically excluded limits within the stated range. When a stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention.

[0237] Variant cytokine and receptor design Described herein are variant cytokine and receptor pairs for selectively activating variant receptors. The variant receptors of the present disclosure comprise the extracellular domain of G-CSFR; 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.

[0238] Variant G-CSF and variant G-CSFR ECD pair In certain embodiments, 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 embodiments, 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, 2A, 4, or 6.

[0239] In certain embodiments, at least one mutation on the variant receptor in the Site II interface region is located at an amino acid position in 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).

[0240] In certain embodiments, at least one mutation on the variant G-CSF site II interface region is located at an amino acid position of G-CSF selected from the group consisting of amino acid positions 12, 16, 19, 20, 104, 108, 109, 112, 115, 116, 118, 119, 122, and 123 of G-CSF (SEQ ID NO: 1).

[0241] In certain aspects, the at least one mutation on the variant receptor site II interface region is selected from the group of mutations in the G-CSFR extracellular domain consisting of R141E, R167D, K168D, K168E, L171E, L172E, Y173K, Q174E, D197K, D197R, M199D, D200K, D200R, V202D, R288D, and R288E.

[0242] In certain embodiments, the at least one mutation on the variant G-CSF site II interface region is selected from the group of G-CSF mutations consisting of K16D, R, S12E, S12K, S12R, K16D, L18F, E19K, E19R, Q20E, D104K, D104R, L108K, L108R, D109R, D112R, D112K, T115E, T115K, T116D, Q119E, Q119R, E122K, E122R, and E123R.

[0243] In certain embodiments, at least one mutation on the variant Site III interface region is selected from the group of mutations in the G-CSFR extracellular domain selected from the group consisting of amino acid positions 30, 41, 73, 75, 79, 86, 87, 88, 89, 91, and 93 of SEQ ID NO:2.

[0244] In certain embodiments, at least one mutation on the variant Site III interface region is selected from the group of G-CSF mutations selected from the group consisting of amino acid positions 38, 39, 40, 41, 46, 47, 48, 49, and 147 of SEQ ID NO:1.

[0245] In certain aspects, at least one mutation on the variant receptor site III interface region is selected from the group of mutations in the G-CSFR extracellular domain consisting of S30D, R41E, Q73W, F75K, S79D, L86D, Q87D, I88E, L89A, Q91D, Q91K, and E93K.

[0246] In certain embodiments, the at least one mutation on the variant G-CSF site III interface region is selected from the group of G-CSF mutations consisting of T38R, Y39E, K40D, K40F, L41D, L41E, L41K, E46R, L47D, V48K, V48R, L49K, and R147E.

[0247] The variant cytokine and receptor pairs described herein can contain mutations in the site II region only, the site III region only, or in both the site II and site III regions.

[0248] The variant cytokine and receptor pairs described herein can have any number of Site II and / or Site III mutations described herein. In certain embodiments, the variant G-CSF and receptor have the mutations described in Table 4. In certain embodiments, the variant receptor and / or variant G-CSF can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations described herein. In certain embodiments, the variant cytokines described herein share at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to the variant cytokines described herein. In certain embodiments, the variant cytokines described herein share at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to a variant cytokine in Table 21.

[0249] In certain embodiments, the variant receptors described herein comprise a G-CSFR ECD domain that shares at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity with the G-CSFR ECD of a SEQ ID NO: 2, 3, 6, or 8. In certain embodiments, the chimeric receptor comprises an ECD of a G-CSFR having the amino acid sequence of SEQ ID NO: 2, 3, 6, or 8.

[0250] In certain embodiments, 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 to the G-CSFR ECD of SEQ ID NO:1.

[0251] 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.

[0252] Variant cytokines and / or receptors can be recombinantly produced not only directly but also as fusion polypeptides with heterologous polypeptides, such as a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. Generally, the signal sequence may be a component of the vector or may be part of a coding sequence inserted into the vector. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For mammalian cell expression, the native signal sequence may be used, or other mammalian signal sequences, such as signal sequences from secreted polypeptides of the same or closely related species, and viral secretory leaders, may be suitable. In certain embodiments, the signal sequence is the G-CSFR or GM-CSFR signal sequence. In certain embodiments, the signal sequence is SEQ ID NO: 11 or SEQ ID NO: 12.

[0253] In certain aspects, the variant receptors and / or variant G-CSFs are modified, either naturally or synthetically, to enhance stability (e.g., sugar groups and polyethylene glycol (PEG)). For example, in certain embodiments, the variant cytokines are fused to the Fc domain of IgG, albumin, or other molecules to enhance half-life, e.g., by PEGylation, glycosylation, etc., as known in the art. In certain embodiments, the variant cytokines described herein are modified by chemical PEGylation. In certain embodiments, the variant G-CSF cytokines in Table 21 are PEGylated. In certain embodiments, the variant G-CSF cytokines corresponding to SEQ ID NOs: 83, 83-1, 83-2, 83-3, 83-4, 83-5, and 84 are modified by PEGylation. In certain embodiments, the variant G-CSF cytokines and / or chimeric cytokine receptors described herein are modified by the addition of PEG to the N-terminus and / or C-terminus of the protein. In certain embodiments, the variant G-CSF cytokine and / or chimeric cytokine receptor described herein is modified by the addition of a compound comprising PEG. In certain embodiments, the PEG or PEG-containing compound is about 20 kDa or less. In certain embodiments, the PEG or PEG-containing compound is 20 kDa or less, 15 kDa or less, 10 kDa or less, 5 kDa or less, or 1 kDa or less.

[0254] Fc fusions can also promote alternative Fc receptor-mediated properties in vivo. An "Fc region" can be a naturally occurring or synthetic polypeptide homologous to the IgG C-terminal domain, which is generated by digesting IgG with papain. IgG Fc has a molecular weight of approximately 50 kDa. Variant cytokines may contain 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. Furthermore, full-length or fragmented Fc regions can be variants of the wild-type molecule.

[0255] Upon binding of a variant cytokine to a variant receptor, the variant receptor activates signaling transduced through native cellular components, providing biological activity that mimics the native response, but is specific to the cell engineered to express the variant receptor. In certain aspects, the variant receptor and G-CSF pair do not bind their native wild-type G-CSF or native wild-type G-CSFR. Thus, in certain embodiments, the variant receptor does not bind to its endogenous counterpart cytokine, including the variant cytokine's native counterpart, while the variant cytokine does not bind to any endogenous receptor, including the variant receptor's native counterpart. In certain embodiments, the variant cytokine binds to the native receptor with significantly reduced affinity compared to the binding of the native cytokine to the native cytokine receptor. In certain embodiments, the affinity of the variant cytokine for the native receptor is less than 10-fold, less than 100-fold, less than 1,000-fold, or less than 10,000-fold greater than the affinity of the native cytokine for the native cytokine receptor. In certain embodiments, the variant cytokine binds to a native receptor with a significantly reduced affinity. -4 Larger than M, 1X10 -5 Larger than M, 1X10 -6 Larger than M; 1X10 -7 Larger than M, 1X10 -8 Larger than M or 1X10 -9 K is bigger than M D In certain embodiments, the variant cytokine receptor binds to the native cytokine with significantly reduced affinity compared to the binding of the native cytokine receptor to the native cytokine. In certain embodiments, the variant cytokine receptor binds the native cytokine less than 10-fold, less than 100-fold, less than 1,000-fold, or less than 10,000-fold that of the native cytokine to the native cytokine receptor. In certain embodiments, the variant cytokine receptor binds the native cytokine with an affinity of less than 1X10 -4 Larger than M, 1X10 -5 Larger than M, 1X10-6 Larger than M or 1X10 -7 Larger than M, 1X10 -8 Larger than M or 1X10 -9 K is bigger than M D In some embodiments, the affinity of the variant cytokine for the variant receptor is comparable to the affinity of the native cytokine for the native receptor, e.g., has an affinity that is at least about 1%, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 100% of the affinity of the native cytokine-receptor pair, and may be higher, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more, than the affinity of the native cytokine for the native receptor. Affinity can be determined by any number of assays familiar to those of skill in the art. For example, affinity can be determined by competitive binding experiments in which receptor binding is measured using a single concentration of labeled ligand in the presence of varying concentrations of unlabeled ligand. Typically, the concentration of unlabeled ligand is varied over at least six orders of magnitude. Competitive binding experiments can provide an IC 50 As used herein, "IC 50 " refers to the concentration of unlabeled ligand required for 50% inhibition of the association between the receptor and the labeled ligand. 50 is an indicator of ligand-receptor binding affinity. Low IC 50 indicates high affinity, while high IC 50 indicates low affinity.

[0256] Binding of a variant cytokine to a variant cytokine receptor expressed on the surface of a cell may or may not affect the function of the variant cytokine receptor (compared to native cytokine receptor activity); native activity is not necessary or desired in all cases. In certain embodiments, binding of a variant cytokine to a variant cytokine receptor will elicit one or more characteristics of native cytokine signaling. In certain embodiments, binding of a variant cytokine to a variant cytokine receptor expressed on the surface of a cell triggers a cellular response selected from the group consisting of proliferation, viability, persistence, cytotoxicity, cytokine secretion, memory, and enhanced activity.

[0257] [Table 1]

[0258] [Table 1A]

[0259] The G-CSFR amino acid sequence set forth in SEQ ID NO: 2 in Table 1 corresponds to the G-CSFR amino acid positions of the mutation numbers used herein. The G-CSFR sequence set forth in SEQ ID NO: 101 listed in Table 1A is identical to SEQ ID NO: 2 except that one amino acid (glutamic acid) has been removed from the N-terminus. Thus, the G-CSFR amino acid positions of the mutation numbers used herein correspond to amino acids 2 to 308 of SEQ ID NO: 101.

[0260] [Table 2] TIFF2024515577000004.tif153165

[0261] [Table 3]

[0262] [Table 4] TIFF2024515577000007.tif138165

[0263] [Table 4A]

[0264] Chimeric receptors In certain embodiments, the variant receptors described herein are chimeric receptors. Chimeric receptors can comprise any of the variant G-CSFR ECD domains described herein. In certain embodiments, the chimeric receptor further comprises at least a portion of the intracellular domain (ICD) of a different cytokine receptor. The intracellular domains of different cytokine receptors may be selected from the group consisting of gp130 (glycoprotein 130, a subunit of the interleukin-6 receptor or IL-6R), IL-2Rβ or IL-2Rb (interleukin-2 receptor beta), IL-2Rγ or γc or IL-2RG (interleukin-2 receptor gamma), IL-7Rα (interleukin-7 receptor alpha), IL-12Rβ2 (interleukin-12 receptor beta 2), IL-21R (interleukin-21 receptor), IL-4R (interleukin-4 receptor), EPOR (erythropoietin receptor), IFNAR (interferon alpha / beta receptor), or IFNγR (interferon gamma receptor). In certain embodiments, at least a portion of the intracellular domain comprises an amino acid sequence that shares at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to the amino acid sequence of a cytokine receptor ICD described herein. In certain embodiments, at least a portion of the cytokine receptor ICD shares at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 4, 7, or 9. In certain embodiments, at least a portion of the intracellular domain comprises an amino acid sequence that shares at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to the amino acid sequence of a cytokine receptor ICD of a cytokine receptor listed in Table 26.

[0265] In certain embodiments, described herein are chimeric cytokine receptors comprising the extracellular domain (ECD) of G-CSFR (granulocyte colony-stimulating factor receptor) operably linked to a second domain, wherein the second domain comprises at least a portion of the intracellular domain (ICD) of a multisubunit cytokine receptor, e.g., IL-2R. In certain aspects, the chimeric cytokine receptor comprises a portion of an ICD of Table 15A, Table 15B, Table 23, and Table 24. 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 the Box 1 and Box 2 regions of Table 15A, Table 15B, Table 16, Table 23, and Table 24.

[0266] In certain aspects, the chimeric cytokine receptor comprises at least one signaling molecule binding site of Table 15A, Table 15B, Table 16, Table 23, Table 24, and Table 27. In some embodiments, the signaling molecule binding site(s) of the ICD comprise a sequence set forth in SEQ ID NOs: 118-146. In some embodiments, the signaling molecule binding site(s) of the ICD comprise a sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence set forth in at least one of SEQ ID NOs: 118-146.

[0267] In certain aspects, the chimeric receptors described herein comprise the amino acid sequence in N-terminal to C-terminal order of the sequences disclosed in each of Tables 17-20, 23, 24, 26, and 32. In certain aspects, the chimeric receptors described herein comprise the nucleic acid sequence in 5' to 3' order of the sequences disclosed in each of 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 to the amino acid sequence in N-terminal to C-terminal order of the amino acid sequences disclosed in each of Tables 17-20, and 23, 24, 26, and 32. In certain embodiments, the chimeric cytokine receptor shares at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleic acid identity to the nucleic acid sequence in 5' to 3' order of the nucleic acid sequences disclosed in each of Tables 17-20.

[0268] In certain embodiments, the chimeric receptors described herein comprise at least a portion of the ICD of a cytokine receptor that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid or nucleic acid sequence identity to an ICD SEQ ID NO: described herein. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of IL-2Rβ having the amino acid sequence of SEQ ID NO: 26, 29, 31, 39, 41, 43, 45, 47, or 49. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of IL-2Rβ, i.e., IL-2Rb, having the nucleic acid sequence of SEQ ID NO: 54, 57, 59, 67, 69, 71, 73, 75, or 77. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of IL-7Rα having the amino acid sequence of SEQ ID NO: 51 or the nucleic acid sequence of SEQ ID NO: 79. In certain embodiments, a chimeric receptor comprises at least a portion of the ICD of IL-7R having the amino acid sequence of SEQ ID NO:53 or the nucleic acid sequence of SEQ ID NO:81. In certain embodiments, a chimeric receptor comprises at least a portion of the ICD of IL-21R having the amino acid sequence of SEQ ID NO:35 or 37. In certain embodiments, a chimeric receptor comprises at least a portion of the ICD of IL-21R having the nucleic acid sequence of SEQ ID NO:63 or 65. In certain embodiments, a chimeric receptor comprises at least a portion of the ICD of IL-12Rβ2 having the amino acid sequence of SEQ ID NO:33, 42, or 46. In certain embodiments, a chimeric receptor comprises at least a portion of the ICD of IL-12Rβ2 having the nucleic acid sequence of SEQ ID NO:61, 70, or 74. In certain embodiments, a chimeric receptor comprises at least a portion of the ICD of G-CSFR having the amino acid sequence of SEQ ID NO:30, 32, 34, 36, 38, 40, 44, 50, or 52. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of G-CSFR having the nucleic acid sequence of SEQ ID NO: 58, 60, 62, 64, 66, 68, 72, 78, or 80. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of gp130 having the amino acid sequence of SEQ ID NO: 28 or 48. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of gp130 having the nucleic acid sequence of SEQ ID NO: 56 or 76.In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of IL-2Rγ (i.e., IL-2RG, IL-2Rgc, γc, or IL-2Rγ) having the amino acid sequence of SEQ ID NO: 27. In certain embodiments, the chimeric receptor comprises at least a portion of the ICD of IL-2Rγ (i.e., IL-2RG, IL-2Rgc, γc, or IL-2Rγ) having the nucleic acid sequence of SEQ ID NO: 55.

[0269] In certain embodiments, at least a portion of the ICD described herein comprises at least one signaling molecule binding site. In certain embodiments, the at least one signaling molecule binding site is a STAT3 binding site for G-CSFR; a STAT3 binding site for gp130; a SHP-2 binding site for gp130; an 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-7Rβ; α STAT5 binding site of IL-7R α phosphatidylinositol 3-kinase (PI3K) binding site of IL-12R β STAT5-binding site of IL-12Rβ2; STAT4-binding site of IL-12Rβ2; STAT3-binding site of IL-12Rβ2; STAT5-binding site of IL-21R; STAT3-binding site of IL-21R; and STAT1-binding site of IL-21R. In certain embodiments, at least one signaling molecule binding site comprises a sequence further comprising an amino acid set forth in Table 16.

