Chimeric cytokine receptors and methods of use

Abstract

Nucleic acids are provided that encode chimeric cytokine receptors (CCRs) capable of signal transduction in the absence of a cognate cytokine. In some embodiments, one or more nucleic acids are provided that encode a first subunit of the chimeric cytokine receptor and a second subunit of the chimeric cytokine receptor. The first subunit comprises a first heterodimerization domain and a first cytokine receptor intracellular signaling domain (ICD). The second subunit comprises a second heterodimerization domain homologous to the first heterodimerization domain and a second cytokine receptor ICD. CCRs find use in a variety of contexts, including, but not limited to, increasing the persistence of therapeutic cells. Accordingly, methods of administering cell-based therapy are also provided, comprising administering therapeutic cells (e.g., CAR-T cells) expressing a CCR to a subject in need thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 401,437, filed August 26, 2022, which is incorporated herein by reference in its entirety.

[0002] Incorporation by reference of a sequence listing provided as a sequence listing XML file The Sequence Listing is provided herewith as Sequence Listing XML "STAN-1985WO_SEQ_LIST", created on August 24, 2023, and having a size of 109,370 bytes. The contents of the Sequence Listing XML file are incorporated herein by reference in their entirety.

[0003] Introduction Cytokines are intercellular signaling molecules that support intercellular communication in the immune response and stimulate cell migration toward sites of inflammation, infection, and trauma. The downstream effects of a particular cytokine result from its high-affinity binding to its receptor expressed on the surface of target cells. This action can occur in an autocrine (acting on the same cell), paracrine (acting on nearby cells), or endocrine (acting on distant cells) manner. Receptor engagement triggers intracellular signaling cascades, causing changes in gene expression in target cells and resulting in biological effects. Cytokines can be divided into several categories, including interleukins (ILs), transforming growth factors (TGFs), interferons (IFNs), colony-stimulating factors (CSFs), tumor necrosis factors (TNFs), and chemokines.

[0004] Interleukins (ILs) are a group of cytokines expressed and secreted by white blood cells (leukocytes) and several other somatic cells. Interleukins and related cytokines function as a means of communication between innate and adaptive immune cells as well as non-immune cells and tissues. All IL-1 family members share a conserved beta-trefoil structure and bind to members of the IL-1 receptor (IL-1R) family. IL-1R family members contain an extracellular Ig-like domain and mediate signal transduction via an intracellular Toll / IL-1R (TIR) ​​domain.

[0005] The four-helical bundle cytokine superfamily is subdivided into class I and class II cytokine receptor families. Ligands for the class I cytokine receptor family include short-helical and long-helical cytokines. The short-helical cytokine family includes members of the common gamma chain and common beta chain families of cytokines. The common beta chain and common gamma chain cytokine families include cytokines such as IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-15, IL-21, and GM-CSF. Members of the common beta chain family signal through heterodimeric receptor complexes containing a common beta chain subunit, while members of the common gamma chain family signal through heterodimeric or heterotrimeric receptor complexes containing a common gamma chain subunit. The common gamma chain (γc) family consists of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 and was named for the binding of these factors to the γc receptor (CD132). They function primarily as growth and proliferation factors for precursor and mature cells and also have a role in lineage-specific cell differentiation.

[0006] Ligands for the class II cytokine receptor family include type I, type II, and type III interferons, as well as IL-10 family cytokines. Members of the IL-10 family cytokines share structural similarities and signal through heterodimeric receptor complexes with common subunits. Members of the type I, type II, and type III interferon families include IFN-alpha, IFN-beta, IFN-omega, IFN-epsilon, IFN-kappa, IFN-gamma, IL-28A, IL-28B, IL-29, and IFN-lambda 4. These cytokines primarily have antiviral, antiproliferative, and immunomodulatory effects.

[0007] IL-17 family cytokines belong to the cysteine ​​knot superfamily and bind to members of the IL-17 receptor family, which are primarily involved in promoting proinflammatory immune responses.

[0008] Members of the tumor necrosis factor (TNF) superfamily form homotrimers, or in some cases heterotrimers, and share a common extracellular domain known as the TNF homology domain (THD). Cytokines in the TNF superfamily bind to oligomeric type I or type II transmembrane proteins with multiple extracellular cysteine-rich domains. Many members of the TNF superfamily regulate apoptosis and / or immune cell functions, such as T cell costimulation, natural killer cell activation, and B cell homeostasis. Additionally, they can regulate cell-type-specific responses and play important roles in regulating the pathogenesis of certain diseases, including chronic inflammation, cancer, and autoimmune diseases.

[0009] Four short-helical bundle cytokines signal through class III receptor tyrosine kinases: M-CSF, SCF, Flt-3 ligand, and IL-34. The receptors for these cytokines, like the IL-1R family, contain extracellular Ig-like domains but possess cytoplasmic domains with tyrosine kinase activity. The long-helical cytokine family includes the IL-6 family cytokines, G-CSF, erythropoietin, thrombopoietin, growth hormone, prolactin, and leptin.

[0010] The four subfamilies of chemokines are the C, CX3C, CC, and CXC subfamilies, based on the number and spacing of conserved cysteine ​​residues located at their amino termini. Chemokines bind with some degree of promiscuity to conventional G protein-coupled seven-transmembrane receptors and play important roles in regulating cell migration during development and under homeostatic and inflammatory conditions. Summary of the Invention

[0011] Nucleic acids encoding chimeric cytokine receptors (CCRs) capable of signaling in the absence of a cognate cytokine are provided. In some embodiments, one or more nucleic acids encoding a first subunit of the chimeric cytokine receptor and a second subunit of the chimeric cytokine receptor are provided. The first subunit comprises a first heterodimerization domain and a first cytokine receptor intracellular signaling domain (ICD). The second subunit comprises a second heterodimerization domain homologous to the first heterodimerization domain and a second cytokine receptor ICD. According to some embodiments, signaling by the CCR is regulatable, for example, using a regulatable protease; in the absence of an inhibitor of the protease, the ICD is cleaved from one or both subunits of the CCR, thereby preventing signaling by the CCR. The CCRs find use in a variety of contexts, including, but not limited to, increasing the persistence of therapeutic cells. Accordingly, also provided are methods of administering cell-based therapy, comprising administering therapeutic cells expressing a CCR (such as, for example, CAR-T cells) to a subject in need thereof. [Brief explanation of the drawings]

[0012] [Figure 1]A: Receptors of the IL-2 family, consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. The receptors contain a common cytokine receptor gamma chain (CD132, γc). IL-13R shares the IL-4Rα with IL-4, and TSLPR shares the IL-7R with IL-7. B: Receptors for IL-3, IL-5, and GM-CSF are heterodimers of a unique α chain and a common β chain (βc, CD131) subunit. C: Receptors for IL-4 and IL-13 consist of two receptor chains, IL-4Rα (CD124) and γc. IL-4 and IL-13 bind to IL-4R, which consists of the IL-4Rα and IL-13Rα1 chains. The IL-13R consists of two subunits, IL-13Rα1 and IL-13Rα2, and signaling occurs via the IL-4R complex type II (composed of IL-4Rα and IL-13Rα). D: Based on their intron-exon structure, conserved secondary protein structure, and similarities with receptors of similar types, the following cytokines (IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28, and IL-29) have been classified as IL-10 family members. They share a common receptor subunit, as shown. E: TNF-α binds to TNFRI and TNFR2, and TGF-β binds to a heterodimeric receptor composed of TGF-βR1 and TGF-βR2. F: IL-12R consists of two subunits, IL-12Rβ1 and IL-12Rβ3. A heterodimer of IL-12Rβ1 and IL-23R binds IL-23. IL-12Rβ2 shows homology to the gp130 subunit of IL-27R. G: IFN-α and IFN-β bind to heterodimeric receptors consisting of IFNAR1 and IFNAR2. In addition, IFN-β binds to IFNAR1, and IFN-γ binds to the IFN-γR1 and IFN-γR2 heterodimer. Adapted from Akdis et al. (2016) J Allergy Clin Immunol. 138(4):984-1010. [Figure 2] Schematic diagram of a leucine zipper chimeric cytokine receptor (CCR). [Figure 3] Flow plot showing cell surface expression of CCR components for various common gamma chain receptors. [Figure 4] Schematic diagram of CCR engineering (transition domains shown for γC1 / γC3 and γC2 / γC4 are SEQ ID NOs: 54 and 55, respectively) and flow plots showing improved cell surface expression of CCRs. [Figure 5] Plot showing proliferation of primary human T cells transduced with the various CCRs shown in Figure 4. [Figure 6] Data showing that constitutive IL-2 CCR is a potent driver of proliferation in vivo and produces lethal toxicity in NSG mice. [Figure 7] Schematic diagram of a drug-regulated chimeric cytokine receptor (CCR) system. A protease cleavage site is incorporated between the transmembrane and intracellular signaling domains of the CCR. Coexpression of a protease specific to the cleavage site causes cleavage of the CCR due to the proteolytic activity of the protease at the cleavage site, resulting in inactivation of the CCR (receptor off). Addition of a protease inhibitor (drug) inhibits this cleavage event, turning the CCR into an on-state (receptor on). Thus, CCR signaling becomes dependent on the presence of the drug. The regulated CCR can avoid the toxicity associated with constitutive CCRs by modulating cell proliferation to fall within the therapeutic window. [Figure 8] A series of flow plots of primary human T cells transduced with a regulated form of the IL-2R CCR, as depicted in FIG. 7, showing cell surface expression of the HA / FLAG tag and phosphorylated STAT5 (pSTAT5), a cytokine signaling molecule downstream of IL-2R signaling (cleavage site indicated is SEQ ID NO: 56). [Figure 9] Amino acid sequence and data showing that insertion of a linker containing a cleavage site (SEQ ID NO: 57) upstream of the gamma chain ICD allows drug-regulated CCR signaling. [Figure 10]Flow plot of primary human T cells transduced with a regulated version of the IL-2R CCR showing phosphorylated STAT5 (pSTAT5). [Figure 11] Data showing drug control of cell expansion in vitro. [Figure 12] Schematic representation of CAR and CCR constructs and data showing that drug-modulated IL-2 CCR enhances in vivo antitumor activity after rechallenge. [Figure 13] Schematic of CAR and CCR constructs and data showing that drug-modulated IL-2 CCR expands CAR-T cells in vivo without toxicity. [Figure 14] Schematic of CAR and CCR constructs and data showing that CAR-T cells expressing drug-regulated IL-2 CCRs from two separate vectors have enhanced expansion in vivo in the presence of grazoprevir. [Figure 15] Flow plot showing the phosphorylation levels of STAT5 from primary human T cells transduced with an IL-2 CCR with or without the leucine zipper component (leucine zipper IL-2 CCR) or without (CD8a H / Tm IL-2 CCR). [Figure 16] Flow plot of IL-21R CCR showing phosphorylation of STAT3 (pSTAT3), a cytokine signaling molecule downstream of IL-21R signaling. [Figure 17] Flow plots of constitutive or protease-regulated IL-2R, IL-7R, and IL-9R CCRs expressed in primary human T cells. [Figure 18] Data showing that constitutive IL-2 CCR, IL-9 CCR, and IL-7 CCR induce lethal toxicity in mice due to unregulated proliferation of CAR-T cells. [Figure 19] Data showing controlled growth of CAR-T cells expressing protease-regulated CCRs. [Figure 20]Schematic diagram of an approach to reduce the "leaky" activity of protease-regulated CCRs through double-strand cleavage. A: Schematic diagram of the original drug-regulated chimeric cytokine receptor (CCR) system. A protease cleavage site is incorporated between the transmembrane domain and the intracellular signaling domain of one of the CCR chains. Coexpression of a protease specific to the cleavage site causes cleavage of the CCR due to the proteolytic activity of the protease at the cleavage site, resulting in inactivation of the CCR (receptor off). Addition of a protease inhibitor (drug) inhibits this cleavage event, turning the CCR into an on-state (receptor on). Thus, CCR signaling becomes dependent on the presence of the drug. Modulated CCRs can avoid the toxicity associated with constitutive CCRs by modulating cell growth to fall within the therapeutic window. B: Schematic diagram of the double-cleavage drug-regulated chimeric cytokine receptor (CCR) system, whereby both chains of the CCR are cleaved. Protease cleavage sites are incorporated between the transmembrane domain and the intracellular signaling domain of both chains / subunits of the CCR. Co-expression of a cleavage site-specific protease leads to cleavage of the CCR due to the proteolytic activity of the protease at the cleavage site, resulting in inactivation of the CCR (receptor off). Addition of a protease inhibitor (drug) inhibits this cleavage event, turning the CCR into an on-state (receptor on). Thus, CCR signaling becomes dependent on the presence of the drug. Modulated CCRs are expected to avoid the toxicity associated with constitutive CCRs by regulating cell proliferation to fall within the therapeutic window. [Figure 21]A: Schematic of a regulatable CCR with a protease fused to one of the CCR subunits. B: Flow plot showing STAT5 phosphorylation levels from primary human T cells transduced with an IL-2 CCR with the HCV NS3 protease fused directly to the gamma chain ICD ("cis"). A short or long peptide linker was placed between the gamma chain ICD and the protease. "Trans" protease refers to the original protease configuration shown in Figure 7. T cells were cultured in medium lacking IL2 for 24 hours before analysis of STAT5 phosphorylation levels. [Figure 22] Flow plot showing CD22.BBz CAR surface expression levels from primary human T cells transduced with various CCRs. The data show that all configurations result in similar levels of surface CAR expression. "Constit." and "Reg." refer to constitutive and regulatable CCRs, respectively. [Figure 23] NSG mice were inoculated with Nalm6 leukemia and then treated with CD22.BBz CAR-T cells engineered with various IL2 CCR constructs. GPV was administered to the mice daily (++), every 48 hours (+), or not at all (-). Mock untransduced T cells, conventional CD22.BBz CAR-T cells, and CD22.BBz CAR-T cells engineered with a CCR lacking the ICD (ΔICD) served as controls. A: Quantification of tumor progression. B: Quantification of T cell expansion. [Figure 24] NSG mice were inoculated with Nalm6 leukemia and then treated with CD22.BBz CAR-T cells engineered with various IL-9 CCR constructs. Mock untransduced T cells and conventional CD22.BBz CAR-T cells served as controls. A: Quantification of tumor progression. B: Quantification of T cell expansion. [Figure 25] Data showing drug control of cell expansion of CD22.BBz CAR-T cells in vitro. T cells were cultured in medium lacking IL-2. [Figure 26]Data showing drug regulation of pSTAT5 levels in HER2.BBz CAR-T cells in vitro. T cells were cultured in medium lacking IL-2. [Figure 27] NSG mice were inoculated with Nalm6 leukemia and then treated with CD22.BBz CAR-T cells engineered with only the IL-2RB component of the IL2 CCR. Mock untransduced T cells and conventional CD22.BBz CAR-T cells served as controls. Data show quantification of tumor progression. [Figure 28] A: Flow plot showing cell surface expression of CCRs with IL-18RA and IL-18RB ICDs. B: NSG mice were inoculated with Nalm6 leukemia and then treated with CD22.BBz CAR-T cells engineered with either regulated or constitutive IL-18CCR constructs. For the regulated IL-18CCR group, mice were administered GPV daily (+GPV) or not at all (-GPV). Mock untransduced T cells and CD22.BBzCAR-T cells engineered with a CCR lacking an ICD (ΔICD) served as controls. Data show quantification of tumor progression. DETAILED DESCRIPTION OF THE INVENTION

[0013] Before the nucleic acids, chimeric cytokine receptors, and methods of the present disclosure are described in more detail, it is to be understood that the nucleic acids, chimeric cytokine receptors, and methods are not limited to the specific embodiments described, as such may, of course, vary. Furthermore, it is to be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, since the scope of the nucleic acids, chimeric cytokine receptors is limited only by the appended claims.

[0014] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limit of that range, to the tenth of the unit of the lower limit, and any other stated or intervening value within this stated range, is encompassed by the nucleic acids, chimeric cytokine receptors, and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed by the nucleic acids, chimeric cytokine receptors, and methods, subject to any specifically excluded limits in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the nucleic acids, chimeric cytokine receptors, and methods.

[0015]

[0023] In this specification, certain ranges are presented with the term "about" before the numerical values. In this specification, the term "about" is used to provide literal support for the exact number it precedes, as well as a number that is near or approximately the number it precedes. In determining whether a number is near or approximately a specifically recited number, the near or approximately unrecited number may be a number that, in the context provided, provides substantial equivalence to the specifically recited number.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the nucleic acids, chimeric cytokine receptors, and methods belong. Although any nucleic acids, chimeric cytokine receptors, and methods similar or equivalent to those described herein can also be used in the practice or testing of the present nucleic acids, chimeric cytokine receptors, and methods, representative and exemplary nucleic acids, chimeric cytokine receptors, and methods are described below.

[0017] All publications and patents cited herein are incorporated by reference to disclose and describe the materials and / or methods in connection with which the publications are cited, to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the nucleic acids, chimeric cytokine receptors, and methods of the invention are not entitled to antedate such publication, as the dates of publication provided may be different from the actual publication dates which may need to be separately confirmed.

[0018] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a precedent for the use of exclusive terminology such as "solely," "solely," and the like or the use of "negative" limitations in connection with the recitation of claim elements.

[0019] It should be understood that, for clarity, certain features of the nucleic acids, chimeric cytokine receptors, and methods that are described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the nucleic acids, chimeric cytokine receptors, and methods that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of embodiments are specifically encompassed by the present disclosure, and to the extent such combinations encompass operable processes and / or compositions, each and every combination is disclosed herein as if it were individually and explicitly disclosed. In addition, all subcombinations listed in embodiments describing such variables are also specifically encompassed by the present nucleic acids, chimeric cytokine receptors, and methods, and are disclosed herein as if each and every such subcombination were individually and explicitly disclosed.

