Chimeric cytokine receptors and methods of use thereof

Abstract

A nucleic acid encoding a chimeric cytokine receptor (CCR) capable of signaling in the absence of its homologous cytokine is provided. In some embodiments, one or more nucleic acids encoding a first subunit of a chimeric cytokine receptor and a second subunit of a 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. The CCR is useful for a variety of uses, including, but not limited to, increasing the persistence of therapeutic cells. Accordingly, also provided are methods of administering a cell-based therapy comprising administering to a subject in need thereof a CCR-expressing therapeutic cell (e.g., a CAR-T cell, etc.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 401,437, filed on August 26, 2022, which is incorporated herein by reference in its entirety.

[0003] Incorporation by Reference into the Sequence Listing provided as a Sequence Listing XML file

[0004] The Sequence Listing is hereby provided as a Sequence Listing XML, "STAN-1985WO_SEQ_LIST", which was created on August 24, 2023 and has a size of 109,370 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in its entirety. Background Art

[0005] Cytokine is an intercellular signaling molecule that contributes to the intercellular communication in the immune response, and stimulates cells to move toward the site of inflammation, infection and trauma. The downstream effect of specific cytokines occurs by its high-affinity binding to the receptor expressed on the surface of target cells. This effect may occur in the form of autocrine (acting on the same cell), paracrine (acting on nearby cells) or endocrine (acting on distant cells). Receptor engagement triggers intracellular signaling cascades, resulting in gene expression changed in target cells, thereby resulting in biological effects. Cytokines can be divided into several categories, including interleukins (IL), transforming growth factors (TGF), interferons (IFN), colony stimulating factors (CSF), tumor necrosis factors (TNF) and chemokines.

[0006] Interleukins (IL) are a group of cytokines expressed and secreted by leukocytes (white blood cells) and some other body cells. Interleukins and associated cytokines serve as a means of communication for innate and adaptive immune cells as well as non-immune cells and tissues. All IL-1 family members share a conserved β-trefoil structure and bind to members of the IL-1 receptor (IL-1R) family. Members of the IL-1R family contain extracellular Ig-like domains and mediate signal transduction through intracellular Toll / IL-1R (TIR) ​​domains.

[0007] The four-helix bundle cytokine superfamily is subdivided into class I and class II cytokine receptor families. The ligands of the class I cytokine receptor family include short-chain helical cytokines and long-chain helical cytokines. The short-chain helical cytokine family includes members of the common γ chain and common β chain families of cytokines. The common β chain and common γ 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. The members of the common β chain family send signals through heterodimer receptor complexes containing common β chain subunits, while the members of the common γ chain family send signals through heterodimer or heterotrimer receptor complexes containing common γ chain subunits. The common γ chain (γc) family is composed of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, and is named because these factors are combined with γc receptors (CD132). They primarily act as growth and proliferation factors for progenitor and mature cells and also have roles in lineage-specific cell differentiation.

[0008] The ligands of the class II cytokine receptor family include IL-10 family cytokines, and type I, type II and type III interferons. The members of the IL-10 family cytokines have structural similarities and send signals through heterodimeric receptor complexes with common subunits. Members of the type I, type II and type III interferon families include IFN-α, IFN-β, IFN-ω, IFN-ε, IFN-κ, IFN-γ, IL-28A, IL-28B, IL-29 and IFN-λ4. These cytokines mainly have antiviral, antiproliferative and immunomodulatory effects.

[0009] IL-17 family cytokines belong to the cysteine-knot superfamily and bind to members of the IL-17 receptor family. They are primarily involved in promoting pro-inflammatory immune responses.

[0010] 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). The cytokines in the TNF superfamily are bound 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 co-stimulation, natural killer cell activation, and B cell homeostasis. In addition, they can regulate cell type-specific responses and can play an important role in regulating the pathogenesis of certain diseases, including chronic inflammation, cancer, and autoimmune diseases.

[0011] Four short-helix bundle cytokines that signal through class III receptor tyrosine kinases include M-CSF, SCF, Flt-3 ligand, and IL-34. The receptors for these cytokines contain extracellular Ig-like domains, similar to the IL-1R family, but they have cytoplasmic domains with tyrosine kinase activity. The long-helix cytokine family includes the IL-6 family cytokines, G-CSF, erythropoietin, thrombopoietin, growth hormone, prolactin, and leptin.