[0270] In certain embodiments, the chimeric receptor comprises at least one signaling molecule binding site derived from the intracellular domain of a cytokine receptor. In certain embodiments, the chimeric receptor comprises at least one signaling molecule binding site derived from the intracellular domain of Table 24. In certain embodiments, the at least one signaling molecule binding site is selected from the group consisting of the SHC binding site of interleukin (IL)-2Rβ; the STAT5 binding site of IL-2Rβ, the IRS-1 or IRS-2 binding site of IL-4Rα, the STAT6 binding site of IL-4Rα, the SHP-2 binding site of gp130, the STAT3 binding site of gp130, the SHP-1 or SHP-2 binding site of erythropoietin receptor (EPOR), the STAT5 binding site of EPOR, the STAT1 or STAT2 binding site of interferon alpha and beta receptor subunit 2 (IFNAR2), and the STAT1 binding site of interferon gamma receptor 1 (IFNγR1), or a combination thereof.

[0271] In certain embodiments, the chimeric receptor ICD further comprises at least one Box 1 region and at least one Box 2 region of at least one protein selected from the group consisting of G-CSFR, gp130, EPOR, and interferon gamma receptor 2 (IFNγR2), or a combination thereof.

[0272] In certain aspects, at least a portion of an ICD described herein comprises the Box1 and Box regions of gp130 or G-CSFR. In certain aspects, the Box1 region comprises a sequence of amino acids set forth in Table 2. In certain aspects, the Box1 region comprises an amino acid sequence that is greater than 50% identical to a Box1 sequence set forth in Table 16.

[0273] In certain embodiments, the ICD comprises (a) the amino acid sequence of one or both of SEQ ID NOs: 90 or 91, or (b) the amino acid sequence of one or both of SEQ ID NOs: 90 or 92, or (c) the amino acid sequence of SEQ ID NO: 93, or (d) the amino acid sequence of SEQ ID NO: 94, or (e) the amino acid sequence of one or both of SEQ ID NOs: 95 or 96, or (f) the amino acid sequence of SEQ ID NO: 97 or 98, or (g) the amino acid sequence of SEQ ID NO: 99 or 100.

[0274] In certain embodiments, the intracellular domain of the different cytokine receptor is a wild-type intracellular domain.

[0275] In certain aspects, the chimeric variant receptors described herein further comprise at least a portion of the transmembrane domain (TMD) of G-CSFR. In certain aspects, the chimeric variant receptors described herein further comprise at least a portion of the transmembrane domain (TMD) of a different cytokine receptor. The TMD of the different cytokine receptor can be selected from the group consisting of G-CSFR, gp130 (glycoprotein 130), IL-2Rβ (interleukin-2 receptor beta), IL-2Rγ or γc (IL-2 receptor gamma), IL-7Rα (interleukin-7 receptor alpha), IL-12Rβ2 (interleukin-12 receptor beta2), and IL-21R (interleukin-21 receptor). In certain embodiments, at least a portion of the TMD comprises an amino acid sequence that shares at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to the amino acid sequence of a cytokine receptor TMD described herein. In certain embodiments, at least a portion of the cytokine receptor TMD shares at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO:4, 5, 7, or 9.

[0276] In certain aspects, the chimeric receptors described herein comprise a G-CSFR ECD domain, a transmembrane domain (TMD), and at least a portion of one ICD arranged in N-terminal to C-terminal order as shown in the chimeric receptor designs in Figures 20, 23, and 24.

[0277] In certain embodiments, the chimeric receptor comprises a G-CSFR ECD of SEQ ID NO: 3, a portion of the gp130 TMD and ICD of SEQ ID NO: 4, and a portion of the IL-2Rβ ICD of SEQ ID NO: 5. In certain embodiments, the chimeric receptor comprises a G-CSFR ECD of SEQ ID NO: 6 and a portion of the IL-2Rβ ICD of SEQ ID NO: 7. In certain embodiments, the chimeric receptor comprises a G-CSFR ECD of SEQ ID NO: 8 and a portion of the IL-2Rγ ICD of SEQ ID NO: 9.

[0278] Binding of a variant or wild-type cytokine to a chimeric cytokine receptor expressed on the surface of a cell may or may not affect the function of the variant cytokine receptor (compared to native cytokine receptor activity); native activity is not necessary or desired in all cases. In certain embodiments, binding of a variant cytokine to a chimeric cytokine receptor will elicit one or more characteristics of native cytokine signaling. In certain embodiments, binding of a variant cytokine to a chimeric cytokine receptor expressed on the surface of a cell triggers a cellular response selected from the group consisting of proliferation, viability, persistence, cytotoxicity, cytokine secretion, memory, and enhanced activity.

[0279] Receptor containing the ECD of IL-7Rα In certain aspects, disclosed herein is a chimeric receptor comprising: (i) the extracellular domain (ECD) of interleukin-7 receptor alpha (IL-7Rα); (ii) a transmembrane domain (TMD); and (iii) an intracellular domain (ICD) of a cytokine receptor that differs from the wild-type human IL-7Rα intracellular signaling domain set forth in SEQ ID NO: 109, wherein the ECD and TMD are each operably linked to the ICD. In some embodiments, the carboxy-terminus (C-terminus) of the ECD is linked to the amino-terminus (N-terminus) of the TMD, and the C-terminus of the TMD is linked to the N-terminus of the ICD. In some embodiments, the ECD is the ECD of native human IL-7Rα. In some embodiments, the TMD is the TMD of IL-7Rα. In some embodiments, the TMD is the TMD of native human IL-7Rα. In some embodiments, the ICD comprises at least one signaling molecule binding site derived from the intracellular domain of a cytokine receptor, and optionally the at least one signaling molecule binding site comprises: (a) a JAK1 binding site (Box 1 and 2 regions) of IL-2Rβ, IL-4Rα, IL-7Rα, IL-21R, or gp130; (b) an SHC binding site of IL-2Rβ; (c) a STAT5 binding site of IL-2Rβ or IL-7Rα; (d) a STAT3 binding site of IL-21R or gp130; (e) a STAT4 binding site of IL-12Rβ2; (f) a STAT6 binding site of IL-4Rα; (g) an IRS-1 or IRS-2 binding site of IL-4Rα; (h) an SHP-2 binding site of gp130; (i) a PI3K binding site of IL-7Rα; or a combination thereof. In some embodiments, when the ICD is part of its native receptor, the ICD comprises at least the intracellular signaling domain of the receptor, which is activated by heterodimerization with the common gamma chain (γc). In some embodiments, the ICD of the chimeric receptor does not heterodimerize with the common gamma chain. In some embodiments, the ICD of the chimeric receptor homodimerizes upon activation.In some embodiments, the ICD comprises at least the intracellular signaling domain of a cytokine receptor selected from the group consisting of IL-2Rβ (interleukin-2 receptor beta), IL-4Rα (interleukin-4 receptor alpha), IL-9Rα (interleukin-9 receptor alpha), IL-12Rβ2 (interleukin-12 receptor), IL-21R (interleukin-21 receptor), and glycoprotein 130 (gp130), and combinations thereof.

[0280] In a particular aspect, a chimeric receptor comprises an ECD and a TMD of IL-7Rα operably linked to an ICD, wherein the ICD comprises: (i) (a) Box1 and Box2 regions of IL-2Rβ; (b) the SHC binding site of IL-2Rβ, and (c) the STAT5 binding site of IL-2Rβ, or (ii) (a) Box1 and Box2 regions of IL-7Rα; (b) the SHC binding site of IL-2Rβ, and (c) the STAT5 binding site of IL-2Rβ, or (iii) (a) Box1 and Box2 regions of IL-2Rβ; (b) SHC binding site of IL-2Rβ; (c) the STAT5 binding site of IL-2Rβ, and (d) the STAT4 binding site of IL-12Rβ2, or (iv) (a) Box1 and Box2 regions of IL-7Rα; (b) SHC binding site of IL-2Rβ; (c) the STAT5 binding site of IL-2Rβ, and (d) the STAT4 binding site of IL-12Rβ2, or (v) (a) the Box1 and Box2 regions of IL-21R, and (b) the STAT3 binding site of IL-21R, or (vi) (a) the Box1 and Box2 regions of IL-7Rα, and (b) the STAT3 binding site of IL-21R, or (vii) (a) Box1 and Box2 regions of IL-21R; (b) the STAT3 binding site of IL-21R, and (c) STAT5 and PI3 kinase binding sites of IL-7Rα; (viii) (a) Box1 and Box2 regions of IL-7Rα; (b) the STAT3 binding site of IL-21R, and (c) STAT5 and PI3 kinase binding sites of IL-7Rα; (ix) (a) Box1 and Box2 regions of IL-2Rβ; (b) SHC binding site of IL-2Rβ; (c) the STAT5 binding site of IL-2Rβ, and (d) the STAT3 binding site of IL-21R, or (x) (a) Box1 and Box2 regions of IL-7Rα; (b) SHC binding site of IL-2Rβ; (c) the STAT5 binding site of IL-2Rβ, and (d) the STAT3 binding site of IL-21R, or (xi) (a) Box1 and Box2 regions of IL-2Rβ; (b) SHC binding site of IL-2Rβ; (c) STAT5 binding site of IL-2Rβ; (d) STAT4 binding site of IL12Rβ2, and (e) the STAT3 binding site of IL-21R, or (xii) (a) Box1 and Box2 regions of IL-7Rα; (b) SHC binding site of IL-2Rβ; (c) STAT5 binding site of IL-2Rβ; (d) STAT4 binding site of IL12Rβ2, and (e) the STAT3 binding site of IL-21R, or (xiii) (a) Box1 and Box2 regions of IL-4Rα; (b) the IRS-1 or IRS-2 binding site of IL-4Rα, and (c) the STAT6 binding site of IL-4Rα, or (xiv) (a) Box1 and Box2 regions of IL-7Rα; (b) the IRS-1 or IRS-2 binding site of IL-4α, and (c) the STAT6 binding site of IL-4α, or (xv) (a) Box1 and Box2 regions of gp130; (b) the SHP-2 binding site of gp130, and (c) the STAT3 binding site of gp130, or (xvi) (a) Box1 and Box2 regions of IL-7Rα; (b) the SHP-2 binding site of gp130, and Described herein are chimeric receptors comprising (c) a STAT3 binding site of gp130. In some embodiments, (b) is N-terminal to (c), or (c) is N-terminal to (b), or (c) is N-terminal to (d), or (d) is N-terminal to (c), or (d) is N-terminal to (e), or (e) is N-terminal to (d).

[0281] In some embodiments, the ICD described herein comprises the Box 1 and Box 2 regions of gp130 or G-CSFR. In certain aspects, the Box 1 region comprises a sequence of amino acids listed in Table 16. In certain 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.

[0282] In some embodiments, the ICD comprises a sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in at least one of SEQ ID NOs: 192-214.

[0283] In some embodiments, the signaling molecule binding site(s) of the ICD comprise a sequence set forth in SEQ ID NOs: 118-146. In some embodiments, the signaling molecule binding site(s) of the ICD comprise a sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence set forth in at least one of SEQ ID NOs: 118-146. In certain aspects, the chimeric cytokine receptor shares at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to the amino acid sequence, in N-terminal to C-terminal order, of the amino acid sequences disclosed in Table 32.

[0284] System containing chimeric receptor and IL-7 In certain aspects, the present disclosure describes a system for selectively activating a receptor expressed on a cell surface, the system comprising: (a) a chimeric receptor described herein; and (b) IL-7.

[0285] In certain aspects, the present disclosure describes a system for selectively activating immune cells, the system comprising: (a) a chimeric receptor described herein; (b) IL-7; and (c) an antigen-binding signaling receptor.

[0286] In certain aspects, the present disclosure describes a system for selectively activating immune cells, comprising: (a) a chimeric receptor described herein; (b) IL-7; and (c) at least one or more additional agonist or antagonist signaling protein(s), optionally wherein the one or more additional agonist or antagonist signaling protein(s) comprise one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent. In some embodiments, the cytokine or chemokine is selected from the group consisting of IL-18, IL-21, interferon alpha, interferon beta, interferon gamma, IL-17, IL-21, TNF-alpha, CXCL13, CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CD40 ligand, B-cell activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, CCL19, NKG2D, and combinations thereof. In some embodiments, the cytokine is IL-18. In some embodiments, the cytokine is human.

[0287] In some embodiments, the system further comprises at least one antigen-binding signaling receptor. In some embodiments, the at least one antigen-binding signaling receptor comprises at least one receptor selected from the group consisting of a natural T cell receptor, an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a natural B cell receptor, an engineered B cell receptor (BCR), a stress ligand receptor, a pattern recognition receptor, and combinations thereof. In some embodiments, the at least one antigen-binding signaling receptor is a CAR.

[0288] Agonist or antagonist signaling proteins In certain aspects, the systems and methods described herein include (a) at least one additional agonist or antagonist signaling protein(s), optionally wherein the one or more additional agonist or antagonist signaling protein(s) comprises one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s); and (b) at least one antigen-binding signaling receptor(s). In some embodiments, the at least one additional cytokine(s) or chemokine(s) comprises at least one of interleukin (IL)-18, IL-21, interferon alpha, interferon beta, interferon gamma, IL-17, IL-21, TNF-alpha, CXCL13, CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CD40 ligand, B-cell activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, CCL19, and receptor NKG2D, and combinations thereof.

[0289] Antigen-binding signaling receptors In certain aspects, the systems and methods described herein include an antigen-binding signaling receptor(s). In some embodiments, the antigen-binding signaling receptor(s) include at least one of a natural T cell receptor, an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a natural B cell receptor, an engineered B cell receptor (BCR), a stress ligand receptor, a pattern recognition receptor, and combinations thereof. Any CAR known in the art can be used, including, but not limited to, a mesothelin CAR or any of the CARs described herein. Any TCR known in the art can be used, including, but not limited to, any of the TCRs described herein.

[0290] Nucleic acids encoding variant cytokines and receptors Included in the present disclosure are nucleic acids encoding any one of the receptors and variant G-CSFs described herein.

[0291] In addition to being produced directly recombinantly, variant receptors or variant G-CSFs can also be produced as fusion polypeptides with heterologous polypeptides, such as other polypeptides that have a signal sequence or a specific cleavage site at the N-terminus of the mature protein or polypeptide. Generally, the signal sequence may be a component of the vector or may be part of the coding sequence inserted into the vector. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For mammalian cell expression, the native signal sequence may be used, or other mammalian signal sequences, such as signal sequences from secreted polypeptides of the same or related species, and viral secretory leaders, may be suitable. In certain embodiments, the signal sequence may 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 embodiments, the signal sequence may be the amino acid sequence MARLGNCSLTWAALIILLLPGSLE (SEQ ID NO: 11).

[0292] Included in the present disclosure are nucleic acids encoding any one of the receptors, IL-7, and variant G-CSFs described herein.

[0293] The variant receptor, or wild-type or variant IL-7, or variant G-CSF can be produced directly recombinantly or as a fusion polypeptide with a heterologous polypeptide, e.g., a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. Generally, the signal sequence may be a component of the vector or part of the coding sequence inserted into the vector. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For mammalian cell expression, the native signal sequence may be used, or other mammalian signal sequences, such as signal sequences from secreted polypeptides of the same or related species, and viral secretory leaders, may be suitable. In certain embodiments, the signal sequence may 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 embodiments, the signal sequence may be the amino acid sequence MTILGTTFGMVFSLLQVVSG (SEQ ID NO: 84). In a particular embodiment, the signal sequence can be the amino acid sequence MARLGNCSLTWAALIILLLPGSLE (SEQ ID NO: 11).

[0294] Expression vectors encoding variant cytokines or receptors Also described herein are expression vectors and kits of expression vectors that include one or more nucleic acid sequence(s) encoding one or more of the variant receptors, variant G-CSF, or wild-type or variant IL-7 described herein.