[0020] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual elements and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the method. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0021] Chimeric cytokine receptors and nucleic acids Embodiments of the present disclosure include chimeric cytokine receptors (CCRs) and nucleic acids encoding them. The CCR platform of the present disclosure allows for constitutive or regulatable signaling of cytokine receptors in the absence of cognate cytokines. CCRs find use in a variety of contexts. For example, cells expressing the CCRs of the present disclosure find use in cell therapy. Cell therapies such as CAR-T cell therapy have shown significant clinical benefits in hematological malignancies. However, impaired T cell function and poor persistence limit clinical responses, particularly in solid tumors. As shown herein, the CCRs of the present disclosure, when expressed on cells, are potent drivers of cell proliferation in vitro and in vivo and result in improved in vivo anti-tumor activity compared to control cells lacking the CCR. Thus, CCRs and cells expressing the CCRs find use in cell therapy, non-limiting examples of which include immune cell therapy (e.g., immune cells lacking antigen-binding receptors (e.g., tumor-infiltrating lymphocytes)), CAR-T cell therapy, TCR therapy, CAR-NK cell therapy, and the like. Details regarding the CCRs and nucleic acids of the present disclosure are now provided.

[0022] In certain embodiments, one or more nucleic acids encoding a chimeric cytokine receptor first subunit and a chimeric cytokine receptor second subunit are provided. As used herein, the term "chimeric" refers to a molecule, e.g., a cytokine receptor, composed of portions of different origins. A chimeric molecule as a whole is non-naturally occurring (e.g., synthetic or recombinant), but the portions comprising the chimeric molecule may be naturally occurring. A "chimeric cytokine receptor" or "CCR" refers to a cytokine receptor comprising one or more subunits that contain one or more heterologous domains, where the receptor is capable of constitutive or regulatable signaling in the absence of its cognate cytokine. "Heterologous," as used in the context of a nucleic acid or polypeptide domain, generally means that the domain is derived from a different source (e.g., a molecule of different sequence, a different species origin, and / or the like) from that to which the nucleic acid or polypeptide is associated or connected, such that the nucleic acid or polypeptide is not found in nature.

[0023] The CCR may be a chimeric embodiment of any cytokine receptor of interest (e.g., any human cytokine receptor of interest), wherein the CCR is capable of constitutive or regulatable signaling in the absence of its cognate cytokine. In certain embodiments, the CCR is a chimeric embodiment of a member of the class I cytokine receptor family, a chimeric embodiment of a member of the class II cytokine receptor family, a chimeric embodiment of a member of the TNF receptor family, a chimeric embodiment of a member of the IL-1 receptor family, a chimeric embodiment of a member of the tyrosine kinase receptor family, or a chimeric embodiment of a member of the chemokine receptor family.

[0024] According to some embodiments, the first and second subunits of the CCR are chimeric versions of the first and second subunits (e.g., human subunits) of an interleukin (IL) receptor, a transforming growth factor (TGF) receptor, an interferon (IFN) receptor, a colony-stimulating factor (CSF) receptor, a tumor necrosis factor (TNF) receptor, or a chemokine receptor.

[0025] In certain embodiments, the first and second subunits of the CCR are chimeric embodiments of first and second subunits independently selected from any of the cytokine receptor subunits shown in FIG. 1.

[0026] According to some embodiments, the first and second subunits of the CCR are chimeric embodiments of the first and second subunits of receptors (e.g., human receptors) for IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-26, IL-27, IL-28, IL-29, IL-35, TSLP, GM-CSF, TNFα, TGFβ, IFNα, IFNβ. The subunits of the receptors for these cytokines are shown in Figure 1.

[0027] The amino acid sequences of cytokine receptor subunits (e.g., human cytokine receptor subunits) and their ICDs, as well as suitable nucleotide sequences for encoding and expressing such subunits and their ICDs, are known and readily available from databases such as UniProt, NCBI Protein, and GenBank. Non-limiting examples include human IL-2Rβ (UniProt KB-P14784), human IL-4Rα (UniProt KB-P24394), human IL-7Rα (UniProt KB-P16871), human IL-9R (UniProt KB-Q01113), human IL-10Rβ (UniProt KB-Q08334), human IL-12Rβ1 (UniProt KB-P42701), human IL-21R (UniProt KB-Q9HBE5), human IL-22Rα1 (UniProt KB-Q8N6P7), human IL-23R (UniProt KB-Q5VWK5), and human common gamma chain (γc) (UniProt KB P31785).

[0028] In certain embodiments, a CCR of the present disclosure retains binding activity for its cognate cytokine. For example, a CCR may comprise an extracellular cytokine-binding portion of a cytokine receptor found in nature, or a variant thereof that retains binding activity for a cognate cytokine. According to some embodiments, a CCR of the present disclosure is engineered such that the extracellular cytokine-binding portion of a cytokine receptor found in nature is absent or modified such that the CCR no longer retains binding activity for its cognate cytokine.

[0029] According to some embodiments, a CCR of the present disclosure comprises a first subunit comprising a first heterodimerization domain and a first cytokine receptor intracellular signaling domain (ICD), and a second subunit comprising a second heterodimerization domain homologous to the first heterodimerization domain and a second cytokine receptor ICD. In this context, "cognate" means that the second dimerization domain can dimerize with the first dimerization domain when the first and second subunits are co-expressed on the surface of a cell, which in turn results in the formation of a dimer comprising the first and second subunits. When the first and second subunits are co-expressed on the surface of a cell, the first and second heterodimerization domains can be extracellular dimerization domains, transmembrane dimerization domains, or intracellular dimerization domains.

[0030] Any suitable heterodimerization domain may be used for the first and second subunits of CCR. In certain embodiments, the first and second heterodimerization domains each comprise a leucine zipper domain, a BTB (BR-C, ttk, and bab) domain, a POZ (poxvirus and zinc finger) domain, a coiled-coil domain, or a PDZ domain. According to some embodiments, the first and second heterodimerization domains comprise the constant heavy chain and constant light chain of an IgG, IgE, or IgD antibody, respectively, or dimerization fragments thereof.

[0031] According to some embodiments, when the first and second subunits are co-expressed on the surface of a cell, the first and second heterodimerization domains are transmembrane dimerization domains. For example, the first subunit of a CCR may comprise a heterologous transmembrane domain that is the first heterodimerization domain, and the second subunit of a CCR may comprise a heterologous transmembrane domain that is the second heterodimerization domain. Examples of such heterologous transmembrane domains include, but are not limited to, CD8α (UniProt-P01732) transmembrane domain, CD28 (UniProt-P10747) transmembrane domain, HER2 (UniProt-Q9UK79), EGFR (UniProt-P00533), etc.

[0032] In certain embodiments, the first and second subunits comprise transmembrane domains independently selected from HER2, EGFR, IL-2Rβ, IL-7R, IL-21R, IL-4R, IL-9R, IL15Rα, common gamma chain (γc), Eph receptor, VEGF receptor, ErbB receptor, FGF receptor, ROR1, ROR2 PDGF receptor, MET receptor, CD35, CD3ζ, CD3γ, CD3δ, CD4, CD5, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD-1 transmembrane domains. The amino acid sequences of such receptors (e.g., human receptors) and their transmembrane domains, as well as suitable nucleotide sequences encoding and expressing such transmembrane domains, are known and readily available from databases such as UniProt, NCBI Protein, and GenBank.

[0033] As summarized above, the CCRs of the present disclosure comprise first and second subunits that comprise first and second cytokine receptor intracellular signaling domains (ICDs), respectively. According to some embodiments, the first cytokine receptor ICD, the second cytokine receptor ICD, or both, is selected from the group consisting of IL-2Rβ ICD, IL-3Rα ICD, IL-4R ICD, IL-5Rα ICD, IL-6Rα ICD, IL-7R ICD, IL-9R ICD, IL-10Rα ICD, IL-10Rβ ICD, IL-11Rα ICD, IL-12Rβ1 ICD, IL-12Rβ2 ICD, IL-13Rα1 ICD, IL-13Rα2 ICD, IL-20Rα ICD, IL-20Rβ, IL-21R ICD, IL-22Rα1, IL-22Rα2, IL-23R ICD, IL-27Rα ICD, IL-28RA ICD, IL-31Rα ICD, TGFβR1 ICD, TGFβR2 ICD, TGFβR3 ICD, IFNAR1 ICD, IFNAR2 ICD, IFNGR1 ICD, IFNGR2 ICD, IFNLR ICD, CSF2RB ICD, gp130 ICD, CD9 ICD, OSMR ICD, CSF-1R ICD, or any combination thereof.

[0034] In certain embodiments, the first cytokine receptor ICD is an IL-22Rα1 ICD and the second cytokine receptor ICD is an IL-10Rβ ICD. Such CCRs find use, for example, when it is desirable for a cell to exhibit constitutive or regulatable IL-22 signaling in the absence of IL-22. According to some embodiments, the first cytokine receptor ICD is an IL-23R ICD and the second cytokine receptor ICD is an IL-12Rβ1 ICD. Such CCRs find use, for example, when it is desirable for a cell to exhibit constitutive or regulatable IL-23 signaling in the absence of IL-23.

[0035] According to some embodiments, the first cytokine receptor ICD comprises an ICD from the common cytokine receptor gamma chain family. γc functions as a shared signaling receptor for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. In certain embodiments, the ICD from the common cytokine receptor gamma chain family is an IL-2Rβ ICD, an IL-4R ICD, an IL-7R ICD, an IL-9R ICD, or an IL-21R ICD. When the first cytokine receptor ICD comprises an ICD from the common cytokine receptor gamma chain family, in some embodiments, the second cytokine receptor ICD is a common gamma chain (γc) ICD.

[0036] In certain embodiments, the first subunit, the second subunit, or both, comprise a linker sequence between the domains of the subunits. Such subunits may comprise 1, 2, 3, 4, 5, or more linkers. In specific embodiments, the linker length is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acids in length. Non-limiting examples of linkers that can be incorporated between the domains of the first and / or second subunits include serine-glycine linkers, disulfide linkers (e.g., to enhance association between the first and second subunits), and the like, including any of the linker sequences provided in Table 1 below.

[0037] According to some embodiments, the first subunit, the second subunit, or both, comprise a transition domain disposed between the transmembrane domain and the ICD. As will be understood with the benefit of this disclosure, such a transition domain may be incorporated into the first and / or second subunit to enhance CCR expression compared to the subunit in the absence of the transition domain, as demonstrated in the experimental section below. For example, in some embodiments, a subunit comprising a γc ICD may be engineered to have a transition domain disposed between the transmembrane domain and the γc ICD. Suitable transition domains include, but are not limited to, transition domains comprising the juxtamembrane (JM) region of a cell surface receptor. In some embodiments, the JM region of CD8α or IL-15Rα is used as the transition domain between the transmembrane domain and the ICD, e.g., the γc ICD. A non-limiting example of a CD8α transition domain (sometimes referred to herein as a transition peptide) is a domain comprising the amino acid sequence NHRNRRRVCKCPRPVV (SEQ ID NO: 54). A non-limiting example of an IL-15Rα transition domain is the domain comprising the amino acid sequence KSRQTPP (SEQ ID NO: 55). Variants of such domains are also provided by the present disclosure.

[0038] In certain embodiments, the first subunit and the second subunit are encoded by a single nucleic acid. When the first subunit and the second subunit are encoded by a single nucleic acid, the nucleic acid may be configured to allow polycistronic expression of the first subunit and the second subunit. That is, two or more (e.g., each) of the proteins encoded by the single nucleic acid can be expressed as separate proteins from the same promoter. In certain embodiments, the single nucleic acid includes a ribosome skipping site to allow polycistronic expression of two or more (e.g., each) protein-coding regions. A non-limiting example of a suitable ribosome skipping site that can be incorporated into such a single nucleic acid is the P2A ribosome skipping site from porcine teschovirus. According to other embodiments, the first subunit is encoded by a first nucleic acid and the second subunit is encoded by a second nucleic acid.

[0039] According to some embodiments, the first subunit, the second subunit, or both, comprise a protease cleavage site located between the transmembrane domain and the ICD. The term "cleavage site" refers to a bond (e.g., a scissile bond) cleaved by an agent, e.g., a protease. A cleavage site for a protease comprises a specific amino acid sequence recognized by the protease during proteolytic cleavage and may include surrounding amino acids (e.g., 1 to 6 amino acids) on either side of the scissile bond that are necessary for binding to the active site of the protease and recognition as a substrate. In some embodiments, the cleavage site is provided as a cleavable linker, and "cleavable linker" refers to a linker comprising a protease cleavage site. A cleavable linker is typically cleavable under physiological conditions.

[0040] According to some embodiments, the protease cleavage site is a viral protease cleavage site. Non-limiting examples of viral protease cleavage sites include cleavage sites of potyvirus family proteases. Potyvirus family proteases of interest include tobacco etch virus (TEV) protease, plum pox virus protease (PPVp), soybean mosaic virus protease (SbMVp), sunflower mild mosaic virus protease (SuMMVp), tobacco vein mottling virus protease (TVMVp), and West Nile virus protease (WNVp). In certain embodiments, the viral protease cleavage site is a TEV protease cleavage site. The amino acid sequence of an exemplary TEV protease cleavage site is ENLYFQS (SEQ ID NO: 58). The amino acid sequence of an exemplary TEV protease is as follows: GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPEEPFQPVKEATQLMN (SEQ ID NO: 59)

[0041] In some embodiments, the protease is a TEV protease comprising the amino acid sequence set forth above, or a functional (proteolytic) variant thereof having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more amino acid sequence identity to such a sequence, and / or a functional (proteolytic) fragment thereof, such as a fragment having a length of 100-125, 125-150, 150-175, 175-200, 200-225, or 225-235 amino acids.

[0042] According to some embodiments, the viral protease cleavage site is for HCV protease. In certain embodiments, the viral protease cleavage site is for a viral protease derived from HCV nonstructural protein 3 (NS3). NS3 consists of an N-terminal serine protease domain and a C-terminal helicase domain. "Derived from HCV NS3" means that the protease is the serine protease domain of HCV NS3 or a proteolytically active variant thereof that can cleave the cleavage site for the serine protease domain of HCV NS3. The NS3 protease domain forms a heterodimer with HCV nonstructural protein 4A (NS4A) to activate its proteolytic activity. The protease derived from HCV NS3 may comprise the entire NS3 protein or a proteolytically active fragment thereof, and may further comprise a cofactor polypeptide, such as an activated NS4A region, derived from HCV nonstructural protein 4A (NS4A). NS3 protease is highly selective and can be inhibited by several non-toxic, cell-permeable drugs, which are currently available for human use. NS3 protease inhibitors that can be used include, but are not limited to, asunaprevir (ASV), danoprevir (DPV), simeprevir (SPV), grazoprevir (GPV), glecaprevir, voxilaprevir, and any combination thereof. Non-limiting examples of proteases derived from HCV NS3 are provided below.

[0043] Examples of proteases derived from HCV NS3 APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTD (SEQ ID NO: 60) APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIMSTATQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDGRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTD (SEQ ID NO: 61) APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTD (SEQ ID NO: 62)

[0044] In some embodiments, the protease comprises one of the above sequences or is a functional (proteolytic) variant thereof having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more amino acid sequence identity to one of such sequences, and / or a functional (proteolytic) fragment thereof, such as a fragment having a length of 100-185, 120-185, 140-185, 160-185, 170-185, 180-185, 182-185, or 184-185 amino acids.

[0045] The NS3 protease cleavage sites can include the four junctions between the nonstructural (NS) proteins of the HCV polyprotein that are normally cleaved by the NS3 protease during HCV infection, including the NS3 / NS4A, NS4A / NS4B, NS4B / NS5A, and NS5A / NS5B junction cleavage sites. For a description of representative sequences of NS3 proteases and their cleavage sites for various HCV strains, see, e.g., Hepatitis C Viruses: Genomes and Molecular Biology (SLTan ed., Taylor & Francis, 2006), Chapter 6, pp. 163-206 (the disclosure of which is incorporated herein by reference in its entirety).

[0046] In some embodiments, the protease is derived from HCV NS3 and engineered to contain one or more amino acid substitutions compared to the HCV NS3 protease amino acid sequence described above. For example, the protease can contain a substitution at a position corresponding to position 54 of the amino acid sequence APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTD (SEQ ID NO: 60). In some embodiments, such a substitution is a threonine to alanine substitution.

[0047] The NS3 nucleic acid and protein sequences can be derived from HCV, including any isolate of HCV having any genotype (e.g., genotypes 1-7) or subtype. Numerous NS3 nucleic acid and protein sequences are known and are described, for example, in U.S. Ser. No. 15 / 737,712, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Additional representative NS3 sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, the following NCBI entries: Accession numbers YP_001491553, YP_001469631, YP_001469632, NP_803144, NP_671491, YP_001469634, YP_001469630, YP_001469633, ADA68311, ADA68307, AFP99000, AFP98987, ADA68322, AFP99033, ADA68330, AFP99056, AFP99041, CBF60982, CB F60817, AHH29575, AIZ00747, AIZ00744, ABI36969, ABN05226, KF516075, KF516074, KF516056, AB826684, AB826683, JX171009, JX171008, JX171000, EU847455, EF154714, GU085487, JX171065, and JX171063 (all of these sequences are incorporated herein by reference). Any of these sequences, or functional variants thereof having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity to any one of these sequences, or proteolytic fragments thereof may be used.