[0012] The four subfamilies of chemokines are the C, CX3C, CC, and CXC subfamilies and are based on the number and spacing of conserved cysteine ​​residues located in the amino terminus. Chemokines bind to seven transmembrane receptors that are conventionally G protein-coupled, are somewhat promiscuous, and have key roles in regulating cell migration during development and under homeostatic and inflammatory conditions. Summary of the invention

[0013] A nucleic acid encoding a chimeric cytokine receptor (CCR) is provided, and the chimeric cytokine receptor can perform signal transduction in the absence of its homologous cytokine. In some embodiments, one or more nucleic acids are provided, which encode the first subunit of a chimeric cytokine receptor and the 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 a second heterodimerization domain homologous to the first heterodimerization domain and a second cytokine receptor ICD. According to some embodiments, the signal transduction by CCR is adjustable, for example, using an adjustable protease, wherein in the absence of a protease inhibitor, ICD is cut from one or two subunits of CCR, thereby preventing the signal transduction by CCR. CCR is used for a variety of purposes, including but not limited to increasing the persistence of therapeutic cells. Therefore, a method for administering a cell-based therapy is also provided, the method comprising administering a therapeutic cell (e.g., CAR-T cell, etc.) expressing CCR to a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1: A: Receptors for the IL-2 family, which consists of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. The receptors contain a common cytokine receptor γ chain (CD132, γc). IL-13R shares IL-4Rα with IL-4, and TSLPR shares IL-7R with IL-7. B: The receptors for IL-3, IL-5 and GM-CSF are heterodimers of unique α chain and common β chain (βc, CD131) subunits. C: The receptors for IL-4 and IL-13 are composed of 2 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. IL-13R consists of 2 subunits, IL-13Rα1 and IL-13Rα2, and signals through the type II IL-4R complex, which consists of IL-4Rα and IL-13Rα. D: Based on the similarity of their intron-exon structure, conserved secondary protein structure, and similar types of receptors, the following cytokines are classified as IL-10 family members: IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28, and IL-29. As shown, they share common receptor subunits. 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 2 subunits: IL-12Rβ1 and IL-12Rβ3. Heterodimers of IL-12Rβ1 and IL-23R bind IL-23. IL-12Rβ2 shows homology to the gp130 subunit of IL-27R. G: IFN-α and IFN-β bind to heterodimeric receptors composed of IFNAR1 and IFNAR2; in addition, IFN-β binds to IFNAR1, and IFN-γ binds to heterodimers of IFN-γR1 and IFN-γR2. Adapted from Akdis et al. (2016) J Allergy Clin Immunol. 138(4):984-1010.

[0015] Figure 2 : Schematic representation of a leucine zipper-based chimeric cytokine receptor (CCR).

[0016] Figure 3 : Flowchart showing the cell surface expression of the CCR components of various common gamma chain receptors.

[0017] Figure 4: Schematic representation of CCR engineering (the transition domains depicted in γC1 / γC3 and γC2 / γC4 are SEQ ID NOs: 54 and 55, respectively) and a flow chart demonstrating improved cell surface expression of CCR.

[0018] Figure 5 :shows the use Figure 4 Figure 2. Proliferation of primary human T cells transduced with various CCRs shown in Figure 2.

[0019] Figure 6 : Data showing that constitutive IL-2 CCR is a strong driver of proliferation in vivo and causes lethal toxicity in NSG mice.

[0020] Figure 7 : Schematic representation of a chimeric cytokine receptor (CCR) system regulated by a drug. A protease cleavage site is integrated between the transmembrane domain and the intracellular signaling domain of the CCR. Due to the proteolytic activity of the protease at the cleavage site, the co-expression of a protease specific to the cleavage site results in the cleavage of the CCR, thereby causing the inactivation of the CCR (receptor closure). Adding a protease inhibitor (drug) inhibits this cleavage event and places the CCR in an open state (receptor opening). Therefore, the signal transduction of the CCR becomes dependent on the presence of the drug. The regulated CCR can circumvent the toxicity associated with the constitutive CCR by regulating the proliferation of the cell to fall within the therapeutic window.

[0021] Figure 8 :Use as Figure 7 A series of flow diagrams of primary human T cells transduced with regulated forms of the IL-2R CCR described in , which demonstrates the cell surface expression of the HA / FLAG tag and phosphorylated STAT5 (pSTAT5), a cytokine signaling molecule downstream of IL-2R signaling (the cleavage site depicted is SEQ ID NO:56).

[0022] Fig. 9 : Amino acid sequence and data showing that insertion of a linker containing a cleavage site (SEQ ID NO:57) upstream of the γ chain ICD allows drug-regulated CCR signaling.