[0295] In certain embodiments, the nucleic acid encoding the variant receptor or variant G-CSF is inserted into a replicable vector for expression. Such vectors can be used to introduce the nucleic acid sequence(s) into host cells to express the variant receptor or cytokine described herein. Many such vectors are available. Vector components generally include, but are not limited to, one or more of an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Vectors include viral vectors, plasmid vectors, integrating vectors, 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).

[0296] Expression vectors usually contain a selection gene, also called a selectable marker. A selectable marker gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with a vector containing a selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement an auxotrophic deficiency, or (c) supply a critical nutrient unavailable from complex media.

[0297] In certain embodiments, the expression vector contains a promoter recognized by the host organism and operably linked to the variant protein-coding sequence. Promoters are untranslated sequences located upstream (5') of the start codon of structural genes (generally within about 100-1000 bp) that control the transcription and translation of specific nucleic acid sequences to which they are operably linked. Such promoters are typically divided into two classes: inducible promoters and constitutive promoters. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Numerous promoters recognized by a variety of potential host cells are known.

[0298] Transcription from vectors in mammalian host cells can be controlled by promoters derived from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus (e.g., murine stem cell virus), hepatitis B virus, and most preferably, simian virus 40 (SV40), heterologous mammalian promoters such as the actin promoter, PGK (phosphoglycerate kinase), or immunoglobulin promoter, or heat shock promoters, provided that such promoters are compatible with the host cell system. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.

[0299] Transcription in higher eukaryotes is often increased by inserting an enhancer sequence into a vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, that act on a promoter to promote its transcription. Enhancers are relatively orientation- and position-independent and are found 5' and 3' to transcription units, within introns, and within the coding sequence itself. Many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin). However, enhancers from eukaryotic viruses are typically used. Examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Enhancers may be spliced ​​into expression vectors at either a 5' or 3' position from the coding sequence, but are preferably located at a 5' site from the promoter.

[0300] Expression vectors used in eukaryotic host cells also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. Construction of suitable vectors containing one or more of the above components employs standard techniques.

[0301] In certain aspects, disclosed herein are lentiviral vectors encoding the chimeric receptors disclosed herein. In certain aspects, the lentiviral vector comprises an HIV-1 5'LT and 3'LTR. In certain aspects, the lentiviral vector comprises an EF1a promoter. In certain aspects, the lentiviral vector comprises an SV40 polyterminator sequence. In certain aspects, the vector is psPAX2, Addgene® 12260, pCMV-VSV-G, or Addgene® 8454.

[0302] Also described in some embodiments are nucleic acid and polypeptide sequences that have high sequence identity to the sequences described herein, e.g., 95, 96, 97, 98, 99% or greater sequence identity. The term percent sequence "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotide or amino acid residues that are identical when compared and aligned for maximum correspondence, as determined using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those of skill in the art) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequences being compared, e.g., over a functional domain, or over the entire length of the two sequences being compared.

[0303] For sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence(s) relative to the reference sequence based on the designated program parameters.

[0304] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0305] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0306] Cells expressing variant receptors and variant cytokines Also described herein are cells that express the variant receptors. Host cells, including engineered immune cells, can be transfected or transduced with the above-described expression vectors for variant cytokine or receptor expression.

[0307] In certain embodiments, the present disclosure provides cells comprising one or more of the variant receptors or variant cytokines described herein. The cells may comprise a nucleic acid or vector encoding the variant receptor or variant cytokine described herein. The present disclosure also provides methods for producing cells expressing a variant receptor. In certain aspects, the cells are produced by introducing a nucleic acid or expression vector described herein into the cells. The nucleic acid or expression vector can be introduced into the cells by any process, including, but not limited to, transfection, viral vector transduction, transposition, or gene editing. Any gene editing technology known in the art can be used, including, but not limited to, technologies consisting of clustered regularly interspaced short batch repeats (CRISPR-Cas) systems, zinc finger nucleases, transcription activator-like effector-based nucleases, and meganucleases.

[0308] 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, a naive CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, helper T cells, e.g., T H 1. T H 2. T H 9. T H 11. T H 22, T FH ;regulatory T cells, e.g. R 1, Natural T Reg , inducible T Regmemory T cells, including, but not limited to, central memory T cells, effector memory T cells, NKT cells, γδT cells, etc. In certain embodiments, the cell is a B cell, including, but not limited to, naive B cells, germinal center B cells, memory B cells, cytotoxic B cells, cytokine-producing B cells, regulatory B cells (Bregs), centroblasts, centrocytes, antibody-secreting cells, plasma cells, etc. In certain embodiments, the cell is an innate lymphoid cell, including, but not limited to, an NK cell, etc. In certain embodiments, the cell is a myeloid cell, including, but not limited to, a macrophage, a dendritic cell, a myeloid-derived suppressor cell, etc.

[0309] 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.

[0310] In some embodiments, the cells are genetically modified in an ex vivo procedure before being transplanted into a subject. The cells may be provided in a unit dose for treatment and can be allogeneic, autologous, etc. with respect to the intended recipient.

[0311] T cells or T lymphocytes are a type of lymphocyte that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of T cell receptors (TCRs) on their cell surface. There are various types of T cells, as summarized below.

[0312] T helper cells (Th cells) assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. Th cells express CD4 on their surface. Th cells become activated when peptide antigens are presented by MHC class II molecules on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes, including Th1, Th2, Th3, Th17, Th9, or Tfh, which secrete different cytokines to promote different types of immune responses.

[0313] Cytolytic T cells (TC cells or CTLs) destroy virus-infected and tumor cells and also participate in transplant rejection. Most CTLs express CD8 on their surface. These cells recognize targets by binding to antigens associated with MHC class I (present on the surface of all nucleated cells).

[0314] Memory T cells are a subset of antigen-specific T cells that persist long-term, even after recovery from infection. Upon re-exposure to their cognate antigen, memory T cells rapidly expand into large numbers of effector T cells, providing the immune system with a "memory" of past infections. Memory T cells include three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Typically, memory T cells express the cell surface protein CD45RO.

[0315] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are essential for maintaining immune tolerance. Their primary role is to shut down T cell-mediated immunity towards the end of an immune response and suppress autoreactive T cells that have escaped the process of negative selection in the thymus. Two major classifications of CD4+ Treg cells have been described: endogenous Treg cells and adaptive Treg cells.

[0316] Endogenous Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and are involved in interactions between both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells activated by TSLP and developing T cells. Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3.

[0317] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can occur during 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 immune signals from virus-infected cells in an MHC-independent manner.

[0318] In certain aspects, cells expressing a variant receptor or variant cytokine described herein are tumor-infiltrating lymphocytes (TILs) or tumor-associated lymphocytes (TALs). In certain aspects, TILs or TALs include CD4+ T cells, CD8+ T cells, natural killer (NK) cells, and combinations thereof.

[0319] In certain embodiments, the T cells described herein are chimeric antigen receptor T cells (CAR-T cells) genetically engineered to produce an artificial T cell receptor for use in immunotherapy. In certain aspects, the CAR-T cells are derived from T cells in the patient's own blood (i.e., autologous). In certain aspects, the CAR-T cells are derived from T cells of another healthy donor (i.e., allogeneic). In certain aspects, the CAR-T cells are derived from or synthesized from a non-immune cell type, such as a pluripotent stem cell.

[0320] In certain embodiments, the T cells described herein are engineered T cell receptor (eTCR-T cells), genetically engineered to produce a specific T cell receptor for use in immunotherapy. In certain aspects, the eTCR-T cells are derived from T cells in the patient's own blood (i.e., autologous). In certain aspects, the eTCR-T cells are derived from T cells of a donor (i.e., allogeneic). In certain aspects, the eTCR-T cells are derived from or synthesized from a non-immune cell type, such as a pluripotent stem cell.

[0321] In certain aspects, the cells expressing the chimeric cytokine receptors described herein are NK cells. NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGLs) and constitute a third type of cell differentiated from a common lymphoid progenitor that generates B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, from where they then enter the circulatory system. In certain aspects, NK cells are derived from NK cells in the patient's own blood (i.e., autologous). In certain aspects, NK cells are derived from NK cells of a donor (i.e., allogeneic). In certain aspects, NK cells are derived from or synthesized from non-immune cell types, such as pluripotent stem cells.

[0322] In certain embodiments, the cells expressing the chimeric cytokine receptors described herein are B cells. B cells include, but are not limited to, naive B cells, germinal center B cells, memory B cells, cytotoxic B cells, cytokine-producing B cells, regulatory B cells (Bregs), germinal center cells, centrocytic cells, antibody-secreting cells, and plasma cells. In certain embodiments, the B cells are derived from B cells in the patient's own blood (i.e., autologous). In certain embodiments, the B cells are derived from B cells of a donor (i.e., allogeneic). In certain embodiments, the B cells are derived from or synthesized from a non-immune cell type, such as a pluripotent stem cell.

[0323] In certain embodiments, the cells expressing the chimeric cytokine receptors described herein are myeloid cells, including but not limited to macrophages, dendritic cells, myeloid-derived suppressor cells, etc. In certain embodiments, the myeloid cells are derived from bone marrow cells in the patient's own blood (i.e., autologous). In certain embodiments, the myeloid cells are derived from bone marrow cells of a donor (i.e., allogeneic). In certain embodiments, the myeloid cells are derived from or synthesized from non-immune cell types, such as pluripotent stem cells.

[0324] Cells expressing the variant receptors or cytokines described herein can be of any cell type. In certain embodiments, cells expressing the variant receptors or cytokines described herein are cells of the hematopoietic system. Immune cells (e.g., T cells or NK cells) according to the present invention can be generated ex vivo from a patient's own peripheral blood (first party), in the setting of hematopoietic stem cell transplantation from donor peripheral blood (second party), or from peripheral blood from an unrelated donor (third party). Alternatively, immune cells described herein can be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells into immune cells. Alternatively, immortalized immune cell lines that retain effector function and can act as therapeutic agents can be used (e.g., T cell or NK cell lines that retain lytic function, plasma cell lines that retain antibody-producing function, or dendritic cell lines or macrophages that retain phagocytic and antigen-presenting functions, etc.). In all of these embodiments, variant receptor-expressing cells are generated by introducing DNA or RNA encoding each variant receptor(s) by one of a number of means, including transduction with a viral vector or transfection with DNA or RNA.

[0325] The cells described herein may be immune cells derived from a subject that have been engineered ex vivo to express a variant receptor and / or a variant cytokine. The immune cells may be derived from a peripheral blood mononuclear cell (PBMC) sample or a tumor sample. The immune cells may be activated and / or expanded, for example, by treatment with anti-CD3 monoclonal antibody and / or IL-2, before being transduced with a nucleic acid encoding a molecule that provides a variant receptor or variant cytokine according to the first aspect of the invention. The immune cells of the invention may be produced by (i) isolating an immune cell-containing sample from a subject or other source as described above, and (ii) transducing or transfecting the immune cells with one or more nucleic acid sequence(s) encoding the variant receptor or variant cytokine.

[0326] Cells can be cultured in conventional nutrient media, modified as needed for inducing promoters, selecting transformants, or amplifying the gene encoding the desired sequence. Mammalian host cells can be cultured in a variety of media. Commercially available media, such as Ham's F10 (Sigma), minimal essential medium (MEM, Sigma), RPMI 1640 (Sigma), and Dulbecco's modified Eagle's medium (DMEM, Sigma), are suitable for culturing host cells. Any of these media may be supplemented, as needed, with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those of skill in the art. Culture conditions, such as temperature and pH, will be those previously used for the host cell selected for expression and will be apparent to those of skill in the art.

[0327] The immune cells can then be selected for purification, e.g., based on expression of the antigen-binding domain of the antigen-binding polypeptide. In certain embodiments, the cells are selected by expression of a selectable marker (e.g., a protein, fluorescent marker, or epitope tag) or by any method known in the art for selecting, isolating, and / or purifying cells.

[0328] kit 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 aspects, kits are described herein comprising cells encoding a chimeric receptor described herein, where the cells are optionally immune cells, and instructions for use, and optionally the kits comprise IL-7 and / or variant G-CSF. In certain aspects, kits are described herein comprising one or more expression vector(s) comprising nucleic acid sequence(s) encoding a chimeric receptor described herein, and instructions for use, and optionally the kits comprise IL-7 and / or variant G-CSF. In certain embodiments, the kits comprise at least one expression vector encoding at least one variant receptor and instructions for use. In certain aspects, the kits further comprise at least one variant cytokine in a pharmaceutical formulation, or an expression vector encoding a variant G-CSF that binds to at least one of the variant receptors described herein. In certain embodiments, the kits comprise cells comprising an expression vector encoding a variant receptor described herein.

[0329] In certain embodiments, the kits include cells comprising an expression vector encoding a chimeric antigen receptor (CAR) / engineered T cell receptor (eTCR), etc. (e.g., an engineered non-native TCR receptor). In certain embodiments, the kits include an expression vector encoding a chimeric antigen receptor (CAR) / engineered T cell receptor (eTCR), etc. In certain embodiments, the kits include an expression vector encoding a variant receptor and chimeric antigen receptor (CAR) / engineered T cell receptor (eTCR), etc., as described herein.

[0330] In certain aspects, the kits described herein further comprise a variant cytokine. In certain embodiments, the kits further comprise at least one additional variant cytokine. In certain aspects, the kits further comprise at least one variant cytokine in a pharmaceutical formulation. In certain aspects, the kits described herein further comprise at least one cytokine (e.g., IL-7 and / or variant G-CSF). In some embodiments, the kits include at least one or more additional agonist or antagonist signaling protein(s), optionally wherein the one or more additional agonist or antagonist signaling protein(s) include one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent. In some embodiments, the kit includes one or more expression vector(s) encoding at least one or more additional agonist or antagonist signaling protein(s), optionally the one or more additional agonist or antagonist signaling protein(s) include one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s).

[0331] In certain embodiments, the ingredients are provided in liquid or solid dosage forms in any convenient packaging.

[0332] In some embodiments, the kit comprises one or more expression vectors encoding at least one additional agonist or antagonist signaling protein(s), optionally the at least one or more additional agonist or antagonist signaling protein(s) comprises one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s). In certain embodiments, the kit further comprises one or more expression vector(s) encoding cytokine(s) or chemokine(s) selected from the group consisting of IL-18, IL-21, interferon alpha, interferon beta, interferon gamma, IL-17, IL-21, TNF-alpha, CXCL13, CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CD40 ligand, B-cell activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, or CCL19, or the receptor NKG2D, and combinations thereof. In certain embodiments, the kit further comprises one or more expression vector(s) encoding at least one antigen-binding receptor(s). In certain embodiments, the at least one antigen binding receptor(s) is selected from the group consisting of a natural T cell receptor, an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a natural B cell receptor, an engineered B cell receptor (BCR), a stress ligand receptor, a pattern recognition receptor, and combinations thereof. In certain embodiments, the kit further comprises an expression vector encoding the chimeric antigen receptor.

[0333] In some embodiments, the cells further comprise one or more expression vector(s) encoding at least one cytokine(s) or chemokine(s) selected from the group consisting of IL-18, IL-21, interferon alpha, interferon beta, interferon gamma, IL-17, IL-21, TNF-alpha, CXCL13, CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CD40 ligand, B-cell activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, or CCL19, or the receptor NKG2D, and combinations thereof. In some embodiments, the cells further comprise one or more expression vector(s) encoding at least one antigen-binding signaling receptor(s).

[0334] In certain aspects, the at least one antigen-binding signaling receptor(s) is selected from the group consisting of a natural T cell receptor, an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a natural B cell receptor, an engineered B cell receptor (BCR), a stress ligand receptor, a pattern recognition receptor, and combinations thereof. In some embodiments, the cells further comprise one or more expression vector(s) encoding at least one CAR(s), optionally wherein the CAR is a mesothelin CAR. In certain embodiments, the components are provided in liquid or solid dosage forms in any convenient packaging.