[0048] The NS4A nucleic acid and protein sequences can be derived from HCV, including any isolate of HCV having any genotype (e.g., seven genotypes 1-7) or subtype. A number of NS4A nucleic acid and protein sequences are known. Representative NS4A sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, the following NCBI entries: Accession Nos. NP_751925, YP_001491554, GU945462, HQ822054, FJ932208, FJ932207, FJ932205, and FJ932199 (all of these sequences (entered as of the filing date of this application) are incorporated herein by reference). Any of these sequences, or functional variants thereof having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more amino acid sequence identity to any one of these sequences, or proteolytic fragments thereof may be used.

[0049] HCV polyprotein nucleic acid and protein sequences can be derived from HCV, including any isolate of HCV having any genotype (e.g., genotypes 1-7) or subtype. Many HCV polyprotein nucleic acid and protein sequences are known. Representative HCV polyprotein sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, the following NCBI entries: Accession numbers YP_001469631, NP_671491, YP_001469633, YP_001469630, YP_001469634, YP_001469632, NC_009824, NC_004102, NC_009825, NC_009827, NC_009823, NC_009826, and EF108306 (all of these sequences (entered up to the filing date of this application) are incorporated herein by reference). Any of these sequences, or functional variants thereof having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more amino acid sequence identity to any one of these sequences, or proteolytic fragments thereof may be used.

[0050] According to some embodiments, when the protease cleavage site is a viral protease cleavage site, the viral protease cleavage site is a human immunodeficiency virus (HIV) protease cleavage site. In certain embodiments, when the protease cleavage site is a viral protease cleavage site, the viral protease cleavage site is a SARS-CoV-2 protease cleavage site.

[0051] In certain embodiments, the protease cleavage site is a human protease cleavage site, non-limiting examples of which include cleavage sites for human kallikrein (KLK) protease, human enterokinase protease, human thrombin, human matrix metalloproteinase (MMP), human urokinase-type plasminogen activator receptor (uPAR), human plasmin, or human cathepsin. According to some embodiments, the protease cleavage site is a cleavage site for human kallikrein (KLK) proteases, non-limiting examples of which include human KLK3 (UniProtKB-Q546G3), human KLK4 (UniProtKB-Q9Y5K2), human KLK6 (UniProtKB-Q92876), human KLK8 (UniProtKB-O60259), human KLK11 (UniProtKB-Q9UBX7), human KLK13 (UniProtKB-Q9UKR3), human KLK14 (UniProtKB-Q9P0G3), and human KLK15 (UniProtKB-Q9H2R5). Any of these sequences, or functional variants thereof having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity to any one of these sequences, or proteolytic fragments thereof may be used.

[0052] In certain embodiments, the protease cleavage site is selected from the group consisting of acrosin (ACR), AGBL carboxypeptidase 1 (AGBL1), AGBL carboxypeptidase 2 (AGBL2), AGBL carboxypeptidase 3 (AGBL3), AGBL carboxypeptidase 4 (AGBL4), AGBL carboxypeptidase 5 (AGBL5), ATP / GTP-binding carboxypeptidase 1 (AGTPBP1), asparaginase and isoaspartyl peptidase 1 (ASRGL1), astacin-like metalloendopeptidase (ASTL), ATP23 metallopeptidase and ATP synthase assembly factor homolog (ATP23), ataxin 3 (ATXN3), ataxin 3-like (ATXN3L), azurocidin 1 (AZU1), beta-secretase 1 (BACE1), beta-secretase 2 (BACE2), bone morphogenetic protein 1 (BMP1), BRCA1 / BRCA2-containing complex subunit 3 (BRCC3), calpain 14 (CAPN14), calpain 3 (CAPN3), caspase recruitment domain family member 8 (CARD8), caspase 4 (CASP4), chymotrypsin-like elastase 1 (CELA1), chymotrypsin-like elastase 2A (CELA2A), chymotrypsin-like elastase 2B (CELA2B), chymotrypsin-like elastase 3A (CELA3A), chymotrypsin-like elastase 3B (CELA3B), and CUGBP Elav-like family member 3 (CELF3), CUGBP Elav-like family member 4 (CELF4), CUGBP Elav-like family member 5 (CELF5), CUGBP Elav-like family member 6 (CELF6), cell growth regulator with EF-hand domain 1 (CGREF1), charged multivesicular body protein 3 (CHMP3), CLN5 intracellular transport protein (CLN5), chymase 1 (CMA1), collectin subfamily member 11 (COLEC11), COP9 signalosome subunit 5 (COPS5), choline, serine peptidase (CORIN), carboxypeptidase A4 (CPA4), carboxypeptidase vitellogenesis-like (CPVL), cystatin SN (CST1), cystatin 11 (CST11), cystatin C (CST3), cystatin S (CST4), cystatin D (CST5),Cystatin E / M (CST6), cystatin 8 (CST8), cystatin 9 (CST9), cystatin-like 1 (CSTL1), chymotrypsinogen B2 (CTRB2), chymotrypsin-like (CTRL), cathepsin L (CTSL), DNA damage-inducible 1 homolog 2 (DDI2), DAP3-binding cell death enhancer 1 (DELE1), adipsin (DF), Dickkopf WNT signaling pathway inhibitor 2 (DKK2), Dickkopf WNT signaling pathway inhibitor 4 (DKK4), dipeptidase 1 (DPEP1), dipeptidyl peptidase 3 (DPP3), dipeptidyl peptidase 9 (DPP9), FAM111 trypsin-like peptidase A (FAM111A), ficolin 1 (FCN1), ficolin 2 (FCN2), ficolin 3 (FCN3), G3BP stress granule assembly factor 1 (G3BP1), hepsin (HPN), HtrA serine peptidase 1 (HTRA1), insulin-degrading enzyme (IDE), mitochondrial intramembrane peptidase subunit 2 (IMMP2L), Jumonji domain-containing 7 (JMJD7), Josephin domain-containing 2 (JOSD2), kallikrein 1 (KLK1), kallikrein-related peptidase 10 (KLK10), kallikrein-related peptidase 11 (KLK11), kallikrein-related peptidase 12 (KLK12), kallikrein-related peptidase 13 (KLK13), kallikrein-related peptidase 14 (KLK14), kallikrein-related peptidase 15 (KLK15), kallikrein-related peptidase 2 (KLK2), kallikrein-related peptidase 3 (KLK3), kallikrein-related peptidase 4 (KLK4), kallikrein-related peptidase 5 (KLK5), kallikrein-related peptidase 6 (KLK6), kallikrein-related peptidase 7 (KLK7), kallikrein-related peptidase 8 (KLK9), kallikrein-related peptidase 9 (KLK10), kallikrein-related peptidase 10 (KLK11), kallikrein-related peptidase 11 (KLK12), kallikrein-related peptidase 12 (KLK13), kallikrein-related peptidase 14 (KLK14), kallikrein-related peptidase 15 (KLK15), kallikrein-related peptidase 2 (KLK2), kallikrein-related peptidase 3 (KLK3), kallikrein-related peptidase 4 (KLK4), kallikrein-related peptidase 5 (KLK5), kallikrein-related peptidase 6 (KLK6), kallikrein-related peptidase 7 (KLK7), kallikrein-related peptidase 13 (KLK14), kallikrein-related peptidase 14 (KLK15), kallikrein-related peptidase 15 (KLK16), kallikrein-related peptidase 16 (KLK17), kallikrein-related peptidase 17 (KLK18), kallikrein-related peptidase peptidase 8 (KLK8), kallikrein-related peptidase 9 (KLK9), kallikrein pseudogene 1 (KLKP1), lipocalin 2 (LCN2), legumain (LGMN), leishimanolysin-like peptidase (LMLN), MAS1 proto-oncogene-like, G protein-coupled receptor (MAS1L), MBL-associated serine protease 1 (MASP1), MBL-associated serine protease 2 (MASP2), mannose-binding lectin 2 (MBL2),Matrix metallopeptidase 10 (MMP10), matrix metallopeptidase 11 (MMP11), matrix metallopeptidase 13 (MMP13), matrix metallopeptidase 16 (MMP16), matrix metallopeptidase 2 (MMP2), napsin A aspartic peptidase (NAPSA), neurolysin (NLN), NLR family CARD domain-containing 4 (NLRC4), NLR family pyrin domain-containing 1 (NLRP1), aminopeptidase puromycin-sensitive (NPEPPS), opiorphin prepropeptide (OPRPN), OTU deubiquitinase, ubiquitin aldehyde-linked 2 (OTUB2), poly(ADP-ribose) polymerase family member 9 (PARP9), proprotein convertase subtilisin / kexin type 1 (PCSK1), proprotein convertase convertase subtilisin / kexin type 1 inhibitors (PCSK1N), proprotein convertase subtilisin / kexin type 2 (PCSK2), proprotein convertase subtilisin / kexin type 4 (PCSK4), proprotein convertase subtilisin / kexin type 5 (PCSK5), proprotein convertase subtilisin / kexin type 6 (PCSK6), proprotein convertase subtilisin Proprotein convertase subtilisin / kexin type 7 (PCSK7), proprotein convertase subtilisin / kexin type 9 (PCSK9), platelet-derived growth factor C (PDGFC), pepsinogen A3 (PGA3), pepsinogen A4 (PGA4), pepsinogen A5 (PGA5), pyroglutamyl-peptidase I-like (PGPEP1L), PTEN-inducible kinase 1 (PINK1), prolyl endopeptidase-like (PREPL), parkin RBR E3 ubiquitin protein ligase (PRKN), serine protease gene cluster (PRSS), serine protease 2 (PRSS2), serine protease 21 (PRSS21), serine protease 22 (PRSS22), serine protease 23 (PRSS23), serine protease 27 (PRSS27), serine protease 33 (PRSS33), serine protease 46, pseudogene (PRSS46P), serine protease 55 (PRSS55), serine protease 8 (PRSS8), proteinase 3 (PRTN3),Presenilin 2 (PSEN2), PYD and CARD domain-containing (PYCARD), retinoic acid receptor responder 1 (RARRES1), ring finger and FYVE-like domain-containing E3 ubiquitin protein ligase (RFFL), rhomboid-like 2 (RHBDL2), SEC11 homolog A, signal peptidase complex subunit (SEC11A), SEC11 homolog B, signal peptidase complex subunit (SEC11B), SEC11 homolog C, signal peptidase complex subunit (SEC11BC) ), SUMO peptidase family member, NEDD8 specific (SENP8), SET nuclear proto-oncogene (SET), synaptosomal associated protein 25 (SNAP25), secreted phosphoprotein 2 (SPP2), small proline-rich protein 3 (SPRR3), spleen-associated tyrosine kinase (SYK), transcription factor EB (TFEB), transglutaminase 2 (TGM2), toll-like receptor adaptor molecule 1 (TICAM1), tubulointerstitial nephritis antigen-like 1 (TINAGL1), transmembrane serine protease 11D (TMPRSS11) D), transmembrane serine protease 11E (TMPRSS11E), transmembrane serine protease 4 (TMPRSS4), transmembrane serine protease 5 (TMPRSS5), transmembrane serine protease 6 (TMPRSS6), transmembrane serine protease 7 (TMPRSS7), TNF receptor superfamily member 10a (TNFRSF10A), tryptase alpha / beta 1 (TPSAB1), tryptase beta 2 (TPSB2), tryptase delta 1 (TPSD1), tryptase gamma 1 (TPSG1), tryptase pseudoprotein 1 (TPSG1), gene 2 (TPSP2), tyrosylprotein sulfotransferase 1 (TPST1), tyrosylprotein sulfotransferase 2 (TPST2), tyrosylprotein sulfotransferase 2 pseudogene 1 (TPST2P1), thyrotropin-releasing hormone degrading enzyme (TRHDE), thyroid hormone receptor interactor 4 (TRIP4), ubiquitin C-terminal hydrolase L1 (UCHL1), ubiquitin-specific peptidase 27X-conjugating (USP27X), vasohibin 2 (VASH2), valosin-containing protein (VCP),and WAP4 disulfide core domain 1 (WFDC1).

[0053] In some embodiments, the protease is highly selective for its cleavage site. Additionally, protease activity may be inhibitable by known small molecule inhibitors that are cell permeable and non-toxic to the cell or individual being studied or treated. For a discussion of proteases, see, for example, VYH Hook, Proteolytic and cellular mechanisms in prohormone and proprotein processing, R.G. Landes Company, Austin, Texas, USA (1998), N.M. Hooper et al., Biochem. J. 321:265-279 (1997), Z. Werb, Cell 91:439-442 (1997), T.G. Wolfsberg et al., J. Cell Biol. 131:275-278 (1995), T. Berg et al., Biochem. J. 307:313-326 (1995), M.J. Myth and J.A. Trapani, Immunology Today 16:202-206 (1995), R.V. Talanian et al. See, e.g., J. Biol. Chem. 272:9677-9682 (1997), and NAThornberry et al., J. Biol. Chem. 272:17907-17911 (1997), the disclosures of which are incorporated by reference herein in their entireties for all purposes. In some embodiments, the protease used is a sequence-specific non-human protease for which FDA-approved pharmacological inhibitors are available.

[0054] Thus, in some embodiments, the one or more nucleic acids further encode a protease, and the protease cleavage site is a cleavage site for the protease. In one configuration (sometimes referred to herein as a "trans" configuration, in which the protease is expressed as a separate polypeptide with respect to the first and second subunits), the one or more nucleic acids encode a fusion protein comprising the protease and a transmembrane domain. In other cases, the protease may be expressed as part of a fusion protein with the first subunit, the second subunit, or both, sometimes referred to herein as a "cis" configuration.

[0055] The proteolytic activity of the protease can be modulated. In some embodiments, the proteolytic activity of the protease can be modulated via a cell-permeable small molecule. For example, such a cell-permeable small molecule can be a protease inhibitor. Such inhibitors are known and available. When the protease is derived from HCV NS3, in some cases, the protease inhibitor is selected from asunaprevir (ASV), danoprevir (DPV), simeprevir (SPV), grazoprevir (GPV), glecaprevir, voxilaprevir, and any combination thereof. When the protease is an HIV protease, in some cases, the protease inhibitor is selected from atazanavir (Reyataz), darunavir (Prezista), fosamprenavir (Lexiva), indinavir (Crixivan), lopinavir / ritonavir (Kaletra), nelfinavir (Viracept), ritonavir (Norvir), saquinavir (Invirase), tipranavir (Aptivus), atazanavir / cobicistat (Evotaz), darunavir / cobicistat (Prezcobix), and any combination thereof. When the protease is a SARS-CoV-2 protease, in some cases, the protease inhibitor is nilmatrervir (Paxlovid). When the protease is a human renin protease, in some cases, the protease inhibitor is aliskiren.

[0056] The first and / or second subunits can include any additional domains and functionalities as desired. For example, in certain embodiments, the first subunit, the second subunit, or both, include an extracellular protein tag. Such tags find use, for example, in assessing cells for cell surface expression of CCRs. In some cases, the extracellular protein tag is an N-terminal protein tag. Non-limiting exemplary tags that can be incorporated into the first and / or second subunits include a FLAG tag, an HA tag, and the like. Exemplary amino acid sequences of such tags are provided in Table 1 below.

[0057] In certain embodiments, the first and second subunits are each engineered to contain an extracellular cysteine ​​residue, and a disulfide bond between the extracellular cysteine ​​residue of the first subunit and the extracellular cysteine ​​residue of the second subunit stabilizes the association of the first and second subunits when expressed on the surface of a cell. For example, a disulfide linker sequence (e.g., CGG) can be incorporated into the extracellular domains of each of the first and second subunits to stabilize their association.

[0058] Aspects of the present disclosure further include chimeric cytokine receptors (CCRs) encoded by one or more nucleic acids of the present disclosure.

[0059] The amino acid sequences of exemplary CCR components are provided below in Table 1. For each sequence, the domains ordered from N-terminus to C-terminus are listed in the left column. The sequences in the right column indicate domains by alternating underlining. [Table 1-1] [Table 1-2] [Table 1-3]

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

[0060] The present disclosure provides each of the polypeptides provided in Table 1, and each of the individual domains therein, as well as nucleic acids encoding such polypeptides and individual domains. Cells comprising such polypeptides and nucleic acids are also provided. As will be appreciated, the present disclosure also provides variants of any of the polypeptides or individual domains therein, and in some cases, the variant polypeptide or domain thereof comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity to the parent / reference sequence, or a functional fragment thereof, wherein the variant retains the functionality of the parent / reference sequence (e.g., ability to dimerize, intracellular signaling activity, protease activity, cleavability by a protease, transition domain, etc.).

[0061] For example, in certain embodiments, variants of such polypeptides having one or more amino acid substitutions are provided. Conservative substitutions are shown in Table 2 under the heading of "Preferred Substitutions." More substantial changes are provided in Table 2 under the heading of "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions may be introduced into a polypeptide of interest, and the products may be screened for a desired activity, e.g., retained / improved cell surface expression, dimerization, signal transduction, modulation, etc. [Table 2]

[0062] Amino acids can be grouped according to the following common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0063] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0064] Exemplary nucleotide sequences encoding the various domains and polypeptides set forth in Table 1 are provided in SEQ ID NOs: 31-53. Nucleic acids encoding polypeptides or domains thereof that differ from the nucleotide sequences of SEQ ID NOs: 31-53 due to the degeneracy of the genetic code are also encompassed by the present disclosure.