[0023] Fig.10 : Flowchart of primary human T cells transduced with a regulated form of IL-2R CCR displaying phosphorylated STAT5 (pSTAT5).

[0024] Fig.11 : Data on pharmacological control of in vitro cell expansion are presented.

[0025] Fig.12: Schematic representation of CAR and CCR constructs and data showing that drug-modulated IL-2CCR enhances in vivo anti-tumor activity after re-challenge.

[0026] Fig.13 : Schematic representation of CAR and CCR constructs and data showing that drug-regulated IL-2CCR expands CAR-T cells in vivo without toxicity.

[0027] Fig.14 : Schematic representation of CAR and CCR constructs and data showing that CAR-T cells expressing drug-regulated IL-2 CCR from two separate vectors have enhanced expansion in vivo in the presence of grazoprevir.

[0028] Fig.15 : Flowchart showing the phosphorylation levels of STAT5 from primary human T cells transduced with IL-2CCR with (leucine zipper IL-2CCR) or without (CD8a H / Tm IL-2CCR) leucine zipper component.

[0029] Fig.16 : Flowchart showing the IL-21R CCR of phosphorylation of STAT3 (pSTAT3), a cytokine signaling molecule downstream of IL-21R signaling.

[0030] Fig.17 : Flowchart of constitutive or protease-regulated IL-2R, IL-7R, and IL-9R CCRs expressed in primary human T cells.

[0031] Fig.18 : Data showing that constitutive IL-2CCR, IL-9CCR, and IL-7CCR induce lethal toxicity in mice due to unregulated growth of CAR-T cells.

[0032] Fig.19 : Data showing controlled growth of CAR-T cells expressing protease-regulated CCRs.

[0033] Fig. 20: Schematic illustration of a method for reducing the "leakage" activity of a protease-regulated CCR by double-chain cleavage. A: Schematic diagram of a chimeric cytokine receptor (CCR) system regulated by an original drug. The protease cleavage site is integrated between the transmembrane domain and the intracellular signaling domain of one of the chains of the CCR. Due to the proteolytic activity of the protease at the cleavage site, the co-expression of a protease specific to the cleavage site leads to the cleavage of the CCR, thereby leading to the inactivation of the CCR (receptor closure). The addition of a protease inhibitor (drug) inhibits this cleavage event and puts the CCR in an open state (receptor opening). Therefore, the signaling of the CCR becomes dependent on the presence of the drug. The regulated CCR can circumvent the toxicity associated with the constitutive CCR by regulating the proliferation of the cell to fall within the therapeutic window. B: Schematic diagram of a chimeric cytokine receptor (CCR) system regulated by a double-cleavage drug, in which both chains of the CCR are cleaved. The protease cleavage site is integrated between the transmembrane domain and the intracellular signaling domain of the two chains / subunits of the CCR. Due to the proteolytic activity of protease at the cleavage site, the co-expression of the protease specific to the cleavage site causes the cleavage of CCR, thereby causing the inactivation of CCR (receptor closure). Adding protease inhibitors (drugs) suppresses this cleavage event, and makes CCR in an open state (receptor opening). Therefore, the signal transduction of CCR becomes dependent on the presence of drugs. Regulated CCR is expected to avoid the toxicity associated with constitutive CCR by regulating the proliferation of cells to fall into the treatment window.

[0034] Fig.21 : A: Schematic representation of an adjustable CCR with a protease fused to one of the CCR subunits. B: Flowchart showing phosphorylation levels of STAT5 from primary human T cells transduced with IL-2CCR, where the HCV NS3 protease was directly fused to the γ chain ICD ("cis"). Either a short peptide linker or a long peptide linker was placed between the γ chain ICD and the protease. A "trans" protease is one such as Figure 7 The original protease configuration shown in . T cells were cultured in medium lacking IL2 for 24 hours before analyzing STAT5 phosphorylation levels.

[0035] Fig. 22 : Flowchart showing the surface expression levels of CD22.BBz CAR 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 CCR and regulatable CCR, respectively.

[0036] Fig.23: NSG mice were inoculated with Nalm6 leukemia and then treated with CD22.BBz CAR-T cells engineered with various IL2CCR constructs. GPV was administered to mice daily (++), every 48 hours (+), or not at all (-). Mock untransduced T cells, conventional CD22.BBz CAR-T cells, and CD22.BBzCAR-T cells engineered with CCRs lacking ICD (ΔICD) were used as controls. A: Quantification of tumor progression, B: Quantification of T cell expansion.