[0335] Additional reagents can be provided for the growth, selection, and preparation of the cells provided or produced as described herein. For example, the kits can include components for cell culture, growth factors, differentiation agents, reagents for transfection or transduction, etc.

[0336] In certain embodiments, in addition to the above components, the kit can also include instructions for use. The instructions can be provided in any convenient form. For example, the instructions can be provided as printed information on the kit packaging, a package insert, etc. The instructions can also be provided as a computer-readable medium having the information recorded thereon. Additionally, the instructions can be provided as a website address that can be used to access the information.

[0337] Methods for selective activation of variant receptors The present disclosure provides methods for selectively activating a variant receptor expressed on the surface of a cell, the method comprising contacting the variant receptor described herein with a cytokine that selectively activates the chimeric receptor. In certain embodiments, the cytokine that selectively activates the chimeric receptor is a variant G-CSF. The G-CSF can be wild-type G-CSF or G-CSF that contains one or more mutations that result in preferential binding and activation of G-CSF to the variant receptor compared to the native (wild-type) cytokine receptor.

[0338] In certain aspects, selective activation of the variant receptor by cytokine binding to the variant receptor results in homodimerization, heterodimerization, or a combination thereof.

[0339] In certain aspects, activation of the variant receptor results in activation of downstream signaling molecules. In certain aspects, the variant receptor activates signaling molecules or pathways transduced through native intracellular signaling molecules to provide biological activity that mimics the native response, but that is specific to cells engineered to express the variant receptor. In certain embodiments, the active form of the chimeric receptor forms a homodimer, and optionally, activation of the chimeric receptor causes a cellular response including at least one of proliferation, viability, persistence, cytotoxicity, cytokine secretion, memory, and enhanced activity of cells expressing the receptor, and combinations thereof, and optionally, the chimeric receptor is activated upon contact with a cytokine.

[0340] In certain embodiments, activation of downstream signaling molecules includes activation of cell signaling pathways that stimulate cell cycle progression, proliferation, survival, and / or enhanced activity. In certain embodiments, activated signaling pathways or molecules include, but are not limited to, JAK1, JAK2, JAK3, TYK2, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6, Shc, ERK1 / 2, IRS-1, IRS-2, and Akt. In certain embodiments, activation of the variant receptor results in increased cell proliferation following administration of a cytokine that binds to the receptor. In certain embodiments, the degree of proliferation is 0.1-10 times greater than that observed when cells are stimulated with IL-2.

[0341] In certain aspects, described herein are methods of activating one or more chimeric receptor(s) expressed on the surface of a cell, comprising contacting the one or more chimeric receptor(s) with IL-7 to activate the chimeric receptor(s), wherein the chimeric receptor comprises (i) an extracellular domain (ECD) of IL-7Rα, (ii) a transmembrane domain (TMD), and (iii) an intracellular domain (ICD) of a cytokine receptor that differs from the wild-type human IL-7Rα intracellular signaling domain set forth in SEQ ID NO: 109, wherein the ECD and TMD are each operably linked to the ICD. In some embodiments, the chimeric receptor(s) are chimeric receptors comprising an ICD described herein.

[0342] Methods and systems for selectively activating cells In certain aspects, described herein are methods and systems for selectively activating cells, the methods and systems comprising: (i) a receptor comprising a variant extracellular domain (ECD) of granulocyte colony-stimulating factor receptor (G-CSFR); (ii) a variant G-CSF that selectively binds to the receptor of (i); and one or both of: (a) at least one additional agonist or antagonist signaling protein(s), optionally wherein the one or more additional agonist or antagonist signaling protein(s) comprise one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s); and (b) at least one antigen-binding signaling receptor. In some embodiments, the receptor is expressed on an immune cell, optionally the immune cell is a T cell, optionally an NK cell, optionally an NKT cell, optionally a B cell, optionally a plasma cell, optionally a macrophage, optionally a dendritic cell, optionally the cell is a stem cell, optionally the cell is a primary cell, optionally the cell is a human cell. +T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, naive CD8 + The receptor is selected from the group consisting of T cells, helper T cells, regulatory T cells, memory T cells, and γδ T cells. In some embodiments, the at least one additional cytokine(s) or chemokine(s) comprises at least one of interleukin (IL)-18, IL-21, interferon α, interferon β, interferon γ, IL-17, IL-21, TNF-α, CXCL13, CCL3 (MIP-1α), CCL4 (MIP-1β), CD40 ligand, B-cell activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, and CCL19, and the receptor NKG2D, and combinations thereof. In certain embodiments, the receptor comprising a variant ECD of G-CSFR is a chimeric receptor described herein.

[0343] In certain embodiments, the system further comprises an antigen-binding signaling receptor. In certain embodiments, the antigen-binding signaling receptor comprises at least one of a natural T cell receptor, an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a natural B cell receptor, an engineered B cell receptor (BCR), a stress ligand receptor, a pattern recognition receptor, and combinations thereof. Any CAR known in the art may be used, including, but not limited to, a mesothelin CAR.

[0344] In certain aspects, described herein are methods of producing cells expressing a receptor comprising a variant ECD of G-CSFR of the system described herein, and one or both of: (i) at least one additional agonist or antagonist signaling protein(s), where optionally the at least one or more additional agonist or antagonist signaling protein(s) comprises one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s) of the system described herein; and (ii) at least one antigen-binding signaling receptor of the system described herein. In some embodiments, the method comprises introducing into the cell one or more nucleic acid(s) or expression vector(s) encoding the receptor, and one or both of (i) and (ii). In some embodiments, a first population of immune cells expresses a receptor comprising a variant ECD of G-CSFR, and a second population of immune cells expresses a variant G-CSF. In some embodiments, one or both of the first and second population(s) of immune cells further express one or both of: (a) at least one additional agonist or antagonist signaling protein(s), optionally wherein the at least one or more additional agonist or antagonist signaling protein(s) comprises one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s), and (b) at least one antigen-binding signaling receptor.

[0345] In certain aspects, described herein are methods and systems for activating cells comprising a chimeric receptor comprising: (a) the extracellular domain (ECD) of interleukin receptor alpha (IL-7Rα); (b) a transmembrane domain (TMD); and (c) an intracellular domain (ICD) of a cytokine receptor that differs from the wild-type human IL-7Rα intracellular signaling domain set forth in SEQ ID NO: 109, wherein the ECD and TMD are each operably linked to the ICD.

[0346] Adoptive cell transfer methods The present invention provides methods for treating and / or preventing disease, comprising administering to a subject cells (e.g., pharmaceutical compositions described below) that express a variant receptor and / or variant cytokine described herein.

[0347] Methods for treating disease involve the therapeutic use of the cells described herein, e.g., T cells, NK cells, or any other immune or non-immune cells expressing a variant receptor. The cells can be administered to a subject with an existing disease or condition to alleviate, reduce, or ameliorate at least one symptom associated with the disease and / or to slow, reduce, or prevent the progression of the disease. Methods for preventing disease involve the prophylactic use of the cells of the present disclosure. Such cells can be administered to a subject not yet suffering from and / or not exhibiting symptoms of the disease to prevent or impair the cause of the disease or to reduce or prevent the occurrence of at least one symptom associated with the disease. The subject may be considered to be predisposed to or at risk of developing the disease.

[0348] In some embodiments, the subject compositions, methods, and kits are used to enhance an immune response, in some embodiments, against conditions where depletion or modulation of target cells, such as cancer cells, infected cells, immune cells involved in autoimmune diseases, etc., is desirable, by systemic administration (e.g., intramuscular, intraperitoneal, intravenous, etc.) of cytokines.

[0349] The method may include the steps of (i) isolating an immune cell-containing sample, (ii) transducing or transfecting such cells, e.g., with a nucleic acid sequence or vector expressing a variant receptor, (iii) administering (i.e., injecting) the cells of (ii) to a subject, and (iv) administering a variant cytokine that stimulates the infused cells. In certain aspects, the subject has undergone immunoablative therapy prior to administering the cells to the subject. In certain aspects, the subject has not undergone immunoablative therapy prior to administering the cells to the subject. In certain aspects, the subject has undergone immunoablative therapy prior to administering the cells to the subject that is reduced in severity, dose, and / or duration that would be required without the use of the variant receptor described herein.

[0350] In certain embodiments, the method further comprises administering or providing at least one additional active agent, and optionally at least one additional agonist or antagonist signaling protein(s), where optionally the at least one or more additional agonist or antagonist signaling protein(s) comprise one or more cytokine(s), chemokine(s), hormone(s), antibody(ies), or derivative(s) thereof, or other affinity reagent(s). In certain embodiments, the subject is administered two or more cell populations, each expressing a distinct chimeric receptor and each expressing a distinct variant form of a cytokine. In certain embodiments, the cells expressing the chimeric receptor further express at least one antigen-binding signaling receptor. In certain embodiments, the antigen-binding signaling receptor comprises at least one receptor(s) selected from the group consisting of a natural T cell receptor, an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a natural B cell receptor, an engineered B cell receptor (BCR), a stress ligand receptor, a pattern recognition receptor, and combinations thereof. In certain embodiments, the antigen-binding signaling receptor is a CAR. In certain embodiments, the at least one cytokine(s) or chemokine(s) is selected from the group consisting of IL-18, IL-21, interferon alpha, interferon beta, interferon gamma, IL-17, IL-21, TNF-alpha, CXCL13, CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CD40 ligand, B cell-activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, or CCL19, or the receptor NKG2D, and combinations thereof.

[0351] Immune cell-containing samples can be isolated from a subject or other source, for example, as described above. Immune cells can be isolated from the subject's own peripheral blood (first party), in the setting of hematopoietic stem cell transplantation from donor peripheral blood (second party), or from peripheral blood from an unrelated donor (third party). Immune cells can also be derived by in vitro methods, such as by induced differentiation from stem cells or other forms of progenitor cells.

[0352] In some embodiments, immune cells are contacted with the variant cytokine in vivo, i.e., by transferring the immune cells to a recipient and administering an effective amount of the variant cytokine to the recipient to contact the immune cells in their native location, e.g., in a lymph node. In some embodiments, the contacting is performed in vitro. When cells are contacted with the variant cytokine in vitro, the cytokine is added to the cells at a dose and for a duration sufficient to activate signaling from the receptor, which can utilize features of the native intracellular machinery, e.g., accessory proteins, co-receptors, etc. The activated cells can be used for any purpose, including, but not limited to, determining antigen specificity, cytokine profiling, and experimental purposes related to in vivo delivery.

[0353] In certain aspects, a therapeutically effective number of cells are administered to a subject. In certain aspects, a subject is administered or infused with cells expressing a variant receptor on multiple separate occasions. In certain embodiments, at least 1x10 6 Cells / kg, at least 1x10 7 Cells / kg, at least 1x10 8 Cells / kg, at least 1x10 9 Cells / kg, at least 1x10 10Cells / kg, or more, may be administered, which may be limited by the number of cells, e.g., transfected T cells, available during collection. The transfected cells can be infused into the subject in any physiologically acceptable medium, usually intravascularly, but may also be introduced at any other convenient site where the cells can find a suitable site for proliferation.

[0354] In certain embodiments, a therapeutically effective amount of a variant cytokine is administered to a subject. In certain embodiments, a subject is administered the variant cytokine on multiple separate occasions. In certain embodiments, the amount of variant cytokine administered is sufficient to achieve a therapeutically desirable result (e.g., alleviate symptoms of a disease in a subject). In certain embodiments, the amount of variant cytokine administered is sufficient to stimulate cell cycle progression, proliferation, survival, and / or functional activity of cells expressing a variant cytokine receptor described herein. In certain embodiments, the variant cytokine is administered at a dose and / or for a duration required to achieve a therapeutically desirable result. In certain embodiments, the variant cytokine is administered at a dose and / or for a duration sufficient to stimulate cell cycle progression, proliferation, survival, and / or functional activity of cells expressing a variant cytokine receptor described herein. Dosage and frequency may vary depending on the agent, method of administration, nature of the cytokine, etc. It will be understood by those skilled in the art that such guidelines will be tailored to individual circumstances. Dosages can also vary for local administration (eg, intranasal administration, inhalation, etc.) and for systemic administration (eg, intramuscular, intraperitoneal, intravascular, etc.).

[0355] Indications for Adoptive Cell Transfer The present disclosure provides cells expressing a variant receptor described herein for use in the treatment and / or prevention of disease. The present invention also relates to the use of cells expressing a variant receptor described herein in the manufacture of a medicament for the treatment and / or prevention of disease.

[0356] The disease treated and / or prevented by the methods of the present invention may be a cancerous disease such as, but not limited to, bile duct cancer, bladder cancer, breast cancer, cervical cancer, ovarian cancer, colon cancer, endometrial cancer, hematological malignancies, kidney cancer (renal cell), leukemia, lymphoma, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, sarcoma, and thyroid cancer.

[0357] The disease to be treated and / or prevented may be an autoimmune disease. Autoimmune diseases are characterized by T and B lymphocytes aberrantly targeting self-proteins, polypeptides, peptides, and / or other self-molecules, causing damage and / or dysfunction of organs, tissues, or cell types within the body (e.g., the pancreas, brain, thyroid, or gastrointestinal tract), resulting in the clinical symptoms of the disease. Some autoimmune diseases affect specific tissues, while others can affect multiple tissues, depending in part on whether the response is directed against antigens restricted to specific tissues or against antigens that are widely distributed throughout the body. Autoimmune diseases include, but are not limited to, type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, autoimmune thyroid disease, and Graves' disease.

[0358] The disease to be treated and / or prevented may be an inflammatory condition such as cardiac fibrosis. Generally, inflammatory conditions or disorders typically involve the immune system attacking the body's own cells or tissues, potentially causing abnormal inflammation and resulting in chronic pain, redness, swelling, stiffness, and damage to normal tissue. Inflammatory diseases are characterized by or caused by inflammation, and include, but are not limited to, celiac disease, vasculitis, lupus, chronic obstructive pulmonary disease (COPD), irritable bowel disease, arteriosclerosis, arthritis, myositis, scleroderma, gout, Sjogren's syndrome, ankylosing spondylitis, antiphospholipid syndrome, and psoriasis.

[0359] In certain embodiments, the method is used to treat an infectious disease.

[0360] In certain embodiments, the method is used to treat a degenerative disease or condition, including, but not limited to, age-related neurodegenerative diseases and conditions.

[0361] In certain embodiments, the methods are used to generate natural or engineered cells, tissues, or organs for transplantation.

[0362] 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, the allograft rejection is acute allograft rejection.

[0363] The disease to be treated and / or prevented may involve transplantation of cells, tissues, organs, or other anatomical structures into an affected individual. The cells, tissues, organs, or other anatomical structures may be derived from the same individual (autologous or "autologous" transplant) or a different individual (allogeneic or "allogeneic" transplant). The cells, tissues, organs, or other anatomical structures may also be produced using in vitro methods, including cell cloning, induced cell differentiation, or production from synthetic biomaterials.

[0364] The present invention provides methods for treating and / or preventing disease, comprising one or more steps of administering to a subject the variant cytokines and / or cells described herein (e.g., in the pharmaceutical compositions described above).

[0365] Methods for treating and / or preventing disease relate to therapeutic uses of the cells of the present disclosure. Herein, the cells can be administered to a subject with an existing disease or condition to alleviate, reduce, or ameliorate at least one symptom associated with the disease and / or to slow, reduce, or prevent the progression of the disease. Methods for preventing disease relate to prophylactic uses of the cells of the present disclosure. Such cells can be administered to a subject who is not yet afflicted with the disease and / or does not exhibit symptoms of the disease to prevent or impair the cause of the disease or to reduce or prevent the occurrence of at least one symptom associated with the disease. The subject may be considered predisposed to or at risk of developing the disease. The methods may include: (i) isolating an immune cell-containing sample; (ii) transducing or transfecting such cells with a nucleic acid sequence or vector provided by the present invention; (iii) administering the cells of (ii) to the subject; and (iv) administering a variant cytokine that stimulates the infused cells. The immune cell-containing sample can be isolated from a subject or other source, e.g., as described above. Immune cells can be isolated from the subject's own peripheral blood (first party), in the setting of hematopoietic stem cell transplantation from donor peripheral blood (second party), or from peripheral blood from an unrelated donor (third party).