[0065] In addition to one or more nucleic acids and CCRs of the present disclosure described herein, aspects of the present disclosure further include membrane-associated polypeptides and nucleic acids encoding same. In some cases, nucleic acids are provided that encode a membrane-associated polypeptide comprising a transmembrane domain and a cytokine receptor ICD, wherein the polypeptide does not comprise an extracellular cytokine binding domain. Such polypeptides are based in part on the unexpected discovery herein that a single cytokine chain (i.e., not paired with a second cytokine chain) can enhance the anti-tumor efficacy of CAR T cells compared to CAR T cells lacking a single cytokine chain (see, e.g., Example 11). In certain embodiments, the cytokine receptor ICD is an IL-2Rβ ICD, an IL-7R ICD, an IL-9R ICD, or a common gamma chain (γc) ICD. According to some embodiments, the cytokine receptor ICD is an IL-2Rβ ICD. In some cases, the polypeptide comprises a heterodimerization domain, e.g., an extracellular heterodimerization domain. In certain embodiments, the polypeptide comprises a protease cleavage site disposed between the transmembrane domain and the ICD.

[0066] Expression constructs, cells and compositions Expression constructs Aspects of the present disclosure further include one or more expression constructs comprising any one or more nucleic acids of the present disclosure. As used herein, an "expression construct" is a circular or linear polynucleotide (a polymer composed of naturally occurring and / or non-naturally occurring nucleotides) comprising a region encoding the first subunit, the second subunit, or both of a CCR of the present disclosure, operably linked to a suitable promoter, e.g., a constitutive or inducible promoter. In some embodiments, expression of the first subunit, the second subunit, or both is under the control of one or more exogenous (including heterologous) regulatory elements, e.g., promoters, enhancers, etc., present in the expression construct. In some embodiments, expression of the first subunit, the second subunit, or both may be controlled by one or more endogenous regulatory elements, e.g., promoters, enhancers, etc., at or near the genomic locus where the expression construct is inserted.

[0067] In certain embodiments, the promoter may be a single shared promoter between each of the protein coding regions of the expression construct, or at least one of the protein coding regions may be operably linked to a promoter that is not shared with any other protein coding region of the expression construct. The expression construct may be configured to allow polycistronic expression of two or more (e.g., each) of the protein coding regions. That is, two or more (e.g., each) of the proteins encoded by the expression construct may be expressed as separate proteins from the same promoter. In certain embodiments, the expression construct includes a ribosome skipping site to allow polycistronic expression of two or more (e.g., each) of the protein coding regions. A non-limiting example of a suitable ribosome skipping site that may be incorporated into the expression construct is the P2A ribosome skipping site from porcine teschovirus.

[0068] The expression construct (e.g., vector) may be suitable for replication and integration in prokaryotes, eukaryotes, or both. The expression construct may include functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulating expression of the nucleic acid encoding the first subunit, the second subunit, or both. The expression construct optionally contains at least one independent terminator sequence, a sequence that allows replication of the cassette in both eukaryotes and prokaryotes, as found in shuttle vectors, and a generic expression cassette that contains selectable markers for both prokaryotic and eukaryotic systems.

[0069] To obtain high levels of expression of a cloned nucleic acid, it is typical to construct an expression construct containing a strong promoter to direct transcription, a ribosome binding site for translation initiation, and a transcription / translation terminator, each in functional orientation relative to each other and to the protein-coding sequence. Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator regions of the tryptophan biosynthetic pathway of E. coli, the phage lambda (P L ), and the L-arabinose (araBAD) operon. It is also useful to include a selectable marker in a DNA vector transformed in E. coli. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. Expression systems for expressing the first subunit, the second subunit, or both of CCR are available using, for example, E. coli, Bacillus sp., and Salmonella. E. coli systems can also be used. Transducing cells with a nucleic acid (e.g., an expression construct) can involve, for example, incubating lipid microparticles containing the nucleic acid with the cells or incubating a viral vector containing the nucleic acid with cells within the vector's host range.

[0070] In certain embodiments, when one or more expression constructs are delivered to a cell, one or more of the expression constructs are episomal (e.g., extrachromosomal), where "episome" or "episomal" refers to a polynucleotide that replicates independently of the chromosomal DNA of the cell. A non-limiting example of an episome that can be used is a plasmid.

[0071] According to some embodiments, delivery of one or more expression constructs to a cell results in integration of one or more of the expression constructs into the cell's genome. In certain embodiments, one or more of the expression constructs are adapted for site-specific integration into the genome. For example, the expression construct may be adapted for site-specific integration into the genome, where the site-specific integration inactivates a target gene in the cell's genome. By way of example, site-specific integration may knock out a target gene by knocking in the expression construct. Any suitable approach for site-specific gene editing and functional integration may be used. Functional integration of the expression construct may be achieved through various means, including by the use of an integrating vector, including viral vectors and non-viral vectors. In some cases, retroviral vectors, such as lentiviral vectors, may be used. In some cases, non-retroviral integrating vectors may be used. The integrating vector may be contacted with the cells in a suitable transduction medium at a suitable concentration (or multiplicity of infection) for a suitable time period to allow the vector to infect the target cells, promoting functional integration of the expression construct. Non-limiting examples of useful viral vectors include retroviral vectors, lentiviral vectors, adenoviral (Ad) vectors, adeno-associated viral (AAV) vectors, hybrid Ad-AAV vector systems, and the like.

[0072] Strategies for site-specific integration that find use in the methods of the present disclosure include those using homologous recombination, non-homologous end joining (NHEJ), and the like. Such strategies may use non-naturally occurring or engineered nucleases, including, but not limited to, zinc-ringer nucleases (ZNFs), meganucleases, transcription activator-like effector nucleases (TALENs), or CRISPR-Cas systems. Eukaryotic cells utilize two different DNA repair mechanisms in response to DNA double-strand breaks (DSBs): homologous recombination (HR) and non-homologous end joining (NHEJ). Mechanistically, HR is an error-free DNA repair mechanism because it requires a homologous template to repair the damaged DNA strand. Due to its homology-based mechanism, HR has been used as a tool for site-specific manipulation of genomes. Gene targeting by HR requires the use of two homology arms flanking the transgene / target site of interest. HR efficiency can be increased by introducing DSBs at target sites using specific rare-cutting endonucleases. The discovery of this phenomenon has prompted the development of methods to create site-specific DSBs in the genomes of different species. Various chimeric enzymes, namely ZFNs, meganucleases, and TALENs, have been designed for this purpose over the past decade. ZFNs are modular chimeric proteins containing a ZF-based DNA-binding domain (DBD) and a Fokl nuclease domain. The DBD typically consists of three ZF domains, each with a three-base-pair specificity, and the Fokl nuclease domain provides DNA nicking activity targeted by two adjacent ZFNs. The modular nature of the DBD allows them to target any site within the genome. TALENs are similar to ZFNs, except that the DBD is derived from transcription activator-like effectors (TALEs). TALE DBDs are modular and consist of 34-residue repeats, and their DNA specificity is determined by the number and order of the repeats. Each repeat binds to a single nucleotide in the target sequence via only two residues.

[0073] cell Aspects of the present disclosure further include cells comprising one or more nucleic acids of the present disclosure and cells comprising one or more expression constructs of the present disclosure. In certain embodiments, the cells are prokaryotic cells (e.g., bacteria), yeast cells, insect (e.g., Drosophila) cells, amphibian (e.g., frog, e.g., Xenopus) cells, plant cells, etc. According to some embodiments, the cells are mammalian cells. Mammalian cells of interest include human cells, rodent cells, etc.

[0074] In some embodiments, the cells are immune cells. Non-limiting examples of immune cells include T cells, B cells, natural killer (NK) cells, macrophages, monocytes, neutrophils, dendritic cells, mast cells, basophils, and eosinophils. In certain embodiments, the cells are T cells. When the immune cells include T cells, the T cells may be naive T cells (T N ), cytotoxic T cells (T CTL ), memory T cells (T MEM ), T memory stem cells (T SCM ), central memory T cells (T CM ), effector memory T cells (T EM ), tissue-resident memory T cells (T RM ), effector T cells (T EFF ), regulatory T cells (T REG ), helper T cells, CD4+ T cells, CD8+ T cells, virus-specific T cells, alpha-beta T cells (T αβ ), and gamma delta T cells (T γδ ) may be included in any combination.

[0075] According to some embodiments, the cells are stem cells, e.g., mammalian (e.g., human) stem cells. For example, the population of cells can include embryonic stem cells (ES), adult stem cells, hematopoietic stem cells (HSC), induced pluripotent stem cells (iPSC), mesenchymal stem cells (MSC), neural stem cells (NSC), or any combination thereof.

[0076] Approaches for introducing one or more nucleic acids or one or more expression constructs into cells of interest are known and may include contacting a cell population with one or more nucleic acids or one or more expression constructs under conditions such that the one or more nucleic acids or one or more expression constructs are delivered to cells of the cell population. The method may further include selecting cells that exhibit cell surface expression of a CCR.

[0077] The contacting step may include contacting the cell population with one or more nucleic acids or one or more expression constructs, for example, by combining the cells and the one or more nucleic acids or one or more expression constructs in a single mixture under conditions suitable for delivery (e.g., transfection, transduction, etc.) of each of the one or more nucleic acids or one or more expression constructs to cells of the cell population.

[0078] Various suitable approaches and conditions for delivering nucleic acids and expression constructs into cells are known. According to some embodiments, delivery is performed by microinjection, transfection, lipofection, heat shock, electroporation, transduction, gene gun, DEAE-dextran-mediated transcription, etc. In certain embodiments, one or more nucleic acids or one or more expression constructs are introduced into cells of a cell population by AAV transduction. The AAV vector may include ITRs from AAV2 and a serotype from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. According to some embodiments, the AAV vector includes ITRs from AAV2 and a serotype from AAV6. In certain embodiments, one or more nucleic acids or one or more expression constructs are introduced into cells (e.g., T cells) by lentiviral or retroviral transduction. Lentiviral vector backbones can be derived from HIV-1, HIV-2, visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), or simian immunodeficiency virus (SIV). Lentiviral vectors can be integration-competent or integrase-deficient lentiviral vectors (TDLV). In one embodiment, an IDLV vector containing an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is used. Non-limiting exemplary approaches for preparing retroviral expression constructs and transducing cells with such constructs are provided in the experimental section below.

[0079] In certain embodiments, cells of the present disclosure (e.g., T cells, NK cells, etc.) are genetically modified to express a receptor (e.g., a recombinant receptor) on their surface. That is, cells may be engineered to co-express a CCR and an additional receptor, non-limiting examples of which include chimeric antigen receptors (CARs), T cell receptors (TCRs) such as recombinant TCRs, synthetic Notch receptors (synNotch), modular extracellular sensor architecture (MESA) receptors, Tango receptors, ChaCha receptors, general extracellular molecular sensor (GEMS) receptors, growth factor receptors, switch receptors, adhesion molecules, integrins, inhibitory receptors, stimulatory receptors, and immunoreceptor tyrosine-based activating receptors. Immunoreceptors such as ITAM-containing receptors, immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptors, hormone receptors, receptor tyrosine kinases, CD28, CD80, ICOS, CTLA4, PD1, PD-L1, BTLA, HVEM, CD27, 4-1BB, 4-1BBL, OX40, OX40L, DR3, GITR, CD30, SLAM, CD2, 2B4, TIM1, TIM2, TIM3, TIGIT, CD226, CD160, LAG3, LAIR1, B7-1, B7-H1, and B7-H3. body, type I cytokine receptors, such as interleukin-1 receptor, interleukin-2 receptor, interleukin-3 receptor, interleukin-4 receptor, interleukin-5 receptor, interleukin-6 receptor, interleukin-7 receptor, interleukin-9 receptor, interleukin-11 receptor, interleukin-12 receptor, interleukin-13 receptor, interleukin-15 receptor, interleukin-18 receptor, interleukin-21 receptor, interleukin-23 receptor, interleukin-27 receptor, erythropoietin receptor, GM-CSF receptor, G-CSF receptor, growth hormone receptor, prolactin receptor, leptin receptor, oncostatin M receptor, leukemia inhibitory factor, type II cytokine receptors, such as interferon-alpha / beta receptor, interferon-gamma receptor, interferon type III receptor, interleukin-10 receptor, interleukin-20 receptor, interleukin-22 receptor, interleukin-28 receptor,Receptors in the tumor necrosis factor receptor superfamily, e.g., tumor necrosis factor receptor 2 (1B), tumor necrosis factor receptor 1, lymphotoxin beta receptor, OX40, CD40, Fas receptor, Decoy receptor 3, CD27, CD30, 4-1BB, Decoy receptor 2, Decoy receptor 1, Death receptor 5, Death receptor 4, RANK, osteoprotegerin, TWEAK receptor, TACI, BAFF receptor, herpes virus entry mediator, nerve growth factor receptor, B cell maturation antigen, glucocorticoid-inducible TNFR-related, TROY, Death receptor 6, Death receptor 3, ectodysplasiacin A2 receptor, chemokine receptors, e.g., CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXC Examples of receptors include R3, CXCR4, CXCR5, CXCR6, CX3CR1, XCR1, ACKR1, ACKR2, ACKR3, ACKR4, CCRL2, receptors in the epidermal growth factor receptor (EGFR) family, receptors in the fibroblast growth factor receptor (FGFR) family, receptors in the vascular endothelial growth factor receptor (VEGFR) family, rearranged receptors in the transfection (RET) receptor family, receptors in the Eph receptor family, receptors that can induce cell differentiation (e.g., Notch receptors), cell adhesion molecules (CAMs), adhesion receptors such as integrin receptors, cadherins, selectins, and receptors in the discoidin domain receptor (DDR) family, transforming growth factor beta receptor 1, and transforming growth factor beta receptor 2. In some embodiments, such receptors are immune cell receptors selected from T cell receptors, B cell receptors, natural killer (NK) cell receptors, macrophage receptors, monocyte receptors, neutrophil receptors, dendritic cell receptors, mast cell receptors, basophil receptors, and eosinophil receptors. ,

[0080] In certain embodiments, the cells of the present disclosure are engineered to express a chimeric antigen receptor (CAR) in addition to a CCR. According to some embodiments, the cells of the present disclosure are engineered to express a recombinant TCR in addition to a CCR.

[0081] As mentioned above, according to some embodiments, cells (e.g., human T cells) of the present disclosure can be engineered to express a CAR in addition to a CCR. The extracellular binding domain of the CAR can comprise a single-chain antibody. The single-chain antibody can be a monoclonal single-chain antibody, a chimeric single-chain antibody, a humanized single-chain antibody, a fully human single-chain antibody, etc. In one non-limiting example, the single-chain antibody is a single-chain variable fragment (scFv). In some embodiments, the extracellular binding domain of the CAR is a single-chain embodiment (e.g., an scFv embodiment) of an antibody approved by the United States Food and Drug Administration and / or the European Medicines Agency (EMA) for use as a therapeutic antibody. Non-limiting examples of single chain antibodies that can be used when the protein of interest is a CAR include adecatumumab, ascribacumab, cixutumumab, conatumumab, daratumumab, drozitumab, durigotuzumab, durvalumab, dusigitumab, enfortumab, enothiocyanate, and cefotaxime. Mab, fizitumumab, ganitumab, glembatumumab, intetumumab, ipilimumab, iratumumab, icrucumab, lexatumumab, lucatumumab, mapatumumab, narunatumumab, necitumumab, nesbacumab, ofatumumab, olaratumumab, panitumumab, patritumumab, pritumumab, radletumab Radretumab, Ramucirumab, Rilotumumab, Lobatumumab, Seribantumab, Tarextumab, Teprotumumab, Tobetumumab, Vanticumumab, Besencumab, Votumumab, Zalutumumab, Franvotumab, Altumomab, Anatumomab, Arcitumomab, Bectumomab, Blinatumomab, Detumomab b), Ibritumomab, Minletumomab, Mitumomab, Moxetumomab, Naptumomab, Nofetumomab, Pemtumomab, Pintumomab, Racotumomab, Satumomab, Solitomab, Taplitumomab,Tenatumomab, Tositumomab, Tremelimumab, Abagovomab, Igovomab, Oregovomab, Capromab, Edrecolomab, Nacolomab, Amatuximab, Bavituximab, Brentuximab, Cetuximab, Derlotuximab, Dinutuximab, Ensituximab, Futuximab, Direntuximab, Indatuximab uximab, isatuximab, margetuximab, rituximab, siltuximab, ublituximab, ecromeximab, abithuzumab, alemtuzumab, bevacizumab, bivatuzumab, brontiximab, cantuzumab, cantuzumab, sitatuzumab, clivatuzumab, dacetuzumab, demcizumab, dalotuzumab, denintuzumab, elotuzumab, emactuzumab, emibetuzumab, enoblitzumab, etaracizumab, fa -retuzumab, ficlatuzumab, gemtuzumab, imgatuzumab, inotuzumab, labetuzumab, rifastuzumab, lintuzumab, lorvotuzumab, lumuletuzumab, matuzumab, milatuzumab, nimotuzumab, obinutuzumab, ocalatuzumab, otlertuzumab, onartuzumab, opoltuzumab, parsatuzumab, pertuzumab, pinatuzumab, polatuzumab, sibrotuzumab, simtuzumab and single chain embodiments (e.g., scFv embodiments) of antigen-binding variants thereof, such as Imtuzumab, Takatuzumab, Tigatuzumab, Trastuzumab, Tucotuzumab, Bunduzumab, Vanucizumab, Veltuzumab, Borsetuzumab, Sofituzumab, Catumaxomab, Ertumaxomab, Depatuxizumab, Ontuxizumab, Blontuvetmab, Tamtuvetmab, or antigen-binding variants thereof.