[0037] Fig.24 : NSG mice were inoculated with Nalm6 leukemia and then treated with CD22.BBz CAR-T cells engineered with various IL-9CCR constructs. Mock untransduced T cells and conventional CD22.BBz CAR-T cells were used as controls. A: Quantification of tumor progression, B: Quantification of T cell expansion.

[0038] Fig.25 : Data showing in vitro drug control of cell expansion of CD22.BBz CAR-T cells. T cells were cultured in medium lacking IL-2.

[0039] Fig.26 : Data showing in vitro drug control of pSTAT5 levels in HER2.BBz CAR-T cells. T cells were cultured in medium lacking IL-2.

[0040] Fig. 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 were used as controls. Data show quantification of tumor progression.

[0041] Fig.28 :A:Shows a flow chart of cell surface expression of CCR with IL-18RA and IL-18RB ICD, B:Inoculated NSG mice with Nalm6 leukemia, and then treated with CD22.BBz CAR-T cells engineered with regulated IL-18CCR constructs or constitutive IL-18CCR constructs. For the regulated IL-18CCR group, GPV was administered to mice every day (+GPV), or not at all (-GPV). Mock untransduced T cells and CD22.BBz CAR-T cells engineered with CCRs lacking ICD (ΔICD) were used as controls. The data show quantification of tumor progression. DETAILED DESCRIPTION

[0042] Before describing the nucleic acids, chimeric cytokine receptors and methods of the present disclosure in more detail, it should be understood that the nucleic acids, chimeric cytokine receptors and methods are not limited to the specific embodiments described and therefore can certainly vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting, as the scope of the nucleic acids, chimeric cytokine receptors will only be limited by the appended claims.

[0043] In the case of providing a range of values, it is understood that unless the context clearly specifies otherwise, each intermediate value between the upper and lower limits of the range and any other stated value or intermediate value in the stated range, up to one tenth of the lower limit unit, is included in nucleic acids, chimeric cytokine receptors and methods. The upper and lower limits of these smaller ranges can be independently included in the smaller range, and are also included in nucleic acids, chimeric cytokine receptors and methods, subject to any specific exclusion limits in the stated range. In the case where the stated range includes one or two limits, the range that does not include any one or both of these included limits is also included in nucleic acids, chimeric cytokine receptors and methods.

[0044] Certain ranges are given herein where the value is preceded by the term "about". The term "about" is used herein to provide literal support for the exact number that follows it, as well as for numbers that are close to or approximately following the term. In determining whether a number is close to or approximately a specifically recited number, a close or approximately unrecited number may be a number that, in the context in which it appears, provides a substantial equivalent to the specifically recited number.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the nucleic acids, chimeric cytokine receptors and methods belong. Although any nucleic acids, chimeric cytokine receptors and methods that are similar or equivalent to those described herein may also be used to practice or test the nucleic acids, chimeric cytokine receptors and methods, representative illustrative nucleic acids, chimeric cytokine receptors and methods are now described.

[0046] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with the cited publications. 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 present invention are not entitled to antedate such publications, as the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0047] It should be noted that as used herein and in 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 elements. Thus, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0048] It should be understood that, for the sake of clarity, certain features of nucleic acids, chimeric cytokine receptors, and methods described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for the sake of brevity, various features of nucleic acids, chimeric cytokine receptors, and methods described in the context of a single embodiment may also be provided individually or in any suitable subcombination. All combinations of embodiments are specifically encompassed by the present disclosure and disclosed herein as if each and every combination were individually and explicitly disclosed, to the extent that such combinations encompass operable processes and / or compositions. In addition, all subcombinations listed in the embodiments describing such variables are also explicitly encompassed by nucleic acids, chimeric cytokine receptors, and methods of the present invention and disclosed herein as if each and every such subcombination were individually and explicitly disclosed herein.

[0049] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has discrete components and features that may be readily separated or combined with the features of any other several embodiments without departing from the scope or spirit of the methods of the present invention. Any recited method may be performed in the order of events recited or in any other order that is logically possible.

[0050] Chimeric cytokine receptors and nucleic acids

[0051] Aspects of the present disclosure include chimeric cytokine receptors (CCR) and nucleic acids encoding them. The CCR platform of the present disclosure allows constitutive signaling or adjustable signaling of cytokine receptors in the absence of their homologous cytokines. CCR is used for various purposes. For example, cells expressing CCR of the present disclosure are used for cell therapy. Cell therapy such as CAR-T cell therapy has shown significant clinical benefits in malignant hematological diseases. However, T cell dysfunction and poor persistence limit clinical response, especially in solid tumors. As shown herein, when expressed on cells, CCR of the present disclosure is a strong driving force for cell proliferation in vitro and in vivo, and compared with control cells lacking CCR, enhanced in vivo anti-tumor activity is provided. Therefore, CCR and cells expressing it are used for cell therapy, and the non-limiting examples of the cell therapy 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, etc. Details about CCR and nucleic acid of the present disclosure will now be described.