[0366] The treatment may be combined with other active agents, such as, but not limited to, antibiotics, anti-cancer agents, anti-viral agents, and other immunomodulatory agents (e.g., antibodies against the programmed cell death protein-1 [PD-1] pathway or antibodies against CTLA-4), and may include additional cytokines (e.g., interferon gamma, tumor necrosis factor alpha, interleukin 12, etc.).

[0367] Methods of Using Stem Cells Expressing Variant Cytokine Receptors The present invention provides methods for treating and / or preventing a condition or disease comprising administering stem cells expressing a variant receptor and / or variant cytokine described herein. In certain embodiments, the stem cells expressing a variant cytokine receptor and / or variant cytokine described herein are used in regenerative medicine, cell / tissue / organ transplantation, tissue reconstruction, or tissue repair.

[0368] Pharmaceutical Composition The present disclosure also relates to pharmaceutical compositions containing a plurality of cells expressing the variant receptors and / or cytokines described herein. The present disclosure also relates to pharmaceutical compositions containing the variant cytokines described herein. The cells of the present invention can be formulated into pharmaceutical compositions. These compositions can contain, in addition to one or more cells expressing the variant receptors described herein, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The pharmaceutical composition can optionally contain one or more additional pharmaceutically active polypeptides and / or compounds. Such formulations can be in a form suitable for intravenous infusion, for example.

[0369] For cells expressing the variant receptors and variant cytokines described herein according to the present disclosure to be administered to an individual, the administration is preferably in a "therapeutically effective amount" sufficient to show benefit to the individual. A "prophylactically effective amount," if sufficient to show benefit to the individual, can also be administered. The actual amount of cytokine or number of cells administered, as well as the rate and time course of administration, will depend on the nature and severity of the disease being treated. Treatment methods, such as determining dosage, are within the responsibility of general practitioners and other physicians and will usually take into account the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to practitioners. Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0370] The pharmaceutical compositions may be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated. [Example]

[0371] Below are examples of specific embodiments for carrying out the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0372] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, cell culture, adoptive cell transfer, and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry, 3rd Ed. (Plenum Press) Vols. A and B (1992).

[0373] Example 1: Rational design of the exclusive Site II interface of G-CSF:G-CSFR(CRH) Wild type (WT) G-CSF WT :G-CSFR WT The complex is a 2:2 heterodimer. G-CSF has two binding interfaces with the extracellular domain (ECD) of G-CSFR. The larger interface between G-CSF and the extracellular cytokine receptor homology (CRH) domain of G-CSFR is called site II. The smaller interface between G-CSF and the N-terminal Ig-like (Ig) extracellular domain of G-CSFR is called site III (see Figure 1).

[0374] Co-evolved, engineered (E)G-CSF E :G-CSFR ETo design the cytokine:receptor pair, the 2:2 complex of WT G-CSF and the Ig-CRH extracellular domain of G-CSFR (Protein Data Bank ID 2D9Q, Tamada et al., PNAS 2006) was separated into two distinct subcomplexes, consisting of G-CSF:G-CSFR(CRH) and G-CSF:G-CSFR(Ig), respectively, encompassing the Site II and Site III interfaces (see Figure 1). See Tables 1 and 1A for sequences. The G-CSFR amino acid sequence set forth in SEQ ID NO:2 in Table 1 corresponds to the amino acid positions used in the mutation numbering used herein for G-CSFR. The G-CSFR sequence set forth in SEQ ID NO:101 listed in Table 1A is identical to SEQ ID NO:2, except for the removal of one amino acid (glutamic acid) at the N-terminus. Therefore, the amino acid positions for the mutation numbering used herein correspond to amino acids 2-308 of SEQ ID NO:101.

[0375] method Co-evolved exclusive G-CSF E :G-CSFR E To generate the mutation pairs (designs), we employed a computational design workflow (see Figure 2). First, we generated an exclusive design at site II.

[0376] G-CSF WT :G-CSFR(CRH) WTIn silico structural analysis of the Site II interface interactions of G-CSFR(CRH) revealed that most molecular interactions are made by charged residues, accounting for a total attractive AMBER energy of 109.64 kcal / mol at Site II (see Figure 3). Closer inspection revealed that most are electrostatic and hydrogen-bond interactions, such as those between R167 of G-CSFR(CRH) and D112 / D109 of G-CSF, which account for 28% of the total attractive AMBER energy at Site II. Another significant electrostatic interaction exists between R141 of G-CSFR(CRH) and E122 / E123 of G-CSF (see Figure 4), which accounts for 21.4% of the total attractive AMBER energy at Site II (see Figure 3). Similarly, a salt bridge between E19 of the cytokine and R288 of the receptor CRH domain accounts for 17.3%, and an arginine is further stabilized by electrostatic and hydrogen-bond interactions to D200 of the receptor CRH domain.

[0377] Each design at Site II was E and G-CSFR(CRH) E First, a positive design was made at site II through charge reversal, for example by mutating a basic residue on the binding partner to an acidic residue, or vice versa, while maintaining packing and hydrophobic interactions through additional mutations as needed. E :G-CSFR(CRH) E were packed using the mean-field packing workflow in ZymeCAD™. The packed in silico models of Site II designs were visually inspected for structural integrity and evaluated using ZymeCAD™ metrics. Specifically, G-CSF E The mutants are the corresponding G-CSFR(CRH) EMutants were designed to have a ZymeCAD™ in silico dAMBER binding affinity of less than 10 kcal / mol (pairwise interaction). This metric compares AMBER affinity, the sum of Lennard Jones affinity and electrostatic affinity, to the AMBER affinity of the WT:WT cytokine-receptor pair. Designs with a dAMBER_folding greater than 80 kcal / mol were excluded. The dAMBER_folding metric scores the change in the sum of Lennard Jones binding folding and electrostatic folding upon mutation. Designs that scored greater than 400 kcal / mol on dDDRW apostability were also excluded. This metric represents the change in knowledge-based potential stability when a protein is mutated from its apo form.

[0378] Next, G-CSF of each design E The mutants were complexed with WT G-CSFR (and vice versa) by ZymeCAD™. E and G-CSF WT ) and the following two complexes G-CSF WT :G-CSFR(CRH) E and G-CSF E :G-CSFR(CRH) WT The metrics of each positive design were evaluated under mispairing conditions when forming the G-CSF complex. ZymeCAD™ was used to calculate in silico ddAMBER metrics for the mispaired orientation (DdAMBER_affinity_Awt_Bmut is the AMBER affinity of the paired engineered complex compared to the mispaired G-CSF complex). WT :G-CSFR(CRH) E and ddAMBER_affinity_Amut_Bwt is the AMBER affinity of the paired engineered complex minus the AMBER affinity of the mispaired complex G-CSF. E :G-CSFR(CRH) WTThe AMBER affinity of the mispaired ddAMBER binding partner was subtracted from the AMBER affinity of the mispaired ddAMBER binding partner. Designs that minimized the mispaired ddAMBER affinity metric were considered to be selective for their paired binding partner over binding to wild-type cytokine or receptor binding.

[0379] Clustering all site II designs and G-CSF E :G-CSFR(CRH) E , G-CSF WT :G-CSFR(CRH) E , G-CSF E :G-CSFR(CRH) WT The packing metrics of the designs were examined to assess the strength of pairing (positive designs) and selectivity for mispairing with WT G-CSF and G-CSFR(CRH) (negative designs), and the designs were ranked.

[0380] result The designs in Table 2 were synthesized in silico using G-CSF E :G-CSFR(CRH) E The designs had packing metrics in ZymeCAD™ that favored pairing with G-CSF (see Table 5) and exhibited satisfactory interactions in silico upon visual inspection, including the presence of salt bridges, hydrogen bonds, and the absence of severe clashes (see Figure 5). These designs also exhibited high selectivity for mispairing with WT G-CSF or G-CSFR(CRH) (see Table 5).

[0381] Therefore, site II mutations of the same variant G-CSF and receptor designs shown in Table 2 are predicted to exhibit preferential binding over wild-type G-CSFR and G-CSF, respectively.

[0382] [Table 5] TIFF2024515577000010.tif219165TIFF2024515577000011.tif56165

[0383] Example 2: In vitro screening of Site II designs Selected Site II designs were tested for their ability to form Site II complexes when co-expressed in a baculovirus-based insect cell system. E :G-CSFR(CRH) E The results were analyzed by pull-down experiments. E Mutant designs and G-CSFR(CRH) WT Conversely, co-expression of G-CSFR(CRH) E Each mutant design was analyzed using GCSF WT We assessed whether the designs could form mispairing complexes with the WT receptor or cytokines by co-expressing G-CSF E Expression of cytokines alone was confirmed by single infection with the cytokine mutants.

[0384] method Briefly, the site II G-CSF design and the corresponding pair of G-CSFR(CRH) mutants were separately cloned into insect cell transfection vectors. G-CSF WT (residues 1-173, Table 1) and mutants were transfected with an N-terminal secretion signal and a C-terminal TEV-cleavable Twin Strep tag (sequence The CRH domain (residues 98-308, Table 1) with the Site II design mutation was the only receptor extracellular domain containing the N-terminal secretion signal and the sequence TIFF2024515577000013.tif4128 The constructs were cloned into a modified pAcGP67b transfer vector containing an in-frame cleavable hexahistidine tag. All constructs were synthesized and codon-optimized for insect cell expression (Genscript). Transfer vector DNA was prepared by Midi-prep (ThermoScientific, cat. K0481), endotoxin-free, and A260 / 280 The absorbance ratio was 1.8-2.0. Recombinant virus production was achieved by co-transfecting recombinant, linearized baculovirus DNA and vector DNA into Spodoptera frugiperda 9 (Sf9) cells using the attachment method according to the manufacturer's instructions (Expression Systems, California). Approximately 1 hour before transfection, 0.46 × 10 6 cells ml -1 Two milliliters of healthy log-phase Sf9 cells were seeded per well of a 6-well tissue culture plate (Greiner, cat. 657-160) in a 100-well tube. The transfection mixture was prepared as follows: 100 μl of transfection medium (Expression Systems, California, cat. 95-020-100) was placed in each of two sterile 1.5 ml microtubes, A and B. Tube A contained 0.4 μg of recombinant BestBac 2.0Δ v-cath / chiA linearized DNA (Expression Systems, California, cat. 91-002) and 2 μg of vector DNA. Tube B contained 1.2 μl of 5X Express 2TR transfection reagent (Expres2ION, cat. S2-55A-001) was added. Solution A and Solution B were incubated at approximately 24°C for 5 minutes, then combined and incubated for 30 minutes. After incubation, 800 μl of transfection medium was added to each transfection reaction, bringing the volume up to 1 ml. The old ESF921 medium was removed from the wells and replaced with 1 ml of the transfection mixture, dropwise, without disturbing the cell monolayer. The plate(s) were gently rocked back and forth side to side 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(s), and 2 mL of fresh ESF921 insect cell culture medium (Expression Systems, California, cat. 96-001-01) containing gentamicin at 10 μg / mL (cat. 15750-060) was added dropwise. 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 post-transfection, the P1 supernatant was collected, clarified by centrifugation at 5000 rpm for 5 minutes, transferred to a new sterile tube, and stored at 4°C protected from light.

[0385] The recombinant P1 stock generated as described above was further amplified to obtain a high-titer, low-passage P2 stock for protein expression studies. In the following workflow, the P1 seed stock of virus harvested from the co-transfection was used as an inoculum to generate 50-100 mL of virus. 6 cells ml -1Fifty mL of log-phase Sf9 cells at 100°C were seeded into a 250 mL shake flask (Fisher Scientific, cat. PBV 250) and 0.5 mL of P1 virus stock was added. Cells were incubated at 27°C with shaking at 135 rpm and observed for infection. P2 virus supernatant was collected 5–7 days post-infection and clarified by centrifugation at 4000 rpm for 10 minutes. To minimize titer loss, 10% heat-inactivated FBS (VWR, cat. 97068-085) was added, and the P2 virus was stored in the dark at 4°C.

[0386] The P2 virus stock was tested for small-scale protein expression. The P2 virus was used to express G-CSF E Mutants and their corresponding G-CSFR E The mutants were co-infected into Trichoplusia ni (Tni) cells in 12-well plates. WT and GCSFR(CRH) WT Using the P2 stock of each design variant, and G-CSF E Separate coinfections were performed for the mutants only. For each reaction, 2x10 6 cells ml -1 Two mL of healthy log-phase Tni cells at 100°C were inoculated with 20 μl of P2 virus. Plates were incubated at 27°C for approximately 70 hours with shaking at 135 rpm. Supernatants were clarified by centrifugation at 5000 rpm for 3 minutes, and secreted proteins were isolated using a 5000 rpm centrifugation system. E Mutants and G-CSF WTBatch-mode pulldown was performed using streptactin-XT Superflow (IBA Lifesciences, cat. 2-4010-010) via the Twin-Strep Tag (TST) on the nuclease. Briefly, to each 1.8 mL reaction supernatant, 0.2 mL of 10X HEPES-buffered saline (HBS: 20 mM HEPES pH 8, 150 mM NaCl) was added to 20 μl of 1X bed volume (bv) of purified beads, and the reaction was incubated at 24°C for 30 minutes with end-over-end mixing. An additional 20 μl of bv of purified beads was added, followed by another 30-minute incubation. The beads were pelleted by centrifugation at 2200 rpm for 3 minutes, the supernatant removed, and 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, cat. 2-1042-025)), boiled in SDS-PAGE sample buffer, and analyzed under reducing conditions on 12% Bolt Bis-Tris plus, 12-well gels (Thermo Fisher Scientific, cat. NW00122BOX) at 200 V for 30 min.

[0387] result Site II designs #6, 7, 8, 9, 15, 17, 30, 34, 35, and 36 were G-CSF E It shows expression alone and G-CSF E Sufficiently stable paired G-CSF pulled down via the Twin Strep tag on E :G-CSFR E For example, after pulldown of the engineered complex, Site II Design #6, the G-CSF complex was visualized by SDS-PAGE. E Approximately 22 kDa band of the mutant and the corresponding co-expressed G-CSFR(CRH) E The mutant showed a band of approximately 33 kDa (see Figure 6).

[0388] Site II designs #8, 9, 15, and 34 were selective for mispairing with WT G-CSF and WT G-CSFR in coexpression assays (see Figure 7), because they were not pulled down by WT G-CSF (see bottom panel of Figure 7), and because the WT receptor was not pulled down by G-CSF. E This is because the site II G-CSFR mutants were not pulled down (see the upper panel of Figure 7). E Designs 30 and 35 were selective for mispairing with WT G-CSF in the coexpression assay, as they were not pulled down by WT G-CSF (see the bottom panel of Figure 7), but were selective for mispairing with reciprocal G-CSF. E The designs were not selective for mispairing with WT G-CSFR in the coexpression assay because WT G-CSFR was pulled down (see Figure 7 , top panel). Site II designs 6, 7, 17, and 36 were not selective for mispairing with WT G-CSFR or WT G-CSFR in the coexpression assay because they pulled down WT G-CSFR and were pulled down by WT G-CSF (see Figure 7 ).

[0389] The Site II receptor mutant with a mutation at residue R288 was not expressed in the form of a G-CSFR (CRH) receptor chain without an Ig domain. The R288-containing design was combined with a Site III design on the Ig domain and evaluated for paired complex formation by SPR (see Example 6). Thus, a sufficiently stable paired G-CSF was obtained that was selective for mispairing with WT G-CSF and WT G-CSFR. E :G-CSFR E Several site II designs were identified that formed complexes.