[0082] Additional receptors (e.g., CARs) may contain one or more linker sequences between various domains. A "variable region linking sequence" is an amino acid sequence that connects the heavy chain variable region to the light chain variable region and provides a spacer function to accommodate the interaction of the two sub-binding domains so that the resulting polypeptide retains the same specific binding affinity for the same target molecule as an antibody comprising the same light and heavy chain variable regions. A non-limiting example of a variable region linking sequence is a glycine-serine linker, such as the (G4S)3 (SEQ ID NO: 63) linker. In certain embodiments, a linker separates one or more heavy or light chain variable domains, hinge domains, transmembrane domains, costimulatory domains, and / or primary signaling domains. In specific embodiments, a receptor (e.g., CAR) comprises one, two, three, four, five, or more linkers. In specific embodiments, the linker length is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids in length.

[0083] In some embodiments, the antigen-binding domain of a receptor (e.g., a CAR) is followed by one or more spacer domains that distance the antigen-binding domain from the cell surface expressing the receptor to allow for proper cell-cell contact, antigen binding, and / or activation. The spacer domain (and any other spacer domains, linkers, etc. described herein) can be derived from either natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, the spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain can comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. In some embodiments, the spacer domain comprises the CH2 and / or CH3 of IgG1, IgG4, or IgD. Exemplary spacer domains suitable for use in the receptors (e.g., CARs) described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8α and CD4, which can be wild-type hinge regions from these molecules or variants thereof. In certain embodiments, the hinge domain comprises a CD8α hinge region. According to some embodiments, the hinge is a PD-1 hinge or a CD152 hinge. In certain embodiments, the hinge is IgG4.

[0084] A "transmembrane domain" (Tm domain) is a portion of a receptor (e.g., a CAR) that fuses the extracellular binding portion and the intracellular signaling domain and anchors the receptor to the plasma membrane of a cell (e.g., a T cell, e.g., a Treg). The Tm domain can be derived from either natural, synthetic, semi-synthetic, or recombinant sources. In some embodiments, the Tm domain is derived from (e.g., includes at least the transmembrane region or a functional portion thereof) the alpha or beta chain of a T cell receptor, CD35, CD3ζ, CD3γ, CD3δ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, or PD-1.

[0085] In one embodiment, a receptor (e.g., a CAR) comprises a Tm domain derived from CD28. In certain embodiments, the receptor comprises a Tm domain derived from CD28 and a short oligopeptide or polypeptide linker, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that connects the Tm domain and the intracellular signaling domain of the receptor. For example, a glycine-serine linker can be used as such a linker.

[0086] The "intracellular signaling" domain of a receptor (e.g., a CAR) refers to the portion of the receptor that is responsible for transducing a signal from binding to a target molecule / antigen into the interior of the cell to induce a cellular function. Thus, the term "intracellular signaling domain" refers to the portion of the protein that transduces a signal and instructs the cell to perform a specific function. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the full-length intracellular signaling domain, so long as it transmits a signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transduce a signal.

[0087] Signals generated solely through the T cell receptor (TCR) are insufficient for full activation of T cells; secondary or costimulatory signals are also required. Thus, T cell activation is mediated by two distinct classes of intracellular signaling domains: primary signaling domains (e.g., TCR / CD3 complexes) that initiate antigen-dependent primary activation via the TCR, and costimulatory signaling domains that act in an antigen-independent manner to provide secondary or costimulatory signals. Thus, receptors (e.g., CARs) expressed by genetically modified cells can contain intracellular signaling domains that include one or more (e.g., one, two, or more) "costimulatory signaling domains" and "primary signaling domains."

[0088] The primary signaling domain regulates primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (or "ITAMs"). Non-limiting examples of ITAM-containing primary signaling domains suitable for use in the receptors of the present disclosure include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79α, CD79β, and CD66δ. In certain embodiments, the receptor comprises a CD3ζ primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling and costimulatory signaling domains are operably linked to the carboxyl terminus of the transmembrane domain.

[0089] In some embodiments, a receptor (e.g., a CAR) comprises one or more costimulatory signaling domains to enhance the efficacy and expansion of immune effector cells (e.g., T cells) that express the receptor. As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule or an active fragment thereof. Exemplary costimulatory molecules suitable for use with receptors contemplated in specific embodiments include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and ZAP70. In some embodiments, the receptor (e.g., CAR) comprises one or more costimulatory signaling domains selected from the group consisting of 4-1BB (CD137), CD28, and CD134, and a CD3ζ primary signaling domain.

[0090] Receptors (e.g., CARs) expressed by cells genetically modified according to the methods of the present disclosure may comprise any of a variety of suitable domains, including, but not limited to, a leader sequence; a hinge, spacer, and / or linker domain; a transmembrane domain; a costimulatory domain; a signaling domain (e.g., a CD3ζ domain); a ribosomal skip element; a restriction enzyme sequence; a reporter protein domain; and the like.

[0091] According to some embodiments, the extracellular binding domain of the receptor (e.g., CAR) specifically binds to a tumor antigen expressed on the surface of a cancer cell.Non-limiting examples of tumor antigens to which the extracellular binding domain of the receptor can specifically bind include 5T4, AXL receptor tyrosine kinase (AXL), B-cell maturation antigen (BCMA), c-MET, C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), cadherin-6, CD19, CD20, CD22, CD25, CD27L, CD30, CD33, CD37, CD44, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEA), cKit, Cripto protein, CS1, and Dekkerin-1. DLL3, endothelin receptor type B (EDNRB), ephrin A4 (EFNA4), epidermal growth factor receptor (EGFR), EGFRvIII, ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPHA2), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), FMS-like tyrosine kinase 3 (FLT3), folate receptor 1 (FOLR1), GD2 ganglioside ("GD2"), glycoprotein B (G PNMB), guanylate cyclase 2C (GUCY2C), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), integrin alpha, lysosome-associated membrane protein 1 (LAMP-1), Lewis Y, LIV-1, leucine-rich repeat-containing 15 (LRRC15), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), sodium-dependent phosphate transport protein 2B (NaPi2b), nectin-4, NMB, NOTCH3, p-cadherin (p-CAD), programmed cell death receptor 1 These include programmed cell death receptor ligand 1 (PD-L1), programmed cell death receptor ligand 2 (PD-L2), prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), solute carrier family 44 member 4 (SLC44A4), SLIT-like family member 6 (SLITRK6), STEAP family member 1 (STEAP1), tissue factor (TF), T-cell immunoglobulin and mucin protein-1 (TIM-1), Tn antigen, trophoblast cell surface antigen (TROP-2), Wilms' tumor 1 (WT1), and VEGF-A.

[0092] In certain embodiments, the cells of the present disclosure are genetically modified to express an antibody. The term "antibody" (also used interchangeably with "immunoglobulin") encompasses antibodies of any isotype (e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramer composed of two dimers of a heavy chain polypeptide and a light chain polypeptide); fragments of antibodies (e.g., whole antibodies or single-chain antibody fragments) that retain specific binding to an antigen, including, but not limited to, single-chain antibodies (e.g., scFv), single-chain Fv (scFv), Fab, (Fab')2, (scFv')2, and diabodies; chimeric antibodies; monoclonal antibodies, humanized antibodies, human antibodies; and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein.

[0093] Immunoglobulin polypeptides include kappa and lambda light chains, and alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu heavy chains or equivalents in other species. Full-length immunoglobulin "light chains" (usually about 25 kDa or about 214 amino acids) contain a variable region of about 110 amino acids at the NH2-terminus and a kappa or lambda constant region at the COOH-terminus. Full-length immunoglobulin "heavy chains" (about 150 kDa or about 446 amino acids) similarly contain a variable region (about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (about 330 amino acids).

[0094] Immunoglobulin light or heavy chain variable regions (V L and V H) are composed of a "framework" region (FR) interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The extent of the framework region and CDRs has been defined (see E. Kabat et al., Sequences of proteins of immunological interest, 4th ed. USDept. Health and Human Services, Public Health Services, Bethesda, MD (1987), and Lefranc et al. IMGT, the international ImMunoGeneTics information system®. Nucl. Acids Res., 2005, 33, D593-D597). The sequences of framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs primarily contribute to binding to an antigen epitope. All CDRs and frameworks provided by this disclosure are defined according to Kabat above unless otherwise indicated.

[0095] Thus, "antibody" encompasses proteins having one or more polypeptides that may be genetically encodable, for example, by immunoglobulin genes or fragments of immunoglobulin genes. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. In some embodiments, antibodies of the present disclosure are IgG antibodies, e.g., IgG1 antibodies, such as human IgG1 antibodies. In some embodiments, cells express antibodies that comprise a human Fc domain.

[0096] A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of approximately 100-110 or more amino acids that is primarily responsible for antigen recognition. The variable light chain (VLC) L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.

[0097] According to some embodiments, the cells of the present disclosure are not genetically modified to express an engineered receptor other than a CCR.

[0098] As will be understood with the benefit of the present disclosure, cells (e.g., T cells) of the present disclosure can be expanded during the therapeutic cell manufacturing process. "Expanding" or "expanded" means that cells are cultured under conditions in which the cells proliferate. Suitable conditions can vary depending, for example, on the type of cells (e.g., type of T cells) being expanded. Such conditions may include culturing cells (e.g., T cells) in a suitable container (e.g., a cell culture plate or well thereof, or a cassette, tube, bottle, or bag suitable for use in an automated therapeutic cell manufacturing system, e.g., a closed automated therapeutic cell manufacturing system such as the CliniMACS Prodigy® system from Miltenyi Biotec, the Xuri® cell expansion system from Cytiva, the G-Rex® cell expansion system from Wilson Wolf, the Quantum® cell expansion system from Terumo, or the Cocoon® system from Lonza), in a suitable medium (e.g., a cell culture medium such as RPMI, DMEM, IMDM, MEM, or DMEM / F-12), at a suitable temperature (e.g., 32°C to 42°C, e.g., 37°C) and pH (e.g., pH 7.0 to 7.7, e.g., pH 7.4), in an environment having a suitable percentage of CO2, e.g., 3% to 10%, e.g., 5%.

[0099] Methods for activating and expanding therapeutic cells (e.g., therapeutic T cells) are known in the art and are described, for example, in U.S. Pat. Nos. 6,905,874, 6,867,041, and 6,797,514, and PCT Publication No. WO 2012 / 079000, the contents of which are incorporated herein by reference in their entirety. In the example of T cells, such methods may include contacting PBMCs or isolated T cells with stimulatory and costimulatory agents, such as anti-CD3 and anti-CD28 antibodies typically attached to beads or other surfaces, in culture medium, along with appropriate cytokines, such as IL-2. The anti-CD3 and anti-CD28 antibodies attached to the same beads function as "surrogate" antigen-presenting cells (APCs). One example is the Dynabeads® system, a CD3 / CD28 activator / stimulator system for the physiological activation of human T cells. In other embodiments, T cells are activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines, such as using methods described in U.S. Pat. Nos. 6,040,177 and 5,827,642, and PCT Publication No. WO2012 / 129514, the contents of which are incorporated herein by reference in their entireties.

[0100] In certain embodiments, the cells of the present disclosure are expanded using an automated system designed for the production of therapeutic cells. Non-limiting examples of such systems include the CliniMACS Prodigy® system from Miltenyi Biotec, the Xuri® cell expansion system from Cytiva, the G-Rex® cell expansion system from Wilson Wolf, the Quantum® cell expansion system from Terumo, and the Cocoon® system from Lonza. Detailed guidance and protocols for producing therapeutic cells on such systems are available from the providers of such systems.

[0101] composition Compositions are also provided by the present disclosure. According to some embodiments, compositions are provided that include one or more nucleic acid(s), one or more expression construct(s), and / or any of the cells or progeny thereof of the present disclosure.

[0102] Such compositions may include one or more nucleic acids, one or more expression constructs, and / or cells of the present disclosure present in a liquid medium. The liquid medium may be an aqueous liquid medium such as water, a buffer solution, or the like. One or more additives may be present in such compositions, such as salts (e.g., NaCl, MgCl, KCl, MgSO), buffers (Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), solubilizers, detergents (e.g., non-ionic detergents such as Tween-20), nuclease inhibitors, glycerol, chelating agents, etc. In certain embodiments, the liquid medium is a cell culture medium, non-limiting examples of which include Minimum Essential Medium, DMEM, a-MEM, RPMI Media, Clicks, F-12, X-Vivo15, X-Vivo20, Optimizer, etc.

[0103] In certain embodiments, compositions comprising the cells of the present disclosure are provided, wherein the compositions are suitable for administration to a subject, e.g., a human subject. Such compositions may include cells and a pharmaceutically acceptable carrier. The compositions generally include a therapeutically effective amount of cells. A "therapeutically effective amount" refers to a sufficient number of cells to produce a desired result, e.g., a sufficient amount to produce a beneficial or desired therapeutic (including prophylactic) result, such as a reduction in the symptoms of a disease (e.g., cancer) or disorder associated with, e.g., a target cell or population thereof (e.g., cancer cells), compared to a control. An effective amount can be administered in one or more administrations. A therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the cells to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount that is effective to "treat" a subject, e.g., a human subject. When a therapeutic amount is indicated, the exact amount of the composition contemplated in a specific embodiment to be administered can be determined by a physician in light of this specification and taking into account individual differences in age, weight, tumor size, extent of infection or metastasis, and patient (individual) condition. In some embodiments, the composition of the present disclosure is administered in an amount of 1×10 6 ~5×10 10 The cell of the present disclosure.

[0104] The cells of the present disclosure can be incorporated into various formulations for therapeutic administration. For example, the cells of the present disclosure can be formulated into a pharmaceutical composition by combining with a suitable pharmaceutically acceptable excipient or diluent. Preparations of cells suitable for administration to a patient (e.g., suitable for human administration) will generally be sterile and will further contain no detectable pyrogens or other contaminants that would contraindicate administration to a patient according to the selected route of administration.

[0105] The cells may be formulated for parenteral (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intraventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration.

[0106] Aqueous cell formulations may be prepared in a pH buffer solution, for example, at a pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively at about 5.5. Examples of buffers suitable for a pH within this range include phosphate buffer, histidine buffer, citrate buffer, succinate buffer, acetate buffer, and other organic acid buffers. The buffer concentration may be, for example, from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending on the buffer and the desired tonicity of the formulation.

[0107] A tonicity agent may be included in the formulation to adjust the tonicity of the formulation. Exemplary tonicity agents include sodium chloride, potassium chloride, glycerin, and any member from the group of amino acids, sugars, and combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be preferred. The term "isotonic" refers to a solution that has the same tonicity as some other solution, such as physiological salt solution or serum, to which it is being compared. The tonicity agent may be used in an amount of about 5 mM to about 350 mM, for example, 100 mM to 350 mM.

[0108] Surfactants may be added to the formulation to reduce aggregation in the formulation, minimize the formation of fine particles, and / or reduce adsorption. Exemplary surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylene sorbitan fatty acid esters are polysorbate 20 (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Exemplary concentrations of surfactants can range from about 0.001% to about 1% w / v.

[0109] In some embodiments, the compositions comprise the cells of the present disclosure and one or more of the agents identified above (e.g., surfactants, buffers, stabilizers, tonicity agents), and are essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at a concentration ranging from about 0.001 to about 2% (w / v).

[0110] kit Kits are also provided by the present disclosure. In certain embodiments, kits are provided that include any one or more nucleic acids or one or more expression constructs of the present disclosure. In certain embodiments, kits of the present disclosure include transfection / transduction reagents useful for introducing any one or more nucleic acids or one or more expression constructs into cells of interest, such as immune cells (e.g., T cells) or other cells of interest.

[0111] The components of the kit may be in separate containers, or multiple components may be in a single container. For example, one or more nucleic acids or one or more expression constructs may be provided in separate containers or in the same container. Suitable containers include a single tube (e.g., a vial), one or more wells of a plate (e.g., a 96-well plate, a 384-well plate, etc.), etc.

[0112] The kits of the present disclosure may further include instructions for contacting the cell population with one or more nucleic acids or one or more expression constructs under conditions whereby either the one or more nucleic acids or one or more expression constructs are delivered to cells of the cell population.The kits of the present disclosure may further include instructions for selecting cells that exhibit cell surface expression of a CCR.

[0113] The instructions for the kit may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. Thus, the instructions may be present in the kit as a package insert, on labeling of a container of the kit or its components (i.e., associated with the packaging or subpackaging), etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer-readable storage medium, e.g., a portable flash drive, DVD, CD-ROM, diskette, etc. In still other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the Internet, are provided. An example of this embodiment is a kit that includes a web address at which the instructions can be viewed and / or from which the instructions can be downloaded. Like the instructions, the means for obtaining the instructions are recorded on a suitable substrate.

[0114] Treatment method

[0010] Aspects of the present disclosure further include methods of treatment. As shown herein, the CCRs of the present disclosure, when expressed on cells, are potent drivers of cell proliferation in vitro and in vivo, and result in improved anti-tumor activity in vivo compared to control cells lacking the CCR. Accordingly, CCRs and cells expressing CCRs find use in cell therapy, non-limiting examples of which include immune cell therapy (e.g., immune cells lacking antigen-binding receptors (e.g., tumor-infiltrating lymphocytes)), CAR-T cell therapy, TCR therapy, CAR-NK cell therapy, and the like.

[0115] According to some embodiments, there is provided a method of administering a cell-based therapy to a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising the cells of the present disclosure. The cells comprise any one or more expression constructs of the present disclosure such that the cells express a CCR comprising first and second subunits encoded by one or more nucleic acids. The cells may be further modified to express any of the additional receptors described herein, such as a CAR, a recombinant TCR, etc.

[0116] "Cell-based therapy" or "cell therapy" refers to the transfer of autologous or allogeneic cellular material to a subject for medical purposes. Non-limiting examples of cell-based therapies include CAR T cell therapy, engineered T cell therapy (e.g., T cells that express a recombinant T cell receptor (TCR)), therapy involving administering T cells that do not express a recombinant receptor other than a CCR, CAR NK cell therapy, etc.

[0117] In certain embodiments, signaling of the chimeric cytokine receptor is regulatable, for example, in some embodiments, the first subunit, the second subunit, or both, comprises a protease cleavage site disposed between the transmembrane domain and the ICD, the one or more nucleic acids further encode a protease, the protease cleavage site is a cleavage site for a protease, and the proteolytic activity of the protease is regulatable.

[0118] According to some embodiments, the proteolytic activity of the protease can be regulated via a cell-permeable small molecule. For example, the cell-permeable small molecule can be an inhibitor of the protease, and the method further comprises administering an effective amount of the cell-permeable small molecule to the subject when signaling via the CCR is desired. In certain embodiments, such a method further comprises ceasing administration of the cell-permeable small molecule when signaling via the CCR is no longer desired.

[0119] When the cell-based therapy is tunable using a cell-permeable protease inhibitor, the protease inhibitor is selected based on the protease used in the tunable system. For example, in some embodiments, when the protease is derived from HCV NS3, the protease inhibitor can be asunaprevir (ASV), danoprevir (DPV), simeprevir (SPV), grazoprevir (GPV), glecaprevir, voxilaprevir, or any combination thereof. Also by way of example, if the protease is an HIV protease, the protease inhibitor can be atazanavir (Reyataz), darunavir (Prezista), fosamprenavir (Lexiva), indinavir (Crixivan), lopinavir / ritonavir (Kaletra), nelfinavir (Viracept), ritonavir (Norvir), saquinavir (Invirase), tipranavir (Aptivus), atazanavir / cobicistat (Evotaz), darunavir / cobicistat (Prezcobix), or any combination thereof. As an additional example, if the protease is a SARS-CoV-2 protease, the protease inhibitor can be nilmatrervir (Paxlovid). As a further example, if the protease is human renin protease, the protease inhibitor can be aliskiren.

[0120] Therapeutic cells can be self / autologous ("autologous") or non-self ("non-autologous", e.g., allogeneic, syngeneic, or xenogeneic). "Autologous," as used herein, refers to cells obtained from the subject to whom the therapeutic cells are subsequently administered. "Allogeneic," as used herein, refers to cells obtained from a donor other than the subject to whom the therapeutic cells are administered. In some embodiments, the cells (e.g., T cells) are cells obtained from a mammalian subject. In certain embodiments, the mammalian subject is frozen. In some embodiments, the cells are obtained from a human.

[0121] Any of the cell-based therapy methods of the present disclosure can be used to treat a variety of conditions in a subject. In certain embodiments, the subject has cancer. The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell growth / proliferation. The methods can be used to treat a wide variety of cancers. In certain embodiments, the cancer comprises a solid tumor. Examples of solid tumors treatable by the methods of the present disclosure include carcinoma, lymphoma, blastoma, and sarcoma. In certain embodiments, the cancer comprises a hematological malignancy, non-limiting examples of which include leukemia, lymphoma, or multiple myeloma.

[0122] More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bile duct cancer, bladder cancer, hepatoma, breast cancer, colon cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer. In certain embodiments, the individual has a cancer selected from a solid tumor, recurrent glioblastoma multiforme (GBM), non-small cell lung cancer, metastatic melanoma, melanoma, peritoneal cancer, epithelial ovarian cancer, glioblastoma multiforme (GBM), metastatic colorectal cancer, colorectal cancer, pancreatic ductal adenocarcinoma, squamous cell carcinoma, esophageal cancer, gastric cancer, neuroblastoma, fallopian tube cancer, bladder cancer, metastatic breast cancer, pancreatic cancer, soft tissue sarcoma, recurrent squamous cell carcinoma of the head and neck, head and neck cancer, anaplastic astrocytoma, malignant pleural mesothelioma, squamous non-small cell lung cancer, rhabdomyosarcoma, metastatic renal cell carcinoma, basal cell carcinoma (basal cell epithelioma), and gliosarcoma. In certain embodiments, the individual has a cancer selected from melanoma, Hodgkin's lymphoma, renal cell carcinoma (RCC), bladder cancer, non-small cell lung cancer (NSCLC), and head and neck squamous cell carcinoma (HNSCC).

[0123] Treatment refers to at least an amelioration of one or more symptoms associated with a condition (e.g., cancer) in a subject, where amelioration is used broadly to refer to at least a reduction in a parameter associated with the condition being treated, e.g., the magnitude of the symptom. Thus, treatment also includes situations in which a condition (e.g., cancer), or at least one or more symptoms associated therewith, are completely inhibited, e.g., prevented from occurring, or halted, e.g., terminated, so that the host no longer suffers from the condition, or at least the symptoms that characterize the condition. [Example]

[0124] The following examples are offered by way of illustration and not by way of limitation.

[0125] experiment Example 1 - Generation of chimeric cytokine receptors (CCRs) that signal in the absence of cognate cytokine This example describes the generation of a chimeric cytokine receptor (CCR) capable of signaling in the absence of a cognate cytokine. The CCR comprises a first subunit comprising a first heterodimerization domain and a first cytokine receptor intracellular signaling domain (ICD), and a second subunit comprising a second heterodimerization domain homologous to the first heterodimerization domain and a second cytokine receptor ICD. As proof of concept, in this example, the CCR uses leucine zippers as the first and second heterodimerization domains, the first ICD being IL-2RB or IL-21R, and the second ICD being the common gamma chain (γC) ICD.

[0126] Figure 2A shows a schematic diagram of a chimeric cytokine receptor (CCR) system that allows constitutive receptor signaling in the absence of cognate cytokine. Receptor 1 of the CCR is composed of a first dimerization domain, such as a leucine zipper fused to a transmembrane domain, and a first intracellular domain (ICD1). Receptor 2 of the CCR is composed of a second dimerization domain, a homolog of the first dimerization domain, such as a second leucine zipper fused to the transmembrane domain, and a second intracellular domain (ICD2). Association of the two dimerization domains brings the two intracellular signaling domains into close proximity, triggering signal transduction of the receptor complex. Cysteine ​​residues are optionally included in the extracellular domain to stabilize the association of the receptor complex via disulfide bonds. A detection tag is optionally included to facilitate detection of cell surface expression of the receptor components. Figure 2B shows an exemplary signaling domain for a chimeric cytokine receptor based on a gamma chain cytokine receptor.

[0127] Figure 3 is a series of flow plots showing the cell surface expression of CCR receptor components for various common gamma chain receptors. The receptor components consist of an extracellular detection module (HA or Flag tag), a leucine zipper domain, a CD8α hinge and transmembrane domain, and an intracellular signaling domain (IL-2RB, IL-21R, or γC). As can be seen in the data, receptors containing the γC intracellular domain, with or without the GGSGS linker, showed poor cell surface expression. Mock-untransduced T cells, with or without added cytokines, served as negative controls for the FLAG and HA tags.

[0128] Figure 4A shows a schematic diagram of the chimeric cytokine receptor (CCR) system. To improve expression of CCR receptors containing a common gamma chain intracellular domain, we engineered the transition between the transmembrane and intracellular domains. Figure 4B lists various CCR constructs containing a common gamma chain intracellular domain and a transition peptide between the transmembrane and ICD domains. The transition peptides were derived from the juxtamembrane region of CD8α or IL-15Rα. HTM refers to the hinge and transmembrane domain. Figure 4C shows a series of flow plots of IL-2R CCRs, showing the cell surface expression of the HA tag and phosphorylated STAT5 (pSTAT5), a cytokine signaling molecule downstream of IL-2R signaling. As can be seen in the data, including the transition peptide in the juxtamembrane domain improves cell surface expression of the common gamma chain CCR receptor components, allowing for the induction of high levels of IL-2 signaling in the absence of cytokines. Mock-untransduced T cells exposed to exogenously added IL-2 serve as a positive control for pSTAT5.

[0129] Figure 5 shows plots depicting the proliferation of primary human T cells transduced with the various CCRs shown in Figure 4. As can be seen in the data, the CCRs induce T cell proliferation in the absence of added IL-2. Mock-untransduced T cells with added IL-2 serve as a positive control. T cells transduced with only one component of the CCR (CCR IL-2RB only) do not proliferate in the absence of IL-2). The CCRs induce T cell proliferation to varying degrees, and the magnitude of proliferation correlates with the strength of pSTAT5 signaling, as shown in Figure 4.

[0130] Example 2 - Development of a tunable CCR system to address the in vivo toxicity observed with constitutive CCR This example shows that the constitutive CCR described in Example 1 exhibits toxicity in vivo. However, the development of a regulatable CCR system to address such toxicity is demonstrated herein.

[0131] Figure 6A is a series of bioluminescence images showing the expansion of primary human T cells transduced with a HER2 chimeric antigen receptor (CAR) and IL-2 or IL-21 CCR in NSG mice bearing 143B osteosarcoma tumors. As can be seen in the data, the IL-2 CCR is a potent driver of T cell proliferation in vivo. Figure 6B shows quantification of T cell expansion from the bioluminescence images shown in Figure 6A. As can be seen in the data, the IL-2 CCR is a potent driver of T cell proliferation in vivo. Figure 6C is a graph showing tumor size in mice implanted with 143B osteosarcoma cells and treated with the indicated T cells. Figure 6D is a graph of survival curves for mice treated with the indicated T cells. Mice treated with CCR-IL-2 rapidly died from treatment-related toxicity due to uncontrolled proliferation of T cells. Thus, although a constitutive IL-2 CCR is a potent driver of proliferation in vivo, it causes lethal toxicity in NSG mice.

[0132] Figure 7 is a schematic diagram of the drug-regulated chimeric cytokine receptor (CCR) system developed in this example. A protease cleavage site is incorporated between the transmembrane and intracellular signaling domains of the CCR. Coexpression of a protease specific to the cleavage site causes cleavage of the CCR due to the proteolytic activity of the protease at the cleavage site, resulting in inactivation of the CCR (receptor off). Addition of a protease inhibitor (drug) inhibits this cleavage event, turning the CCR into an on-state (receptor on). Thus, CCR signaling becomes dependent on the presence of the drug. The regulated CCR can avoid the toxicity associated with constitutive CCRs by modulating cell proliferation to fall within the therapeutic window.

[0133] Figure 8 shows a series of flow plots of primary human T cells transduced with a regulated version of the IL-2R CCR, as described in Figure 7, showing cell surface expression of the HA / FLAG tag and phosphorylated STAT5 (pSTAT5), a cytokine signaling molecule downstream of IL-2R signaling. The regulated CCR was constructed by incorporating a 33-amino acid linker containing the HCV NS3 protease cleavage site between the transmembrane and intracellular domains of the CCR. HCV NS3 protease was coexpressed intracellularly, allowing receptor cleavage in the absence of a protease inhibitor (3 μM grazoprevir). As can be seen in the data, inclusion of the 33-amino acid linker containing the HCV NS3 cleavage site eliminated CCR signaling in the presence or absence of the drug, and is not an optimal configuration for regulated CCR signaling. Mock-untransduced T cells exposed to exogenously added IL-2 served as a positive control for pSTAT5.

[0134] Figure 9 shows that inserting a linker containing a cleavage site upstream of the gamma chain ICD allows for drug-regulated CCR signaling. Figure 9A is a list showing amino acid linkers containing an HCV NS3 protease cleavage site inserted at various positions within the gamma chain ICD. Figure 9B is a series of flow plots of primary human T cells transduced with a regulated version of the IL-2R CCR, as shown in Figure 9A, showing cell surface expression of an HA / FLAG tag and phosphorylated STAT5 (pSTAT5). The regulated CCR was constructed by incorporating a 19-amino acid linker containing an HCV NS3 protease cleavage site between the transmembrane and intracellular domains of the gamma chain CCR component. HCV NS3 protease was coexpressed intracellularly, allowing receptor cleavage in the absence of a protease inhibitor (3 μM grazoprevir, drug). As can be seen in the data, inclusion of the 19-amino acid linker containing the HCV NS3 cleavage site at position 1 allows for drug-dependent signaling of the CCR. Mock non-transduced T cells exposed to exogenously added IL-2 serve as a positive control for pSTAT5.

[0135] Figure 10 shows a series of flow plots of primary human T cells transduced with a regulated version of the IL-2R CCR, demonstrating phosphorylated STAT5 (pSTAT5). The regulated CCR was constructed by incorporating a 19-amino acid linker containing an HCV NS3 protease cleavage site between the transmembrane and intracellular domains of the gamma chain CCR component, the IL-2RB CCR component, or both. HCV NS3 protease was coexpressed intracellularly to allow receptor cleavage in the absence of a protease inhibitor (drug). The HCV 4a4B or 4b5A cleavage site was used as the HCV cleavage site. "RB" indicates a CCR component containing the IL-2RB ICD. "gc" indicates a CCR component containing the common gamma chain ICD. "Off" and "On" indicate the absence or presence of a protease inhibitor (3 μM grazoprevir), respectively. As can be seen in the data, several configurations allow for drug-regulated control of CCR signaling. Mock non-transduced T cells exposed to exogenously added IL-2 serve as a positive control for pSTAT5.

[0136] Figure 11 shows drug control of in vitro cell expansion. Figure 11A is a series of plots showing the in vitro proliferation of primary human T cells transduced with various CCRs shown in Figure 10 and grown in the presence (Reg CCR On) or absence (Reg CCR Off) of a protease inhibitor (3 μM grazoprevir). The original (OG) constitutive IL-2 CCR and untransduced T cells (mock) serve as positive and negative controls, respectively. Figure 11B shows plots showing the percent viability of primary human T cells transduced with various CCRs and grown in the presence (On) or absence (Off) of a protease inhibitor (3 μM grazoprevir). As can be seen in the data, several configurations allow the survival and proliferation of drug-regulated regulatory T cells.

[0137] Example 3 - CCR enhances anti-tumor activity in vivo. In this example, we demonstrate that CCR enhances in vivo antitumor activity, including after rechallenge.

[0138] Figure 12A is a schematic diagram showing the constructs used in this experiment. A CD22-targeted CAR (CD22 CAR) was constructed by fusing an anti-CD22 scFv to a CD8a hinge, CD8a transmembrane domain, 41BB intracellular domain, and CD3z signaling domain. A constitutive IL-2 chimeric cytokine receptor (Const CCR) was constructed using the IL-2RB and common gamma chain intracellular domains and expressed in a bicistronic vector. A drug-regulated CCR (Reg CCR) was constructed by incorporating a 4b5a HCV cleavage site between the transmembrane domain and the common gamma chain intracellular domain. HCV NS3 protease was co-expressed in a tricistronic vector.

[0139] Figure 12B shows a series of bioluminescence images tracking the growth of Nalm6 leukemia expressing firefly luciferase. On day 0, NSG mice were injected with 1 x 10^6 Nalm6 and then treated with the indicated CAR-T cells. Four days later, 4 x 10^6 CAR-T or control cells were transplanted via tail vein injection. On day 17, mice were re-challenged with 4.6 x 10^6 Nalm6 tumor cells. Mice in the "+GPV" group were also implanted with osmotic drug pumps (Azlet model 2002) containing 54 mg / mL grazoprevir and 0.6 mg / mL ritonavir. These mice were additionally administered 50 mg / kg grazoprevir and 25 mg / kg ritonavir by oral gavage once or twice daily. Mock-untransduced T cells served as a negative control.

[0140] Figure 12C is a graph showing quantification of the bioluminescence images shown in Figure 12B. As can be seen in the data, mice receiving CD22 CAR-T co-transduced with regulated IL-2 CCR (Reg CCR) and administered grazoprevir were able to maintain long-term tumor control, whereas control CD22 CAR-T cells lacking the regulated CCR succumbed to tumor growth. Mice receiving CD22 CAR-T cells co-expressing constitutive IL-2 CCR (Const CCR) died due to treatment-related toxicity.

[0141] Figure 13 shows that the regulated CCR expands CAR-T cells in vivo without toxicity. A schematic diagram showing the constructs used in this experiment is shown in Figure 13A. A CD22-targeted CAR (CD22 CAR) was constructed by fusing an anti-CD22 scFv to a CD8a hinge, CD8a transmembrane domain, 41BB intracellular domain, and CD3z signaling domain. A constitutive IL-2 chimeric cytokine receptor (Const CCR) was constructed using the IL-2RB and common gamma chain intracellular domains and expressed in a bicistronic vector. A drug-regulated CCR (Reg CCR) was constructed by incorporating a 4b5a HCV cleavage site between the transmembrane domain and the common gamma chain intracellular domain. HCV NS3 protease was coexpressed in a tricistronic vector.

[0142] Figure 13B shows a series of bioluminescence images tracking the growth of Antares luciferase-expressing T cells in the mice shown in Figure 12B. On day 0, NSG mice were injected with 1 x 10^6 Nalm6 and then treated with the indicated CAR-T cells. Four days later, 4 x 10^6 CAR-T or control cells were transplanted via tail vein injection. On day 17, mice were re-challenged with 4.6 x 10^6 Nalm6 tumor cells. Mice in the "+GPV" group were also implanted with osmotic drug pumps (Azlet model 2002) containing 54 mg / mL grazoprevir and 0.6 mg / mL ritonavir. These mice were additionally administered 50 mg / kg grazoprevir and 25 mg / kg ritonavir by oral gavage once or twice daily. Mock-untransduced T cells served as a negative control.

[0143] Figure 13C shows a graph depicting quantification of the bioluminescence images shown in Figure 13B. As can be seen in the data, mice given CD22 CAR-T co-transduced with a regulated IL-2 CCR (Reg CCR) and administered grazoprevir expanded and persisted to a greater extent than control CD22 CAR-T cells lacking the regulated CCR. Mice given CD22 CAR-T cells co-expressing a constitutive IL-2 CCR (Const CCR) had rapid, uncontrolled expansion of CAR-T cells, which caused fatal treatment-related toxicity.