[0390] Thus, various site II mutations of the same variant G-CSF and receptor designs shown in Table 2 show preferential binding to wild-type G-CSFR and G-CSF, respectively.

[0391] Example 3: Rational design of the exclusive Site III interface of G-CSF:G-CSFR(Ig) The avidity effect of the Site III receptor Ig domain binding to G-CSF contributes to the formation of a G-CSF:G-CSFR (Ig-CRH) complex with a 2:2 heterodimeric stoichiometry (see Figure 1). The selective design present only in Site II with the WT Site III interface results in a completely exclusive 2:2 G-CSF E :G-CSFR (Ig-CRH) E To prioritize paired coevolutionary design binding that is selective over mispaired binding to the WT cytokine or receptor, the in silico design workflow described in Example 1 was applied to the Site III interface to generate selective Site III designs (see Figure 2).

[0392] method First, we performed a structural analysis of the Site III interface interactions. The Site III interface contributes an AMBER energy of 55.64 kcal / mol to the G-CSF:G-CSFR complex, with a resolution of 692.6 Å. 2 571.5 Å compared to site II in the interfacial region of 2 The Site III interface has an overall smaller interface area of ​​1.25. Closer inspection of the Site III interface reveals fewer electrostatic and hydrogen-bonding interactions compared to the Site II interface (see Figure 8). Important interactions at Site III are, for example, the salt bridge between E46 of G-CSF and R41 of the receptor Ig domain, and between R147 of G-CSF and E93 of the receptor Ig domain (see Figure 9). Both interactions account for 15.9% and 15.4%, respectively, of the total attractive AMBER energy of Site III. Additional interactions are made, for example, through Q87 of the receptor Ig domain, which forms a hydrogen-bonding interaction with a side chain amide to the G-CSF Site III backbone and has Lennard Jones interactions with surrounding side chains of the cytokine, such as E46 and L49. This interaction accounts for 12.3% of the total attractive AMBER energy of Site III.

[0393] Next, G-CSF EThe variants are related to their co-evolved G-CSFR(Ig) E Mutants (pair interactions) were positively designed in silico to have favorable AMBER binding affinity at site III, for example, by charge reversal or shape complementarity alteration while maintaining Lennard Jones and hydrogen bond interactions. E :G-CSFR(Ig) E were packed using the mean-field packing workflow in ZymeCAD™. The in silico models of the packed Site III designs were visually inspected for structural integrity and evaluated using the ZymeCAD™ metrics described in Example 1. Next, the G-CSF of each design was E The mutants were analyzed by ZymeCAD™ to identify WT G-CSFR(Ig) (and its reverse G-CSFR(Ig) E and G-CSF WT ) and metrics were evaluated under conditions of mispairing with the WT cytokine and receptor. For Site III designs using ZymeCAD™, as described above in Example 1 for Site II, ddAMBER affinity metrics (G-CSF WT :G-CSFR(Ig) E , G-CSF E :G-CSFR(Ig) WT ) was calculated (see Table 6).

[0394] Clustered site III design and all three in silico complexes of G-CSF E :G-CSFR(Ig) E , G-CSF WT :G-CSFR(Ig) E , G-CSF E :G-CSFR(Ig) WT Packing metrics were considered in conjunction with visual inspection to assess pairing strength and selectivity against mispairing with WT to rank designs.

[0395] result The designs in Table 3 are G-CSF E :G-CSFR(Ig) E and has in silico packing metrics in ZymeCAD™ that favor pairing with WT G-CSF or G-CSFR(Ig), and is highly selective against mispairing with WT G-CSFR or G-CSFR(Ig).

[0396] Therefore, various site III mutations of the same variant G-CSF and receptor designs shown in Table 3 are predicted to exhibit preferential binding over wild-type G-CSFR and G-CSF, respectively.

[0397] [Table 6]

[0398] Example 4: Combining Sites II and III to Create a Variant, Co-Evolved Cytokine Receptor Switch A fully selective design pair, G-CSF, that allows binding and signaling through a 2:2 heterodimeric engineered complex with reduced or completely eliminated cross-reactivity with wild-type cytokines or receptors. E :G-CSF (Ig-CRH) E Selected Site II and III designs from Examples 1 and 3 were combined (see Table 4) and tested in vitro to develop

[0399] Similar to combining the designs in Table 4, any other combination of the Site II design in Example 1 (Table 2) and the Site III design in Example 3 (Table 3) can produce fully selective G-CSF variants that allow for variant signaling. E :G-CSFR (Ig-CRH) E Combination designs 401 and 402 were used to compare engineered G:CSF in the co-expression assay described in Example 2. E :G-CSFR (Ig-CRH) E The ability to form complexes and to bind WT cytokine or receptor was tested.

[0400] method Coexpression assays involving pull-down via the cytokine TST tag were performed as described in Example 2 above, except that the receptor construct contained Ig and CRH domains (residues 2-308, Table 1), designated G-CSFR(Ig-CRH).

[0401] result Combinatorial designs 401 and 402 were completely selective in co-expression assays in that the design cytokines pulled down their co-evolved, engineered receptors but not the WT receptor, and conversely, WT G-CSF did not pull down the engineered receptor (see Figure 10).

[0402] These results demonstrate that variant G-CSFs, as well as receptors containing variant G-CSFR ECD designs combining selected site II and site III mutants, are capable of specifically binding to the engineered cytokine-receptor pair, respectively, and do not bind to the wild-type receptor or cytokine.

[0403] Example 5: Production of G-CSF and G-CSFR wild-type and mutants To produce wild-type and engineered cytokine and receptor variants and compare their biophysical properties, recombinant proteins were expressed and purified from insect cells.

[0404] method G-CSF E and G-CSF WT was cloned as described above. Preparative-scale production of recombinant protein was performed in 2-4 L of healthy log-phase Tni cells as follows: 2 x 10 6 cells ml -1800 mL of Tni at 100°C was inoculated with 20 μl of cytokine-variant P2 virus per 2 mL of cells and incubated at 27°C for 70 h with shaking at 135 rpm. After incubation, cells were pelleted by centrifugation at 5500 rpm for 15 min, and the supernatant was filtered twice, first through a 1 μm A / E-type glass fiber filter (PALL, cat. 61631) and then through a 0.45 μm PVDF membrane filter (Sigma Aldrich, cat. HVLP04700). Protease inhibitor cocktail III (Sigma Aldrich, cat. 539134) was added, and the supernatant buffer was exchanged into HBS (20 mM HEPES pH 8, 150 mM NaCl) and concentrated to 300 mL by tangential flow. Protein was purified in batch mode using 3 x 3 mL bv Streptactin-XT superflow tubes, incubated at 4°C for 2 x 1 h and 1 x overnight with agitation. The resin was washed with 10 CV of HBS buffer before elution. The protein was eluted with 4 x 5 mL of BXT elution buffer. The eluate was analyzed by nanodrop A280 and reduced SDS-PAGE, concentrated to approximately 2 mL, and the TST purification tag was cleaved by end-over-end incubation with TEV at a TEV:protein ratio of 1:80 overnight at 18°C. The cleaved protein was confirmed by SDS-PAGE before loading onto an SX75 16 / 600 or SX200 16 / 600 size-exclusion column (GE Healthcare, cat. 28-9893-33 or 28-9893-35) equilibrated with 20 mM BisTris pH 6.5, 150 mM NaCl (see Figure 11). Protein-containing fractions were analyzed by reduced SDS-PAGE, pooled, and the concentration determined by nanodrop A280 measurement.

[0405] For protein purification of receptor variants, wild-type and G-CSFR (Ig-CRH) EMutants were cloned as described above, and the receptor construct for purification contained an Ig domain in addition to the CRH domain (residues 3-308 of Uniprot ID Q99062, Table 1). Virus stocks were prepared and used for infection at a 2-4 L scale as described above. The clarified supernatant was buffer-exchanged into Ni-NTA binding buffer (20 mM HEPES pH 8, 1 M NaCl, 30 mM imidazole) and concentrated to 300 mM as described above. The protein was purified in batch binding mode using 3 x 3 mL bv Ni-NTA superflow tubes, incubated at 4 °C for 2 x 1 h and 1 x overnight with agitation. The resin was washed with 10 CV of binding buffer before elution. The protein was eluted with 4 x 5 mL Ni-NTA elution buffer (20 mM HEPES pH 8, 1 M NaCl, 250 mM imidazole). The eluate was analyzed, buffer exchanged into 20 mM Bis-Tris pH 6.5, 150 mM NaCl, concentrated, and cleaved overnight as described above. The cleaved protein was then loaded onto an SX75 16 / 600 or SX200 16 / 600 size-exclusion column (GE Healthcare) equilibrated with 20 mM Bis-Tris pH 6.5, 150 mM NaCl (see Figure 12). Protein-containing fractions were analyzed by reducing SDS-PAGE, pooled, and the concentration determined by nanodrop A280 measurement.

[0406] result Wild type, G-CSF E , and G-CSFR E The variants were greater than 90% pure after SEC as judged by reducing SDS-PAGE (see Figures 11 and 12). G-CSF of Designs 401 and 402 E The yields after SEC per 1 L of culture for Designs 401 and 402 were 2.7 mg and 1.6 mg, respectively. E The post-SEC yields per L of production for the WT G-CSF and WT G-CSFR were 1.7 mg and 1.5 mg, respectively. WT G-CSF was purified to a post-SEC yield of 2.1 mg per L of culture, and WT G-CSFR was purified to a post-SEC yield of 3.1 mg per L of culture.

[0407] These results confirm that the methods used to purify the variant G-CSF and receptor were efficient in obtaining purified proteins that could be used for analysis of biophysical properties in vitro.

[0408] Example 6: Determining the affinity of designs for paired and mispaired binding partners by SPR To determine the affinity of the designed cytokines for the co-evolved receptor variants, we used G-CSFR E G-CSF against E The affinity of G-CSF was also measured. E and G-CSFR WT affinity of G-CSF WT and G-CSFR E The affinity of was measured for a subset of designs (mispaired) by SPR.

[0409] method SPR binding assays were performed on a Biacore T200 instrument (GE Healthcare, Mississauga, ON, Canada) using a PBS-T (PBS + 0.05% (v / v) Tween 20) running buffer at 25°C. CM5 Series S sensor chips, Biacore amine coupling kit (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 immobilization orientations.

[0410] G-CSF E G-CSFR E To determine the binding affinity to G-CSFR E The mutants were captured by standard amine coupling as described by the manufacturer (GE LifeSciences). Briefly, immediately after EDC / NHS activation, G-CSFR 1 was lysed in 10 mM NaOAc, pH 5.0. EA 5 μg / mL solution of G-CSF was injected at a flow rate of 5 μL / min 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 seconds. Single-cycle kinetics was used to quench the corresponding G-CSF starting from 200 nM at six concentrations. E A two-fold dilution series of the variants and a blank buffer control were injected sequentially at 25 μL / min for 300 seconds, with a dissociation phase of 1800 seconds, to obtain a series of sensorgrams with the buffer blank reference. The same sample titration was also performed on a reference cell in which no variants were captured. The chip was regenerated for the next injection cycle with one pulse of 10 mM glycine / HCl, pH 2.0 (30 μL / min for 30 seconds).

[0411] G-CSF WT G-CSFR E To evaluate the binding affinity to G-CSF E were captured on the chip at a density of approximately 700-900 RU as described above. Six concentrations of each G-CSFR were assayed starting at 200 nM using single-cycle kinetics. WT A two-fold dilution series of and a blank buffer control were injected sequentially at 25 μL / min for 300 seconds, with a total dissociation time of 1800 seconds, to obtain a series of sensorgrams with the buffer blank reference. The same sample titration was also performed on a reference cell where no variants were captured and the chip was regenerated as described above.

[0412] G-CSF E G-CSFR WT To assess binding affinity to G-CSFR, purified recombinant G-CSFR was used as described in Example 5. WT were captured as described in this example using single-cycle kinetics at six concentrations of each G-CSF starting at 200 nM. E Two-fold dilutions of the variants and a blank buffer control were injected sequentially as described above. The same sample titrations were also performed in a reference cell where no variants were captured. WT The surface was regenerated as described above.

[0413] As a control, binding of WT G-CSF to WT G-CSFR (Ig-CRH) was assessed in each experiment, and the K within each independent measurement was calculated. D was used to calculate fold changes.

[0414] Double-referenced sensorgrams from two or three replicate injections were analyzed using Biacore™ T200 evaluation software v3.0 and fitted to a 1:1 Langmuir binding model.

[0415] result The kinetically derived affinity constants (K D ) was obtained. The K D The KD fold change ranged from 1.8 to 2.5E-9. When kinetic parameters could not be fitted, we attempted to derive steady-state affinity constants. In these cases, the KD fold change was calculated from the KD derived from the steady-state of the WT G-CSF:WT G-CSFR(Ig-CRH) pair. D and are shown in Table 7.

[0416] Designs 9, 130, 134, 137, 307, 401, and 402 exhibited affinities for their co-evolved binding partners that were within a 2-fold difference of the WT:WT KD (see Table 7 and Figure 13).

[0417] G-CSFR(Ig-CRH) in designs 9, 30, and 34 E The mutants showed over 700-fold weaker affinity for WT G-CSF compared to WT G-CSFR(Ig-CRH). G-CSFR(Ig-CRH) of Design #35 E were less selective for mispairing with the WT cytokine, with affinity for WT G-CSF reduced approximately 19-fold compared to WT:WT affinity. EThe mutant was most selective for mispairing with WT G-CSF and showed no significant binding to WT G-CSF at the concentrations titrated (see Table 8, Figure 13).

[0418] G-CSF in designs 124, 130, 401, 402, 300, 303, 304, and 307 E The mutants did not show significant binding to WT G-CSFR (Ig-CRH) at the titrated concentrations. G-CSF of Designs 9, 30, and 34 E The mutants exhibited at least approximately 20-fold weaker K activity against WT G-CSFR(Ig-CRH) compared to WT:WT KD. D Design #134 G-CSF E showed approximately 500-fold weaker affinity to WT G-CSFR (Ig-CRH) (see Table 9, Figure 13). E The mutants exhibit binding to WT G-CSFR(Ig-CRH) similar to WT:WT KD.

[0419] These results indicate that the selected variant G-CSF designs either do not bind to the wild-type G-CSFR ECD or bind with significantly reduced affinity to the wild-type G-CSFR ECD (at least approximately 20-fold weaker KD).

[0420] [Table 7]

[0421] [Table 8]

[0422] [Table 9]

[0423] Example 7: Design G-CSF E Measurement of mutant thermostability G-CSF compared to WT cytokine and receptor E and G-CSFR E To measure the thermal stability of the mutants, differential scanning calorimetry (DSC) was performed.

[0424] method The thermal stability of the variants was assessed by differential scanning calorimetry (DSC) as follows: 950 mL of purified sample at concentrations of 1–2 mg / mL was used for DSC analysis using a Nano DSC (TA Instruments, New Castle, DE). At the start of each run, a buffer blank was injected to allow baseline stabilization. Each sample was scanned from 25 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), an indicator of thermal stability.

[0425] result The thermal stability of the engineered variants is reported as the difference in the most significant transition (highest enthalpy) between the engineered molecule and the equivalent wild-type molecule measured under the same conditions and experimental settings. The measured Tm of WT G-CSFR varied between 52.2 and 55.4 °C between independent experiments, while the Tm of WT G-CSFR was 50.5 °C. All G-CSFs tested except for designs #15 and #34. E The mutants exhibited Tm's that differed by less than 5°C from that of WT G-CSF (see Table 10 and Figure 14). All receptor mutants tested exhibited the same thermal stability as the WT receptor (see Table 10 and Figure 14).

[0426] These results demonstrate that receptors with variant G-CSF and variant G-CSFR ECD designs have similar thermostabilities to wild-type G-CSF and G-CSFR, and that site II and / or site III mutations do not impair the thermostability of either the G-CSF or G-CSFR ECDs.