[0144] Figure 14 shows that CAR-T cells expressing drug-regulated IL-2 CCRs from two separate vectors exhibit enhanced in vivo expansion in the presence of grazoprevir. Figure 14A is a schematic diagram showing the constructs used in this experiment. The two chains of the regulated IL-2 CCR were expressed on separate vectors, and the IL-2RB component was bicistronic co-expressed with the CD22 CAR. A common gamma chain component containing the 4b5A HCV cleavage site was co-expressed with the HCV NS3 protease on a separate vector.

[0145] Figure 14B shows a series of bioluminescence images tracking the growth of Antares luciferase-expressing T cells. On day 0, NSG mice were injected with 1 x 10^6 Nalm6 and then treated with the indicated CAR-T cells. Four days later, 4 x 10^6 CAR-T cells were implanted via tail vein injection. On day 17, mice were rechallenged with 4.6 x 10^6 Nalm6 tumor cells. Mice in the "+GPV" group were also implanted with osmotic drug pumps (Azlet model 2002) containing 54 mg / mL grazoprevir and 0.6 mg / mL ritonavir. These mice received an additional 50 mg / kg grazoprevir and 25 mg / kg ritonavir by oral gavage once or twice daily.

[0146] Figure 14C is a graph showing quantification of the bioluminescence images shown in Figure 14B. As can be seen in the data, mice receiving CD22 CAR-T cells co-expressing drug-regulated IL-2 CCR (Reg CCR) and administered grazoprevir (+GPV) expand to a greater extent in vivo compared to mice that did not receive GPV.

[0147] Figure 15 shows that the leucine zipper domain enhances STAT5 signaling in IL-2 CCR. Flow plots are shown showing the phosphorylation levels of STAT5 from primary human T cells transduced with IL-2 CCR with or without the leucine zipper component (leucine zipper IL-2 CCR) or without (CD8a H / Tm IL-2 CCR). Mock-untransduced T cells exposed to exogenously added IL-2 serve as a positive control for pSTAT5, while those not exposed to IL-2 serve as a negative control. As can be seen in the data, the inclusion of the leucine zipper component in the IL-2 CCR results in higher levels of phosphorylated STAT5.

[0148] Expression of IL-21 CCR in primary human T cells results in high phosphorylation levels of STAT3. Flow plots of IL-21 CCR showing phosphorylation of STAT3 (pSTAT3), a cytokine signaling molecule downstream of IL-21R signaling, are shown. Mock non-transduced T cells exposed to exogenously added IL-21 serve as a positive control for pSTAT3, while those not exposed to IL-21 serve as a negative control. As can be seen in the data, expression of IL-21 CCR in primary human T cells results in high phosphorylation levels of STAT3.

[0149] Example 4 - IL-7R and IL-9R CCR In this example, the development of additional types of CCRs, specifically CCRs based on the IL-7R and IL-9R signaling domains (ICDs), is described.

[0150] Figure 17 shows flow plots of constitutive (Const.IL-2R, Const.IL-9R, Const.IL-7R) or HCV NS3 protease-regulated (Reg.IL-2R, Reg.IL-9R, Reg.IL-7R) chimeric cytokine receptors expressed on CD22.BBz CAR T cells. "On" and "Off" indicate cell culture in the presence or absence of 3 μM grazoprevir (an HCV NS3 protease inhibitor). The HCV NS3 protease-regulated receptors contained the HCV 4b5a cleavage site between the transmembrane domain and the gamma chain ICD. These data demonstrate constitutive signaling of IL-2R, IL-9R, and IL-7R CCRs, determined by high levels of pSTAT5 and / or pSTAT3. In contrast, the HCV NS3 protease-regulated chimeric receptors Reg.IL-2R and Reg.IL-9R express high levels of pSTAT5 and / or pSTAT3 only upon exposure to the HCV NS3 protease inhibitor grazoprevir. Reg.IL-7R fails to induce pSTAT5 and / or pSTAT3 signaling even in the presence of grazoprevir, suggesting that this specific HCV NS3 protease-regulated chimeric receptor embodiment is not compatible with the IL-7R ICD. Phosphoflow analysis was performed 48 hours after IL-2 was removed from the culture medium.

[0151] Figure 18 provides data showing that constitutive IL-2, IL-9, and IL-7 CCRs induce lethal toxicity in mice due to unregulated proliferation of CAR-T cells. Figure 18A is a series of bioluminescence images tracking the growth of T cells expressing Antares luciferase. On day 0, NSG mice were injected with 1 x 10^6 Nalm6. Four days later, 3 x 10^6 of the indicated CAR+CCR T cells were transferred by tail vein injection. On day 11, mice were re-challenged with 6 x 10^6 Nalm6 tumor cells. Figure 18B is a plot quantifying the bioluminescence of the images in Figure 18A. Figure 18C is a survival curve for the mice shown in Figure 17A. As can be seen in the data, mice receiving constitutive IL2, IL-9, and IL-7 CCRs experienced lethal toxicity due to unregulated proliferation of CAR-T cells.

[0152] Figure 19 shows data demonstrating the controlled growth of CAR-T cells expressing protease-regulated CCRs. Figure 19A shows a series of bioluminescence images tracking the growth of T cells expressing Antares luciferase. On day 0, NSG mice were injected with 1x10^6 Nalm6. Four days later, 3x10^6 of the indicated CAR+CCR T cells were transferred via tail vein injection. On day 11, mice were re-challenged with 6x10^6 Nalm6 tumor cells. Mice in the "On" group were also implanted with osmotic drug pumps (Azlet model 2002) containing 54 mg / mL grazoprevir and 0.6 mg / mL ritonavir. These mice received an additional 50 mg / kg grazoprevir and 25 mg / kg ritonavir by oral gavage once daily. One component of the regulated CCR was delivered in the CD22.BBz CAR vector (i.e., 22-IL-2RB or 22-IL-9R), while a second vector delivered the regulatable gamma chain (Reg gc) component and HCV NS3 protease, as shown in Figure 13A. As can be seen in the data, GPV can induce the growth of T cells expressing the regulatable CCR. The regulatable IL-9 CCR showed significant proliferation even in the absence of GPV, likely due to the signaling ability of the IL-9R ICD even in the absence of the cognate gamma chain ICD. Further iterations of the regulated IL-9 CCR may reduce this "leaky" activity using a dual truncation of the IL-9 CCR ICD in addition to truncation of the gamma chain ICD, as shown schematically in Figure 20.

[0153] Example 5 - Regulatable CCR with a protease fused to one of the CCR subunits This example describes the development of a regulatable CCR that contains a protease fused to one of the CCR subunits.

[0154] Figure 21A is a schematic diagram of a Reg CCR with a protease fused directly to the ICD2 system. The protease cleavage site is incorporated between the transmembrane domain and the intracellular signaling domain 2 (ICD2). The protease is incorporated between the ICD2 and the C-terminus of one of the chains / subunits of the CCR. Expression of the protease as part of the subunit causes cleavage of the CCR due to the proteolytic activity of the protease at the cleavage site, resulting in inactivation of the CCR (receptor off). Addition of a protease inhibitor (drug) inhibits this cleavage event, turning the CCR into an on-state (receptor on). Thus, CCR signaling becomes dependent on the presence of the drug. Regulatable CCRs are expected to avoid the toxicity associated with constitutive CCRs by modulating cell growth to fit within a therapeutic window.

[0155] Figure 21B shows flow plots depicting staining of primary human T cells expressing HCV NS3 protease-regulated IL2CCR T cells. "+GPV" and "-GPV" indicate cell culture in the presence or absence of 3 μM grazoprevir (an HCV NS3 protease inhibitor), respectively. The HCV NS3 protease-regulated receptor contained an HCV 4b5a cleavage site between the transmembrane domain and the gamma chain ICD. HCV NS3 protease was delivered as a direct fusion to the gamma chain ICD with a short linker (cis-protease short L), as a direct fusion to the gamma chain ICD with a long linker (cis-protease long L), or in the original approach with HCV NS3 protease delivered on a separate transmembrane protein (as shown in Figure 7). These data indicate that the HCV NS3 protease-regulated chimeric receptor can be regulated by HCV protease expressed in cis as a direct fusion to one of the chains or in trans on a separate transmembrane protein, with high levels of pSTAT5 induced only upon exposure to the HCV NS3 protease inhibitor grazoprevir (GPV). Phosphoflow analysis was performed 48 hours after IL-2 was removed from the culture medium.

[0156] Example 6 - T cells co-expressing a CD22 CAR and a CCR show similar levels of CAR expression compared to T cells expressing only a CD22 CAR This example shows that T cells co-expressing a CD22 CAR and a CCR exhibit similar levels of CAR expression compared to T cells expressing only a CD22 CAR.

[0157] The data are provided in Figure 22. Flow plots showing CD22.BBz CAR surface expression levels from primary human T cells transduced with various CCRs are shown. The data indicate that all configurations result in similar levels of surface CAR expression. "Constit." and "Reg." refer to constitutive and regulatable CCRs, respectively.

[0158] Example 7 - CCR enhances CAR T cell proliferation and anti-tumor efficacy NSG mice were inoculated with Nalm6 leukemia and then treated with CD22.BBz CAR-T cells engineered with various IL2 CCR constructs (Figure 23). GPV was administered to the mice daily (++), every 48 hours (+), or not at all (-). Mock untransduced T cells, conventional CD22.BBzCAR-T cells, and CD22.BBzCAR-T cells engineered with a CCR lacking the ICD (ΔICD) served as controls. A: Quantification of tumor progression. B: Quantification of T cell expansion.

[0159] In further experiments, NSG mice were inoculated with Nalm6 leukemia and then treated with CD22.BBz CAR-T cells engineered with various IL9 CCR constructs (Figure 24). Mock untransduced T cells and conventional CD22.BBz CAR-T cells served as controls. A: Quantification of tumor progression. B: Quantification of T cell expansion.

[0160] The data show that CCR enhances the proliferation and anti-tumor efficacy of CAR T cells.

[0161] Example 8 - Data showing drug control of cell expansion of CD22.BBz CAR-T cells in vitro. In this example, drug control of CAR-T cell expansion is evaluated. CD22.BBz CAR-T cells were cultured in medium lacking IL2. As shown in Figure 25, CD22 CAR-T cells with IL2 CCR regulated by GPV exhibited enhanced proliferation in vitro, and a significant >40-fold expansion of Reg IL2 CCR CAR-T cells in the presence of GPV was observed compared to cells grown in the absence of GPV.

[0162] Example 9 - Regulatable CCR-expressing HER2 CAR-T cells express high levels of pSTAT5 when cultured in medium lacking exogenous cytokines This example demonstrates drug regulation of pSTAT5 levels in HER2.BBz CAR-T cells in vitro. HER2.BBz CAR-T cells were cultured in medium lacking IL2. As shown in Figure 26, HER2 CAR-T cells engineered with various constitutive and regulatable CCRs express high levels of pSTAT5 when cultured in medium lacking exogenous cytokines.

[0163] Example 11 - CAR T cells expressing membrane-associated IL2RB ICD in the absence of common gamma chain ICD exhibit enhanced anti-tumor efficacy compared to CAR T cells lacking membrane-associated IL2RB ICD In this example, the antitumor efficacy of CAR T cells expressing membrane-associated IL2RB ICD in the absence of a common gamma chain ICD was evaluated. NSG mice were inoculated with Nalm6 leukemia and then treated with CD22.BBz CAR-T cells engineered with only the IL2RB component of the IL2 CCR. Mock untransduced T cells and conventional CD22.BBz CAR-T cells served as controls. The data show quantification of tumor progression (Figure 27). The data unexpectedly demonstrate that CAR T cells expressing membrane-associated IL2RB ICD in the absence of a common gamma chain ICD exhibit enhanced antitumor efficacy compared to CAR T cells lacking membrane-associated IL2RB ICD.

[0164] Example 12 - In vitro expression and in vivo activity of CAR-T cells engineered with non-gamma chain CCRs In this example, the in vitro expression and in vivo activity of CAR-T cells engineered with a non-gamma chain CCR (IL18 CCR in this example as a proof-of-concept) were evaluated. Figure 28A shows a flow plot illustrating cell surface expression of a CCR with IL-18RA and IL-18RB ICD. As shown in Figure 28B, NSG mice were inoculated with Nalm6 leukemia and then treated with CD22.BBz CAR-T cells engineered with either a regulated or constitutive IL-18 CCR construct. For the regulated IL18 CCR group, mice were administered GPV daily (+GPV) or not at all (-GPV). Mock-untransduced T cells and CD22.BBz CAR-T cells engineered with a CCR lacking the ICD (ΔICD) served as controls. Data show quantification of tumor progression.

[0165] method Construction of retroviral plasmid vectors DNA sequences were synthesized as oligonucleotides or gBlocks (Integrated DNA Technologies). DNA sequences were cloned into the MSGV1 retroviral vector using In-Fusion cloning (Takara Bio). The products of the In-Fusion reaction were transformed into chemically competent cells (Stellar Cell, Takara Bio) using the heat shock method. The resulting colonies were sequence-confirmed using Sanger sequencing. Bacterial cultures were grown in LB-ampicillin medium for 16 hours. Plasmid DNA was then extracted from the bacterial cultures using a miniprep kit (QIAprep Spin Miniprep Kit, Qiagen).

[0166] Isolation of primary human T cells from blood donors Primary human T cells were extracted from buffy coats by negative selection using the RosetteSep Human T cell Enrichment Kit (Stem Cell Technologies) and SepMate-50 tubes. T cells were cryopreserved in cryopreservation medium (CryoStor CS10, Stem Cell Technologies) until use.

[0167] Retrovirus production Retroviral supernatant RD114 was prepared using 293GP packaging cells and the RD114 envelope plasmid. Briefly, 11 μg of RD114 and 22 μg of the corresponding MSGV1 transfer plasmid were mixed in Lipofectamine 2000 reagent (Thermo Fisher Scientific) and used to transfect 293GP cells, which were grown on poly-D-lysine cell culture dishes (Corning) to approximately 80% confluency. 293GP cells were cultured in 293GP culture medium (DMEM, 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 100 μg / mL streptomycin, 100 U / mL penicillin; Gibco) at 37°C in a 5% CO2 environment. The medium was refreshed every 24 hours. At 48 and 72 hours post-transfection, retroviral supernatants were collected, centrifuged to remove dead cells and debris, and then stored at -80C until further use.

[0168] T cell retroviral transduction On day 0, primary human T cells were thawed and activated using anti-CD3 / CD28 Human T-Expander Dynabeads (Thermo Fisher) at a bead-to-cell ratio of 3:1. Two days later, retroviral supernatant was spun onto 12- or 24-well cell culture plates precoated with RetroNectin (Takara Bio) according to the manufacturer's instructions. Approximately 0.1–1 mL of retroviral supernatant diluted with DMEM was added to the RetroNectin plate and centrifuged at 3200 RPM at 32°C for approximately 2 hours. The supernatant was then aspirated from the well. Subsequently, 0.25–0.5 × 10 6 T cells prepared at a concentration of 1000 cells / mL were added to 1 mL of T cell medium consisting of AIM V (Thermo Fisher), 5% fetal bovine serum (FBS), 2 mM L-glutamine (Gibco), 100 mg / mL streptomycin (Gibco), 100 U / mL penicillin (Gibco), 10 mM HEPES (Gibco), and 100 U / mL rhIL-2 (Peprotech). The plate was then slowly spun down at 1200 RPM for 2 minutes and then incubated overnight at 37°C, 5% CO2. This transduction process was repeated the following day. On day 4, the Dynabeads were removed by magnetic separation. Cells were transduced at a concentration of 0.4–2 × 10 6 Cells were cultured at a concentration of 1000 cells / mL and expanded for up to several weeks.

[0169] In vitro cell proliferation assay To determine whether CCR-transduced T cells could proliferate in the absence of exogenously added cytokines, cells were harvested from culture, centrifuged, and resuspended in T cell culture medium lacking T cell growth cytokines (i.e., IL-7, IL-2, IL-15, etc.). Cell number and viability were determined using a trypan blue exclusion test for viability and a Cellometer Auto1000 cell counter (Nexcelom Biosciences).

[0170] Antibodies and flow cytometry The following antibodies were used to stain cells: Brilliant Violet 421™ anti-DYKDDDDK Tag Antibody (Biolegend), anti-HA.11 Epitope Tag Antibody (BioLegend), Alexa Fluor® 647 Mouse Anti-Stat5 (pY694) (BD), and PE Mouse Anti-Stat3 (pY705) Clone 4 / P-STAT3 (BD). Recombinant CD22-Fc fluorescently labeled with the DyLight 650 Microscale Antibody Labeling Kit (Thermo Fisher Scientific) was used for CAR detection. Cells were stained according to the antibody manufacturer's instructions. Flow cytometry samples were run using a BD Fortessa instrument. Data were analyzed using FlowJo software (Tree Star). Intracellular phosphoflow cytometry was performed to assess the phosphorylation levels of STAT3 and STAT5 using the Life Technologies Fix&perm kit with methanol modification (Thermo Fisher Scientific).

[0171] Preparation and Administration of Grazoprevir For in vitro experiments, grazoprevir (Acme Bioscience, custom synthesis) was reconstituted as a 1000x stock solution (3 mM) using DMSO and frozen at -80°C. For in vivo mouse experiments, grazoprevir potassium salt (Acme Bioscience, custom synthesis) was reconstituted to 60 mg / mL using 100% PEG300 (Rigaku) ​​by incubation in a 37°C water bath and vortexing. Ritonavir (ACROS Organic), used as a pharmacokinetic enhancer, was similarly reconstituted to 60 mg / mL using a 1:1 solution of ethanol and propylene glycol (MP Biomedicals). Oral formulations were prepared at 50 mg / kg grazoprevir and 25 mg / kg ritonavir in gavage diluent (70% (v / v) PEG300, 5% (w / v) sucrose (Sigma), and 5% (v / v) DPBS (Gibco)) and delivered to mice by oral gavage using a 20-gauge 30 mm feeding tube (Instech) in a total volume of 100 μL per dose.