[0427] [Table 10]

[0428] Example 8: G-CSF by UPLC-SEC E Determining the monodispersity of mutants G-CSF compared to WT G-CSF E To determine the monodispersity of the variants, the variants were analyzed by UPLC-SEC.

[0429] method UPLC-SEC was performed on SEC-purified protein samples using an Acquity BEH125 SEC column (4.6 x 150 mm, stainless steel, 1.7 μm particles) (Waters LTD, Mississauga, ON) set at 30 °C and attached to an Agilent Technologies 1260 Infinity II system equipped with a PDA detector. The analysis consisted of a 7-minute run time at a flow rate of 0.4 mL / min using a running buffer of 150 mM NaCl, 20 mM HEPES pH 8.0 or 150 mM NaCl, 20 mM BisTris pH 6.5. Elution was monitored by UV absorbance in the range of 210–500 nm, and chromatograms were extracted at 280 nm. Peak integration was performed using OpenLAB™ CDS ChemStation™ software.

[0430] result WT G-CSF and G-CSF of designs 34, 35, and 130 E The variants were 100% monodisperse (see Table 11). Variants 8, 9, 15, 117, 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 monodispersity at lower mobile phase pH was due to a shift in pI (e.g., from a calculated pI of 5.41 for WT G-CSF to 8.5 for G-CSF in design #134). E This may be due to the calculated pI of 8.35.

[0431] These results showed that some of the variant G-CSF designs had 100% monodispersity, the same as wild-type G-CSF, suggesting that a subset of site II and / or site III mutants did not impair the monodispersity of G-CSF, whereas other variant G-CSF designs exhibited lower monodispersity that increased at lower pH.

[0432] [Table 11]

[0433] Example 9: Construction of a chimeric G-CSF receptor with an intracellular IL-2 receptor signaling domain Design G-CSF E Cytokine mutant designed G-CSFR (Ig-CRH) E To investigate whether receptor variants can signal through and induce immune cell proliferation, we investigated the G-CSFR ECD (G-CSFR ECD) fused to the gp130 transmembrane (TM) domain and intracellular signaling domain (ICD), as well as the IL-2Rβ intracellular signaling domain (IL-2Rβ). WT We constructed a single-chain chimeric G-CSF receptor using the G-CSFR-ICDgp130-IL-2Rβ complex. We also utilized a chimeric G-CSFR consisting of two subunits designed to co-express as a heterodimeric receptor. 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 subunit consists of a G-CSFR ECD fused to the common gamma chain (γc, IL-2Rγ) TM and ICD.

[0434] method 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 confirmed by Sanger sequencing. The transfer plasmids and lentiviral packaging plasmids (psPAX2, pVSVG) were cotransfected into the lentiviral packaging cell line HEK293T / 17 cells (ATCC) as follows: Cells were plated overnight in DMEM containing 10% fetal bovine serum and penicillin / streptomycin, and the medium was changed 2–4 h before transfection. Plasmid DNA and water were mixed in a polypropylene tube, and CaCl2 (0.25 M) was added dropwise. After 2–5 min of incubation, the DNA was precipitated by mixing 1:1 with 2x HEPES-buffered saline (0.28 M NaCl, 1.5 mM Na2HPO4, 0.1 M HEPES). The precipitated DNA mixture was added to the cells and incubated overnight at 37°C and 5% CO2. The following day, the HEK293T / 17 medium was replaced, and the cells were incubated for an additional 24 h. The following morning, the cell supernatant was collected from the plate, briefly centrifuged to remove debris, and filtered through a 0.45 μm filter. The supernatant was spun at 25,000 rpm for 90 min in the SW-32Ti rotor of a Beckman Optima L-XP ultracentrifuge. The supernatant was removed, and the pellet was resuspended in an appropriate volume of Opti-MEM medium. Viral titers were determined by adding serial dilutions of virus to BAF3 cells (growth medium RPMI containing 10% fetal bovine serum, penicillin, streptomycin, and 100 IU / ml hIL-2).Forty-eight to seventy-two hours after transduction, cells were incubated with anti-human G-CSFR APC-conjugated antibody (1:50 dilution) and eBioscience™ Fixable Viability Dye eFluor™ 450 (1:1000 dilution) for 15 minutes at 4°C, washed, and analyzed using a Cytek Aurora or BD FACS Calibur flow cytometer. The estimated titer determined by this method was used to transduce the 32D-IL-2Rβ cell line (grown in RPMI containing 10% fetal bovine serum, penicillin, streptomycin, and 300 IU / ml hIL-2) with lentiviral supernatant encoding the chimeric receptor construct at an MOI of 0.5. Transduction was performed by adding the appropriate amount of viral supernatant to the cells, incubating for 24 hours, and then replacing the cell medium. Three to four days after transduction, human G-CSFR expression was confirmed by flow cytometry as described above. Cells were grown in G-CSF WT for approximately 14–28 days before BrdU assays were performed.

[0435] As mentioned above, G-CSF WT 32D-IL-2Rβ cells grown in PBS were washed three times with PBS and treated with the relevant assay cytokines (no cytokine, hIL-2 (300 IU / ml), G-CSF WT (30ng / ml) or G-CSF E Cells were replated with fresh medium containing BrdU (30 ng / ml) for 48 hours. The BrdU assay procedure was performed according to the BD Pharmingen™ APC BrdU Flow Kit (557892) instructions, with the addition of the following: cells were co-incubated with BrdU and eBioscience™ Fixable Viability Dye eFluor™ 450 (1:5000) for 30 minutes. Analytical flow cytometry was performed using a Cytek Aurora instrument.

[0436] result In the BrdU assay, the single-chain chimeric receptor construct G-CSFR WT -ICD gp130-IL-2Rβ or heterodimeric receptor construct G-CSFRWT -ICD IL-2Rβ +G-CSFR WT -ICD γc Cells transfected with G-CSF WT (30 ng / ml) showed comparable or superior proliferation compared to hIL-2 (300 IU / ml). Cells did not proliferate in the absence of cytokines (see Figure 15).

[0437] These results indicate that upon stimulation with G-CSF, both single-chain and heterodimeric chimeric receptor constructs can be activated to induce cell proliferation.

[0438] Example 10: G-CSFR of Design 137 probed by BrdU E -ICD IL-2 Transduced with wild-type or engineered G-CSF 137 Proliferation of 32D-IL-2Rβ cells treated with Site II / III combination designs that were sufficiently selective as judged by SPR or coexpression assays were tested in vitro for their ability to induce proliferation of 32D-IL-2Rβ cells.

[0439] method Design 137 point mutations were introduced into the constructs listed in Tables 12-14. 137 -ICD gp130-IL-2Rβ (homodimer) or G-CSFR 137 -ICD IL-2Rβ +G-CSFR 137 -ICD γc Cloning and expression of the (heterodimer) constructs followed the same procedures as described above. Before performing the BrdU assay, cells were incubated with G-CSF. 137 The cells were grown in the presence of HCl for approximately 14 to 28 days.

[0440] G-CSF 137 32D-IL-2Rβ cells grown in G-CSF were analyzed using the BrdU assay procedure described above. 137 (30ng / ml), G-CSF WT(30 ng / ml), hIL-2 (300 IU / ml), or assayed for proliferation in the absence of cytokines.

[0441] result In the BrdU assay, the single-chain chimeric receptor construct G-CSFR 137 -ICD gp130-IL-2Rβ or heterodimeric receptor construct G-CSFR 137 -ICD IL-2Rβ +G-CSFR 137 -ICD γc Cells transduced with G-CSF 137 Cells did not proliferate in the absence of cytokines, and G-CSF (30 ng / ml) induced comparable or superior proliferation compared to hIL-2 (300 IU / ml). WT (30 ng / ml) showed poor growth (Figure 16).

[0442] These results indicate that variant G-CSF specifically activates the engineered receptor; conversely, the engineered receptor is activated by variant G-CSF, but less significantly than wild-type G-CSF. Thus, variant G-CSF can specifically activate the chimeric receptor via the variant G-CSFR ECD and specifically induce proliferation of cells expressing the chimeric receptor.

[0443] Example 11: Wild-type G-CSFR-ICD investigated by BrdU IL-2 Transduced with wild-type or engineered G-CSF E Proliferation of 32D-IL-2Rβ cells treated with Subsequently, a site II / III combination design that was able to restore paired signaling in 32D-IL-2R2Rβ cells was identified using the single-chain chimeric receptor construct G-CSFR. WT -ICD gp130-IL-2Rβ or heterodimeric receptor construct G-CSFR WT -ICD IL-2Rβ +G-CSFR WT -ICD γc The IL-2Rβ transduced 32D-IL-2Rβ cells were then tested for their ability to induce proliferation of IL-2Rβ transduced 32D-IL-2Rβ cells.

[0444] method G-CSFR WT -ICD gp130-IL-2Rβ (homodimer) or G-CSFR WT -ICD IL-2Rβ +G-CSFR WT -ICD γc Cloning and expression of the (heterodimer) constructs followed the same procedures as described above. Before performing the BrdU assay, cells were incubated with G-CSF. WT The cells were grown in the presence of HCl for approximately 14 to 28 days.

[0445] G-CSF WT 32D-IL-2Rβ cells grown in G-CSF were analyzed using the BrdU assay procedure described above. 137 (30ng / ml), G-CSF WT (30 ng / ml), hIL-2 (300 IU / ml), or assayed for proliferation in the absence of cytokines.

[0446] result In the BrdU assay, 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 Cells transduced with G-CSF 137 G-CSF (30ng / ml) WT The cells showed poor proliferation compared to the control (30 ng / ml). Cells did ...

Claims

1. A variant granulocyte colony stimulating factor (G-CSF), 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; the at least one mutation in the Site II interface region comprises at least one of L108K, D112R, E122R, E122K, E123K, and E123R relative to the corresponding amino acid position of the sequence set forth in SEQ ID NO:1; The at least one mutation in the Site III interface region comprises a mutation E46R relative to the corresponding amino acid position in the sequence shown in SEQ ID NO:1; and the variant G-CSF selectively binds to a receptor comprising a variant extracellular domain (ECD) of the granulocyte colony-stimulating factor receptor (G-CSFR); The variant G-CSF.

2. the receptor comprising a 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; Optionally, said at least one mutation in the Site II interface region of said G-CSFR ECD comprises one or both of an R141E or an R167D mutation, and said at least one mutation in the Site III interface region of said G-CSFR ECD comprises an R41E mutation, and said variant G-CSFR mutations correspond to amino acid positions in the sequence set forth in SEQ ID NO:2, and optionally said receptor comprising said variant G-CSFR ECD is a chimeric receptor or said G-CSFR is chemically modified. A variant G-CSF according to claim 1.

3. A system for selectively activating a receptor expressed on a cell surface, comprising: (a) a variant G-CSF according to any one of claims 1 to 2, (b) a receptor comprising a variant ECD of G-CSFR, wherein the variant G-CSF selectively binds to the receptor comprising the variant ECD of G-CSFR; and optionally (c) one or more additional agonist or antagonist signaling proteins or antigen-binding signaling receptors. Including, i) said variant G-CSF comprises the mutations E46R, L108K, D112R, E122R and E123R relative to the corresponding amino acid positions of the sequence shown in SEQ ID NO:1, and said receptor comprising a variant ECD of G-CSFR comprises the mutations R41E, R141E and R167D relative to the corresponding amino acid positions of the sequence shown in SEQ ID NO:2; ii) said variant G-CSF comprises the mutations E46R, L108K, D112R and E122K relative to the corresponding amino acid positions of the sequence shown in SEQ ID NO:1, and said receptor comprising a variant ECD of G-CSFR comprises the mutations R41E, R141E and R167D relative to the corresponding amino acid positions of the sequence shown in SEQ ID NO:2; iii) said variant G-CSF comprises the mutations E46R, L108K, D112R and E123K relative to the corresponding amino acid positions of the sequence shown in SEQ ID NO:1, and said receptor comprising a variant ECD of G-CSFR comprises the mutations R41E, R141E and R167D relative to the corresponding amino acid positions of the sequence shown in SEQ ID NO:2; iv) the variant G-CSF comprises the mutations E46R, L108K, D112R, and E122R relative to the corresponding amino acid positions of the sequence shown in SEQ ID NO:1, and the receptor comprising a variant ECD of G-CSFR comprises the mutations R41E, R141E, and R167D relative to the corresponding amino acid positions of the sequence shown in SEQ ID NO:2; v) said variant G-CSF comprises the mutations E46R, L108K, D112R and E123R relative to the corresponding amino acid positions of the sequence shown in SEQ ID NO:1, and said receptor comprising a variant ECD of G-CSFR comprises the mutations R41E, R141E and R167D relative to the corresponding amino acid positions of the sequence shown in SEQ ID NO:2; vi) said variant G-CSF comprises the mutations E46R, L108K, D112R, E122K, and E123K relative to the corresponding amino acid positions of the sequence shown in SEQ ID NO:1, and said receptor comprising a variant ECD of G-CSFR comprises the mutations R41E, R141E, and R167D relative to the corresponding amino acid positions of the sequence shown in SEQ ID NO:2, or vii) the variant G-CSF corresponds to SEQ ID NO: 83 or 84; The system.

4. Use of the variant ECD of G-CSFR and variant G-CSF of claim 1 or 2 for selectively activating a receptor expressed on the surface of a cell.

5. 3. Use of the variant ECD of G-CSFR and variant G-CSF according to claim 1 or 2 for increasing an immune response in a subject in need thereof or for treating a disease in a subject in need thereof, comprising: administering a cell expressing a receptor comprising a variant ECD of G-CSFR; administering or providing said variant G-CSF; Including, Optionally, the disease is cancer, an inflammatory condition, an autoimmune disease, a degenerative disease, or an infectious disease; The use optionally comprises: a) one or more cell populations each expressing either or both of (i) a distinct chimeric receptor comprising a G-CSFR ECD or (ii) at least one distinct variant form of G-CSF. administering Optionally, at least one population of cells further expresses at least one distinct antigen-binding signaling receptor or an additional agonist or antagonist signaling protein. said administering, or b) administering to the subject one or more additional agonist or antagonist signaling proteins; or c) (i) isolating an immune cell-containing sample; (ii) transducing or transfecting said immune cells with a nucleic acid sequence encoding a receptor comprising a variant ECD of G-CSFR; (iii) administering said immune cells of (ii) to said subject; and (iv) contacting said immune cells with said variant G-CSF that selectively binds to said receptor; or d) administering immune cells that have been contacted in vitro with the variant G-CSF prior to administering the immune cells to the subject. Including, The above uses.

6. A chimeric receptor comprising: (a) an extracellular domain (ECD) operably linked to at least one second domain; wherein the second domain is: (b) an intracellular domain (ICD) comprising at least one signaling molecule binding site derived from the intracellular domain of a cytokine receptor, the at least one signal transduction molecule binding site is selected from the group consisting of an SHC binding site of interleukin (IL)-2Rβ, a STAT5 binding site of IL-2Rβ, an IRS-1 or IRS-2 binding site of IL-4Rα, a STAT6 binding site of IL-4Rα, an SHP-2 binding site of glycoprotein 130 (gp130), a STAT3 binding site of gp130, an SHP-1 or SHP-2 binding site of erythropoietin receptor (EPOR), a STAT5 binding site of EPOR, a STAT1 or STAT2 binding site of interferon alpha and beta receptor subunit 2 (IFNAR2), and a STAT1 binding site of interferon gamma receptor 1 (IFNγR1), or a combination thereof; the ICD further comprises at least one Box 1 domain and at least one Box 2 domain of at least one protein selected from the group consisting of G-CSFR, gp130, EPOR, and interferon gamma receptor 2 (IFNγR2), or a combination thereof; The ICD Including; And (c) at least one third domain comprising a transmembrane domain (TMD), The ECD is N-terminal to the TMD, and the TMD is N-terminal to the ICD. The at least one third domain.