[0172] Nalm6 leukemia mouse model Six to ten week old male or female NSG mice were transduced with 1 × 10 6 Nalm6 leukemia cells were transplanted and tumor progression was monitored. Four days later, 4 x 10^6 CAR-T or control cells were transplanted via tail vein injection. T cells were transduced with Antares luciferase to allow in vivo monitoring. On day 17, mice were re-challenged with 4.6 x 10^6 Nalm6 tumor cells.

[0173] 143B osteosarcoma mouse model 1 x 10 PBS-prepared solution was administered to 6- to 10-week-old male or female NSG mice. 6143B cells were implanted into the tibia periosteum. Four days later, mice were treated with 3 x 106 human T cells expressing the indicated transgenes via tail vein injection. Tumor progression was monitored using caliper measurements. T cells were transduced with Antares luciferase to allow in vivo monitoring.

[0174] In vivo bioluminescence imaging Tumor progression was monitored using a firefly luciferase assay. Mice were administered 200 μL of 15 μg / mL D-luciferin to detect tumor cells expressing firefly luciferase. T cell expansion was monitored using an Antares luciferase assay. Mice were administered 200 μL of a 1:40 dilution of Nano-Glo substrate (Promega, diluted in DPBS) via intraperitoneal injection to detect T cells expressing Antares luciferase. Images were acquired 5 minutes after injection using an IVIS imaging system with a 30-second exposure and medium binning. If pixels were saturated within an image, additional images were acquired using the autoexposure setting. Living Image software was used to calculate the total flux around the region of interest around each mouse's body. Unsaturated images were used to quantify BLI images. BLI images within the same experiment were set to the same scale.

[0175] Accordingly, the foregoing description merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements, not explicitly described or shown herein, which embody the principles of the present invention and are within its spirit and scope. Furthermore, all examples and conditional language set forth herein are intended primarily to aid the reader in understanding the principles of the present invention and the concepts the inventors contributed to furthering the art, and should not be construed as being limited to such specifically described examples and conditions. Furthermore, all statements herein describing principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, such equivalents are intended to include both currently known equivalents and future-developed equivalents, regardless of structure, i.e., any elements developed to perform the same function, regardless of structure. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

1. one or more nucleic acids encoding a first subunit of a chimeric cytokine receptor and a second subunit of a chimeric cytokine receptor, the first subunit comprises a first heterodimerization domain and a first cytokine receptor intracellular signaling domain (ICD); The second subunit comprises one or more nucleic acids, a second heterodimerization domain homolog for the first heterodimerization domain, and a second cytokine receptor ICD.

2. 10. The one or more nucleic acids of claim 1, wherein the first and second heterodimerization domains are extracellular dimerization domains.

3. 3. The one or more nucleic acids of claim 1 or 2, wherein the first and second heterodimerization domains each comprise a leucine zipper domain, a BTB (BR-C, ttk, and bab) domain, a POZ (poxvirus and zinc finger) domain, a coiled-coil domain, or a PDZ domain.

4. 3. The one or more nucleic acids of claim 1 or 2, wherein the first and second heterodimerization domains comprise a constant heavy chain and a constant light chain of an IgG, IgE, or IgD antibody, respectively, or a dimerization fragment thereof.

5. the first subunit comprises a heterologous transmembrane domain that is the first heterologous dimerization domain; 10. The one or more nucleic acids of claim 1, wherein the second subunit comprises a heterologous transmembrane domain that is the second heterologous dimerization domain.

6. 6. The one or more nucleic acids of claim 5, wherein the heterologous transmembrane domains of the first and second subunits are independently selected from the group consisting of a CD8α transmembrane domain, a CD28 transmembrane domain, a HER2 transmembrane domain, and an EGFR transmembrane domain.

7. 5. The one or more nucleic acids of any one of claims 1-4, wherein the first and second subunits comprise transmembrane domains independently selected from HER2, EGFR, IL-2Rβ, IL-7R, IL-21R, IL-4R, IL-9R, IL15Rα, common gamma chain (γc), Eph receptor, VEGF receptor, ErbB receptor, FGF receptor, ROR1, ROR2 PDGF receptor, MET receptor, CD35, CD3ζ, CD3γ, CD3δ, CD4, CD5, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD-1 transmembrane domains.

8. The first cytokine receptor ICD, the second cytokine receptor ICD, or both are selected from the group consisting of IL-2Rβ ICD, IL-3Rα ICD, IL-4R ICD, IL-5Rα ICD, IL-6Rα ICD, IL-7R ICD, IL-9R ICD, IL-10Rα ICD, IL-10Rβ ICD, IL-11Rα ICD, IL-12Rβ1 ICD, IL-12Rβ2 ICD, IL-13Rα1 ICD, IL-13Rα2 ICD, IL-20Rα ICD, IL-20Rβ, IL-21R ICD, IL-22Rα1, IL-22Rα2, IL-23R ICD, IL-27Rα ICD, IL-28RA ICD, IL-31Rα ICD, TGFβR1 8. The one or more nucleic acids of any one of claims 1 to 7, comprising a CD9 ICD, a TGFβR2 ICD, a TGFβR3 ICD, an IFNAR1 ICD, an IFNAR2 ICD, an IFNGR1 ICD, an IFNGR2 ICD, an IFNLR ICD, a CSF2RB ICD, a gp130 ICD, a CD9 ICD, an OSMR ICD, a CSF-1R ICD, or any combination thereof.

9. 8. The one or more nucleic acids of any one of claims 1 to 7, wherein the first cytokine receptor ICD comprises an ICD from the common cytokine receptor gamma chain family.

10. 10. The one or more nucleic acids of claim 9, wherein the ICD from the common cytokine receptor gamma chain family is an IL-2Rβ ICD, an IL-4R ICD, an IL-7R ICD, an IL-9R ICD, or an IL-21R ICD.

11. 11. The one or more nucleic acids of claim 9 or 10, wherein the second cytokine receptor ICD is a common gamma chain (γc) ICD.

12. 12. The one or more nucleic acids of claim 11, wherein the second subunit comprises a transition domain disposed between the transmembrane domain and the γc ICD.

13. 13. The one or more nucleic acids of claim 12, wherein the transition domain comprises a juxtamembrane (JM) region of a cell surface receptor.

14. 14. The one or more nucleic acids of claim 13, wherein the transition domain comprises the JM region of CD8α or IL-15Rα.

15. 15. The one or more nucleic acids of any one of claims 1 to 14, wherein the first subunit and the second subunit are encoded by a single nucleic acid.

16. 16. The one or more nucleic acids of claim 15, wherein the single nucleic acid provides cistronic expression of the first and second subunits.

17. 17. The one or more nucleic acids of claim 16, wherein the single nucleic acid comprises a ribosome skipping element disposed between the region encoding the first subunit and the region encoding the second subunit.

18. 15. One or more nucleic acids according to any one of claims 1 to 14, wherein the first subunit is encoded by a first nucleic acid and the second subunit is encoded by a second nucleic acid.

19. 19. The one or more nucleic acids of any one of claims 1 to 18, wherein the first subunit, the second subunit, or both, comprise a protease cleavage site located between the transmembrane domain and the ICD.

20. 20. The one or more nucleic acids of claim 19, wherein the protease cleavage site is a viral protease cleavage site.

21. 21. The one or more nucleic acids of claim 20, wherein the viral protease cleavage site is a hepatitis C virus (HCV) nonstructural protein 3 (NS3) cleavage site.

22. 22. The one or more nucleic acids of claim 21, wherein the viral protease cleavage site is selected from the group consisting of an NS4A / 4B junction cleavage site, an NS3 / NS4A junction cleavage site, an NS4A / NS4B junction cleavage site, an NS4B / NS5A junction cleavage site, an NS5A / NS5B junction cleavage site, and variants thereof that are cleavable by the viral protease.

23. 21. The one or more nucleic acids of claim 20, wherein the viral protease cleavage site is an HIV protease cleavage site.

24. 21. The one or more nucleic acids of claim 20, wherein the viral protease cleavage site is a SARS-CoV-2 protease cleavage site.

25. 20. The one or more nucleic acids of claim 19, wherein the protease cleavage site is a human protease cleavage site.

26. 26. The one or more nucleic acids of claim 25, wherein the human protease cleavage site is a cleavage site for human renin protease, human kallikrein (KLK) protease, human enterokinase protease, human thrombin, human matrix metalloproteinase (MMP), human urokinase-type plasminogen activator receptor (uPAR), human plasmin, or human cathepsin.

27. 27. The one or more nucleic acids of any one of claims 19 to 26, wherein the one or more nucleic acids further encode a protease and the protease cleavage site is a cleavage site for the protease.

28. 28. The one or more nucleic acids of claim 27, wherein the one or more nucleic acids encode a fusion protein comprising the protease and a transmembrane domain.

29. 28. The one or more nucleic acids of claim 27, wherein the protease is a soluble cytosolic protease.

30. the one or more nucleic acids a protease fused to the first subunit; a protease fused to the second subunit; or 28. One or more nucleic acids according to claim 27, encoding the protease fused to the first subunit and the protease fused to the second subunit.

31. 31. The one or more nucleic acids according to any one of claims 27 to 30, wherein the proteolytic activity of the protease is regulatable.

32. 32. The one or more nucleic acids of claim 31, wherein the proteolytic activity of the protease is modulatable via a cell-permeable small molecule.

33. 33. The one or more nucleic acids of claim 31 or 32, wherein the cell-permeable small molecule is an inhibitor of the protease.

34. 34. The one or more nucleic acids of claim 33, wherein the protease is derived from HCV NS3 and the inhibitor of the protease is selected from the group consisting of asunaprevir (ASV), danoprevir (DPV), simeprevir (SPV), grazoprevir (GPV), glecaprevir, voxilaprevir, and any combination thereof.

35. 34. The one or more nucleic acids of claim 33, wherein the protease is an HIV protease and the inhibitor of the protease is selected from the group consisting of atazanavir (Reyataz), darunavir (Prezista), fosamprenavir (Lexiva), indinavir (Crixivan), lopinavir / ritonavir (Kaletra), nelfinavir (Viracept), ritonavir (Norvir), saquinavir (Invirase), tipranavir (Aptivus), atazanavir / cobicistat (Evotaz), darunavir / cobicistat (Prezcobix), and any combination thereof.

36. 34. The one or more nucleic acids of claim 33, wherein the protease is SARS-CoV-2 protease and the inhibitor of the protease is nilmatrervir (Paxlovid).

37. 34. The one or more nucleic acids of claim 33, wherein the protease is human renin protease and the inhibitor of the protease is aliskiren.

38. 38. The one or more nucleic acids of any one of claims 1 to 37, wherein the first subunit, the second subunit, or both, comprise an extracellular protein tag.

39. 39. The one or more nucleic acids of Claim 38, wherein the extracellular protein tag is an N-terminal protein tag.

40. 40. The one or more nucleic acids of claim 38 or 39, wherein the extracellular protein tag is a FLAG tag or an HA tag.

41. 41. The one or more nucleic acids of any one of claims 1 to 40, wherein the first subunit and the second subunit each comprise an extracellular cysteine ​​residue, and a disulfide bond between the extracellular cysteine ​​residue of the first subunit and the extracellular cysteine ​​residue of the second subunit stabilizes the association of the first subunit and the second subunit when expressed on the surface of a cell.

42. A chimeric cytokine receptor comprising the first and second subunits encoded by one or more nucleic acids of any one of claims 1 to 41.

43. 42. One or more expression constructs comprising one or more nucleic acids of any one of claims 1 to 41 operably linked to one or more promoters.

44. A nucleic acid encoding a membrane-associated polypeptide comprising a transmembrane domain and a cytokine receptor ICD, wherein said polypeptide does not comprise an extracellular cytokine binding domain.

45. 45. The nucleic acid of claim 44, wherein the cytokine receptor ICD is an IL-2Rβ ICD, an IL-7R ICD, an IL-9R ICD, or a common gamma chain (γc) ICD.

46. 46. ​​The nucleic acid of claim 45, wherein the cytokine receptor ICD is IL-2Rβ ICD.

47. 47. The nucleic acid of any one of claims 44 to 46, wherein the polypeptide comprises a heterodimerization domain.

48. 48. The nucleic acid of claim 47, wherein the heterodimerization domain is an extracellular heterodimerization domain.

49. 49. The nucleic acid of claim 47 or 48, wherein the heterodimerization domain is as defined in claim 3 or 4.

50. 50. The nucleic acid of any one of claims 44 to 49, wherein the polypeptide comprises a protease cleavage site located between the transmembrane domain and the ICD.

51. 51. The nucleic acid of claim 50, wherein the protease cleavage site is as defined in any one of claims 20 to 26.

52. A polypeptide encoded by the nucleic acid of any one of claims 44 to 51.

53. 52. An expression construct comprising the nucleic acid of any one of claims 44 to 51 operably linked to a promoter.

54. A cell comprising one or more nucleic acids according to any one of claims 1 to 41.

55. A cell comprising the nucleic acid of any one of claims 44 to 51.

56. 44. A cell comprising one or more expression constructs of claim 43.

57. 54. A cell comprising the expression construct of claim 53.

58. 58. The cell of claim 57, wherein the cell does not express a cytokine receptor subunit containing the common gamma chain (γc) ICD.

59. The cell of any one of claims 54 to 58, wherein the cell is a human cell.

60. The cell of any one of claims 54 to 59, wherein the cell is an immune cell.

61. 61. The system of claim 60, wherein the immune cells are T cells.

62. 62. The cell of any one of claims 54-61, wherein the one or more nucleic acids further encode a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), a synthetic Notch (SynNotch) receptor, a modular extracellular sensor architecture (MESA) receptor, a Tango receptor, a ChaCha receptor, a generalized extracellular molecular sensor (GEMS) receptor, a cytokine receptor, a chemokine receptor, a switch receptor, an adhesion molecule, an integrin, an inhibitory receptor, a stimulatory receptor, an immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor, or an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor.

63. The cell of any one of claims 54 to 61, wherein the one or more nucleic acids further encode a CAR.

64. 64. A composition comprising a population of cells as defined in any one of claims 56 to 63, wherein said composition is suitable for administration to a subject in need thereof.

65. 65. A method of administering a cell-based therapy to a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of claim 64, wherein the cells express the chimeric cytokine receptor comprising the first and second subunits encoded by the one or more nucleic acids, or the membrane-associated polypeptide encoded by the nucleic acids.

66. 66. The method of claim 65, wherein the signaling of the chimeric cytokine receptor or membrane-associated polypeptide is regulatable.

67. the cell expresses the chimeric cytokine receptor comprising the first and second subunits encoded by the one or more nucleic acids; the first subunit, the second subunit, or both, comprise a protease cleavage site located between the transmembrane domain and the ICD; the one or more nucleic acids further encode a protease, and the protease cleavage site is a cleavage site for the protease; 67. The method of claim 66, wherein the proteolytic activity of the protease is regulatable.

68. 67. The method of claim 66, wherein the cell expresses the membrane-associated polypeptide, and the membrane-associated polypeptide comprises a protease cleavage site located between the transmembrane domain and the cytokine receptor ICD.

69. 69. The method of claim 67 or 68, wherein the proteolytic activity of the protease is modulatable via a cell-permeable small molecule.

70. 70. The method of claim 69, wherein the cell-permeable small molecule is an inhibitor of the protease, and the method further comprises administering to the subject an effective amount of the cell-permeable small molecule when signaling by the chimeric cytokine receptor or membrane-associated polypeptide is desired.

71. 71. The method of claim 70, comprising ceasing administration of the cell-permeable small molecule when signaling by the chimeric cytokine receptor or membrane-associated polypeptide is no longer desired.

72. 72. The method of claim 70 or 71, wherein the protease is derived from HCV NS3.

73. 73. The method of claim 72, wherein the protease inhibitor is selected from the group consisting of asunaprevir (ASV), danoprevir (DPV), simeprevir (SPV), grazoprevir (GPV), glecaprevir, voxilaprevir, and any combination thereof.

74. 72. The method of claim 70 or 71, wherein the protease is an HIV protease and the inhibitor of the protease is selected from the group consisting of atazanavir (Reyataz), darunavir (Prezista), fosamprenavir (Lexiva), indinavir (Crixivan), lopinavir / ritonavir (Kaletra), nelfinavir (Viracept), ritonavir (Norvir), saquinavir (Invirase), tipranavir (Aptivus), atazanavir / cobicistat (Evotaz), darunavir / cobicistat (Prezcobix), and any combination thereof.

75. 72. The method of claim 70 or 71, wherein the protease is SARS-CoV-2 protease and the inhibitor of the protease is nilmatrervir (Paxlovid).

76. 72. The method of claim 70 or 71, wherein the protease is human renin protease and the inhibitor of the protease is aliskiren.

77. 77. The method of any one of claims 65-76, wherein the subject in need thereof has cancer and the cell-based therapy is administered to treat the cancer.

78. 78. The method of claim 77, wherein the cancer comprises a solid tumor.

79. 79. The method of claim 78, wherein the solid tumor is a carcinoma, lymphoma, blastoma, or sarcoma.

80. 78. The method of claim 77, wherein the cancer comprises a hematological malignancy.

81. 81. The method of claim 80, wherein the hematological malignancy is leukemia, lymphoma, or multiple myeloma.

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