7. An ECD operably linked to a second domain. A chimeric receptor comprising: The second domain comprises an ICD, the ICD comprising: (i) (a) Box 1 and Box 2 regions of G-CSFR; (b) at least one signaling molecule binding site of IL-4Rα; or (ii) (a) Box 1 and Box 2 regions of gp130; (b) at least one signaling molecule binding site of gp130; or (iii) (a) Box 1 and Box 2 domains of the erythropoietin receptor (EPOR); (b) at least one signal transduction molecule binding site of EPOR; or (iv) (a) Box 1 and Box 2 regions of G-CSFR; (b) at least one signaling molecule binding site for interferon alpha and beta receptor subunit 2 (IFNAR2); or (v) (a) Box 1 and Box 2 regions of interferon gamma receptor 2 (IFNγR2); (b) at least one signaling molecule binding site for interferon gamma receptor 1 (IFNγR1); Including, The ECD is N-terminal to the TMD, and the TMD is N-terminal to the ICD. The chimeric receptor.

8. The active form of the chimeric receptor forms a homodimer, and optionally Activation of the chimeric receptor induces a cellular response comprising at least one of proliferation, viability, persistence, cytotoxicity, cytokine secretion, memory, and enhanced activity of cells expressing the receptor, and optionally the chimeric receptor is activated upon contact with G-CSF, and optionally wherein the G-CSF is a wild-type G-CSF, and optionally the extracellular domain of the G-CSFR is a wild-type extracellular domain; A chimeric receptor according to claim 6 or 7.

9. The chimeric receptor of claim 8, wherein the G-CSF is wild-type G-CSF and the extracellular domain of the G-CSFR is a wild-type extracellular domain.

10. The method of claim 1, wherein the ECD is an ECD of G-CSFR, and optionally the ECD of G-CSFR binds to a variant G-CSF, wherein: (i) 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; the at least one mutation in the Site II interface region comprises at least one of L108K, D112R, E122R, E122K, E123K, and E123R, and combinations thereof, relative to the corresponding amino acid positions of the sequence set forth in SEQ ID NO:1; The at least one mutation in the Site III interface region comprises a mutation E46R relative to the corresponding amino acid position in the sequence shown in SEQ ID NO:1; and the variant G-CSF selectively binds to a receptor comprising a variant extracellular domain (ECD) of the granulocyte colony-stimulating factor receptor (G-CSFR); (ii) 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; Optionally, the at least one mutation in the Site II interface region of the G-CSFR ECD comprises one or both of an R141E or R167D mutation; and the at least one mutation in the Site III interface region of the G-CSFR ECD comprises an R41E mutation; and the mutations in said variant G-CSFR correspond to the amino acid positions in the sequence shown in SEQ ID NO:2, or the TMD is a G-CSFR TMD, or the active form of the chimeric receptor forms a homodimer; or the chimeric receptor is activated upon contact with G-CSF; or the chimeric receptor is expressed in a cell, optionally an immune cell, or The ICD comprises: (a) one or both of the amino acid sequences of SEQ ID NO: 90 or 91; or (b) one or both of the amino acid sequences of SEQ ID NO: 90 or 92; or (c) the amino acid sequence of SEQ ID NO: 93; or (d) the amino acid sequence of SEQ ID NO: 94; or (e) one or both of the amino acid sequences of SEQ ID NO: 95 or 96; or (f) the amino acid sequence of SEQ ID NO: 97 or 98, or (g) the amino acid sequence of SEQ ID NO: 99 or 100. Contains, or The TMD comprises the sequence set forth in SEQ ID NO: 88; or the TMD is a G-CSFR TMD, or The TMD is a wild-type TMD; A chimeric receptor according to claim 6 or 7.

11. A system for selectively activating a receptor expressed on a cell surface, comprising: (a) a variant G-CSF according to claim 6 or 7, (b) the receptor comprising a variant ECD of G-CSFR according to claim 6 or 7; Including, the variant G-CSF preferentially binds to the receptor comprising the variant ECD of the G-CSFR compared to an otherwise identical wild-type G-CSFR ECD, and the receptor comprising the variant ECD of the G-CSFR preferentially binds to the variant G-CSF compared to an otherwise identical wild-type G-CSFR. The system.

12. 1. A method for selectively activating a chimeric receptor expressed on the surface of a cell, preferably an immune cell, comprising: Contacting the chimeric receptor of claim 6 or 7 with a cytokine that selectively binds to said chimeric receptor. and optionally the active form of the chimeric receptor forms a homodimer; or Activation of the chimeric receptor induces a cellular response in a cell expressing the receptor, comprising at least one of proliferation, viability, persistence, cytotoxicity, cytokine secretion, memory, and enhanced activity, and optionally the chimeric receptor is activated upon contact with the cytokine; or the chimeric receptor is activated upon contact with G-CSF; or a first population of immune cells expressing said chimeric receptor and a second population of immune cells expressing a cytokine that binds to said chimeric receptor, and optionally one or both of said populations of immune cells further express at least one distinct antigen-binding signaling receptor, an additional agonist or antagonist signaling protein, or an antigen-binding signaling receptor; or each of the first and second populations of immune cells expressing a distinct chimeric receptor comprising a distinct variant ECD of G-CSFR and a distinct variant G-CSF; The method.

13. 8. Use of the chimeric receptor of claim 6 or 7 for treating a subject in need thereof, comprising:

8. Administering to the subject a cell expressing the chimeric receptor of claim 6 or 7; providing to the subject a cytokine that specifically binds to the chimeric receptor; and optionally the active form of the chimeric receptor forms a homodimer; or Activation of the chimeric receptor results in a cellular response; or the cytokine is G-CSF; Optionally, i) said use comprises administering at least one additional agonist or antagonist signaling protein; or ii) the use comprises administering to the subject: One or more cell populations each expressing a distinct chimeric receptor and each expressing a distinct variant form of a cytokine, and optionally expressing the chimeric receptor further expressing at least one antigen-binding signaling receptor. or iii) said use comprises: (i) isolating an immune cell-containing sample; (ii) introducing a nucleic acid encoding said chimeric cytokine receptor into said immune cells; (iii) administering said immune cells of (ii) to said subject; and (iv) contacting said immune cells with said cytokine that binds to said receptor, optionally with G-CSF. The above uses.

14. A method for treating a cancer cell comprising administering to a patient a therapeutically effective amount of a therapeutically effective amount of a chimeric receptor comprising administering to said patient a therapeutically effective amount of a chimeric receptor for a cancer cell, comprising administering to said patient a therapeutically effective amount of a chimeric receptor for said cancer cell; (b) Instructions for use; A kit comprising: Optionally, the kit comprises: at least one cytokine that binds to said chimeric receptor; or One or more expression vectors encoding one or more cytokines or chemokines, antigen binding receptors, additional agonist or antagonist signaling proteins, antigen binding signaling receptors, CARs, or separate chimeric receptors selected from the group consisting of IL-18, IL-21, interferon alpha, interferon beta, interferon gamma, IL-17, IL-21, TNF-alpha, CXCL13, CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CD40 ligand, B cell activating factor (BAFF), Flt3 ligand, CCL21, CCL5, XCL1, or CCL19, or the receptor NKG2D, and combinations thereof. Including, The kit.

15. A nucleic acid encoding a chimeric receptor according to claim 1, 2, 6 or 7.

16. A vector comprising the nucleic acid described in claim 15.

17. Use of the nucleic acid described in claim 15 for gene editing, comprising introducing the nucleic acid described in claim 15 into the genome of the cell.

18. Use of a chimeric receptor described in claim 1, 2, 6, or 7 for treating cancer in a subject in need thereof.

19. Use of a chimeric receptor described in claim 1, 2, 6, or 7 for treating an autoimmune disease or disorder in a subject in need thereof. (i) a cell encoding a receptor comprising a variant ECD of G-CSFR, a variant G-CSF of claim 1, 2, 6, or 7, and instructions for use; or (ii) one or more nucleic acid sequences encoding a receptor comprising a variant ECD of G-CSFR, a variant G-CSF of claim 1, 2, 6, or 7, and instructions for use. Including the kit.

21. A system for selectively activating cells, optionally immune cells, comprising: (i) a receptor comprising a variant ECD of G-CSFR; and (ii) a variant G-CSF that selectively binds to the receptor of (i), and (iii) Below: (a) at least one additional agonist or antagonist signaling protein, optionally wherein said at least one additional agonist or antagonist signaling protein comprises one or more cytokines, chemokines, hormones, antibodies, or derivatives thereof, or other affinity reagents; and (b) at least one antigen-binding signaling receptor. Either or both of Including, 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; i) said at least one mutation in the Site II interface region is located at an amino acid position of said 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 the sequence set forth in SEQ ID NO:2; or ii) the at least one mutation in the Site II interface region of the G-CSFR ECD is selected from the group consisting of R141E, R167D, K168D, K168E, L171E, L172E, Y173K, Q174E, D197K, D197R, M199D, D200K, D200R, V202D, R288D, and R288E of the sequence set forth in SEQ ID NO:2; or iii) the at least one mutation in the Site III interface region of the G-CSFR ECD is selected from the group consisting of amino acid positions 30, 41, 73, 75, 79, 86, 87, 88, 89, 91, and 93 of amino acids 2 to 308 of the sequence set forth in SEQ ID NO:2; or iv) the at least one mutation in the Site III interface region of the G-CSFR ECD is selected from the group consisting of S30D, R41E, Q73W, F75KF, S79D, L86D, Q87D, I88E, L89A, Q91D, Q91K, and E93K of the sequence set forth in SEQ ID NO:2; The system.

22. 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; i) said at least one mutation in said site II interface region of said 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 the sequence shown in SEQ ID NO: 1, or ii) said at least one mutation in the site II interface region of said variant G-CSF is selected from the group of mutations selected from the group consisting of S12E, S12K, S12R, K16D, L18F, E19K, Q20E, D104K, D104R, L108K, L108R, D109R, D112R, D112K, T115E, T115K, T116D, Q119E, Q119R, E122K, E122R, and E123R, or iii) said at least one mutation in said Site III interface region of said variant G-CSF is selected from the group of mutations selected from the group consisting of 38, 39, 40, 41, 46, 47, 48, 49 and 147 of the sequence shown in SEQ ID NO: 1; or iv) said at least one mutation in the site III interface region of said variant G-CSF is selected from the group of mutations selected from the group consisting of: T38R, Y39E, K40D, K40F, L41D, L41E, L41K, E46R, L47D, V48K, V48R, L49K and R147E; A system for selectively activating cells according to claim 21.

23. (i) the ECD of interleukin receptor alpha (IL-7Rα); and (ii) a TMD; and (iii) a cytokine receptor ICD that is different from the wild-type human IL-7Rα intracellular signaling domain set forth in SEQ ID NO: 109; A chimeric receptor comprising: the ECD and TMD are each operably linked to the ICD; The chimeric receptor.

24. the carboxy terminus (C-terminus) of the ECD is linked to the amino terminus (N-terminus) of the TMD, and the C-terminus of the TMD is linked to the N-terminus of the ICD; and optionally the ECD is the ECD of native human IL-7Rα; or the TMD is a TMD of IL-7Rα; or The TMD is the TMD of native human IL-7Rα, or The ICD comprises at least one signaling molecule binding site derived from an intracellular domain of a cytokine receptor, and optionally the at least one signaling molecule binding site is (a) the Jak1-binding site of IL-2Rβ, IL-4Rα, IL-7Ra, IL-21R, or gp130 (Box 1 and 2 domains); (b) the SHC binding site of IL-2Rβ; (c) the STAT5 binding site of IL-2Rβ or IL-7Ra; (d) the STAT3 binding site of IL-21R or gp130; (e) the STAT4 binding site of IL-12Rβ2; (f) the STAT6 binding site of IL-4Rα; (g) the IRS-1 or IRS-2 binding site of IL-4Rα, and (h) the SHP-2 binding site of gp130; (i) the PI3K binding site of IL-7Rα; or a combination of these or optionally, The ICD comprises at least the intracellular signaling domain of a receptor that is activated by heterodimerization with the common gamma chain (gc), or optionally the ICD comprises at least an intracellular signaling domain of a cytokine receptor selected from the group consisting of IL-2Rβ (interleukin-2 receptor beta), IL-4Rα (interleukin-4 receptor alpha), IL-9Rα (interleukin-9 receptor alpha), IL-12R (interleukin-12 receptor), IL-21R (interleukin-21 receptor), gp130, and combinations thereof; The chimeric receptor of claim 23.

25. A chimeric receptor comprising an ECD and a TMD of IL-7Rα operably linked to an ICD, The ICD comprises: (i) (a) Box 1 and Box 2 regions of IL-2Rβ; (b) the SHC binding site of IL-2Rβ, and (c) the STAT5 binding site of IL-2Rβ, or (ii) (a) Box 1 and Box 2 regions of IL-7Rα; (b) the SHC binding site of IL-2Rβ, and (c) the STAT5 binding site of IL-2Rβ, or (iii) (a) Box 1 and Box 2 regions of IL-2Rβ; (b) the SHC binding site of IL-2Rβ; (c) the STAT5 binding site of IL-2Rβ, and (d) the STAT4 binding site of IL-12Rβ2, or (iv) (a) Box 1 and Box 2 regions of IL-7Rα; (b) the SHC binding site of IL-2Rβ; (c) the STAT5 binding site of IL-2Rβ, and (d) the STAT4 binding site of IL-12Rβ2, or (v) (a) the Box 1 and Box 2 regions of IL-21R, and (b) the STAT3 binding site of IL-21R; or (vi) (a) the Box 1 and Box 2 regions of IL-7Rα, and (b) the STAT3 binding site of IL-21R; or (vii) (a) Box 1 and Box 2 regions of IL-21R; (b) the STAT3 binding site of IL-21R, and (c) STAT5 and PI3 kinase binding sites of IL-7Rα; (viii) (a) Box 1 and Box 2 regions of IL-7Rα; (b) the STAT3 binding site of IL-21R, and (c) STAT5 and PI3 kinase binding sites of IL-7Rα; (ix) (a) Box 1 and Box 2 regions of IL-2Rβ; (b) the SHC binding site of IL-2Rβ; (c) the STAT5 binding site of IL-2Rβ, and (d) the STAT3 binding site of IL-21R; or (x) (a) Box 1 and Box 2 regions of IL-7Rα; (b) the SHC binding site of IL-2Rβ; (c) the STAT5 binding site of IL-2Rβ, and (d) the STAT3 binding site of IL-21R; or (xi) (a) Box 1 and Box 2 regions of IL-2Rβ; (b) the SHC binding site of IL-2Rβ; (c) the STAT5 binding site of IL-2Rβ; (d) the STAT4 binding site of IL12Rβ2, and (e) the STAT3 binding site of IL-21R; or (xii) (a) Box 1 and Box 2 regions of IL-7Rα; (b) the SHC binding site of IL-2Rβ; (c) the STAT5 binding site of IL-2Rβ; (d) the STAT4 binding site of IL12Rβ2, and (e) the STAT3 binding site of IL-21R; or (xiii) (a) Box 1 and Box 2 regions of IL-4Rα; (b) the IRS-1 or IRS-2 binding site of IL-4Rα, and (c) the STAT6 binding site of IL-4Rα, or (xiv) (a) Box 1 and Box 2 regions of IL-7Rα; (b) the IRS-1 or IRS-2 binding site of IL-4α, and (c) the STAT6 binding site of IL-4α, or (xv) (a) Box 1 and Box 2 regions of gp130; (b) the SHP-2 binding site of gp130, and (c) the STAT3 binding site of gp130, or (xvi) (a) Box 1 and Box 2 regions of IL-7α; (b) the SHP-2 binding site of gp130, and (c) STAT3 binding site of gp130 and optionally (b) is on the N-terminal side of (c), or (c) is on the N-terminal side of (b), or (c) is on the N-terminal side of (d), or (d) is on the N-terminal side of (c), or (d) is on the N-terminal side of (e), or (e) is N-terminal to (d); or optionally, The ICD comprises a sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 85-107; The chimeric receptor.

Images