[0052] In certain embodiments, one or more nucleic acids are provided, which encode the first subunit of a chimeric cytokine receptor and the second subunit of a chimeric cytokine receptor. As used herein, the term "chimeric" refers to a molecule composed of parts from different sources, such as a cytokine receptor. As a whole, a chimeric molecule is non-naturally occurring, such as synthetic or recombinant, although the part comprising the chimeric molecule can be naturally occurring. "Chimeric cytokine receptor" or "CCR" means a cytokine receptor comprising one or more subunits, wherein the subunit comprises one or more heterologous domains, wherein the receptor is capable of constitutive signal transduction or adjustable signal transduction in the absence of its homologous cytokine. "Heterologous" as used in the context of a nucleic acid or polypeptide domain generally means that the domain is from different sources (e.g., molecules of different sequences, different species sources, etc.) associated or connected with a nucleic acid or polypeptide, so that the nucleic acid or polypeptide is a nucleic acid or polypeptide not found in nature.

[0053] The CCR can be a chimeric form of any cytokine receptor of interest (e.g., any human cytokine receptor of interest), wherein the CCR is capable of constitutive signaling or regulatable signaling in the absence of its cognate cytokine. In certain embodiments, the CCR is a chimeric form of a member of the class I cytokine receptor family, a chimeric form of a member of the class II cytokine receptor family, a chimeric form of a member of the TNF receptor family, a chimeric form of a member of the IL-1 receptor family, a chimeric form of a member of the tyrosine kinase receptor family, or a chimeric form of a member of the chemokine receptor family.

[0054] According to some embodiments, the first and second subunits of the CCR are chimeric forms 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.

[0055] In certain embodiments, the first and second subunits of the CCR are independently selected from Figure 1 A chimeric form of the first subunit and the second subunit of any cytokine receptor subunit shown in .

[0056] According to some embodiments, the first and second subunits of CCR are chimeric forms of the first and second subunits of a receptor (e.g., a human receptor) 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 Figure 1 Shown in.

[0057] 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 can be readily obtained from databases such as UniProt, NCBI Protein, GenBank, and the like. 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).

[0058] In certain embodiments, the CCR of the present disclosure retains binding activity to its homologous cytokine. For example, the CCR may be included in the extracellular cytokine binding portion of the cytokine receptor found in nature, or a variant thereof that retains binding activity to the homologous cytokine. According to some embodiments, the CCR of the present disclosure is engineered so that the extracellular cytokine binding portion of the cytokine receptor found in nature is missing or modified in such a manner that the CCR no longer retains binding activity to its homologous cytokine.

[0059] According to some embodiments, the CCR of the present disclosure comprises a first subunit and a second subunit, the first subunit comprises a first heterodimerization domain and a first cytokine receptor intracellular signaling domain (ICD), and the second subunit comprises a second heterodimerization domain homologous to the first heterodimerization domain and a second cytokine receptor ICD. "Homologous" in this context means that when the first subunit and the second subunit are co-expressed on the surface of a cell, the second dimerization domain is able to dimerize with the first dimerization domain, which in turn leads to the formation of a dimer comprising the first subunit and the second subunit. After the first subunit and the second subunit are co-expressed on the surface of the cell, the first heterodimerization domain and the second heterodimerization domain can be an extracellular dimerization domain, a transmembrane dimerization domain, or an intracellular dimerization domain.

[0060] Any suitable heterodimerization domain can be used in the first and second subunits of the CCR. In certain embodiments, the first heterodimerization domain and the second heterodimerization domain 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 heterodimerization domain and the second heterodimerization domain respectively comprise the constant heavy chain and constant light chain of an IgG, IgE or IgD antibody or its dimerization fragment.

[0061] According to some embodiments, after the first subunit and the second subunit are co-expressed on the surface of the cell, the first heterodimerization domain and the second heterodimerization domain are transmembrane dimerization domains. For example, the first subunit of the CCR may include a heterologous transmembrane domain, which is the first heterodimerization domain, and the second subunit of the CCR may include a heterologous transmembrane domain, which 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.

[0062] In certain embodiments, the first subunit and the second subunit comprise a transmembrane domain independently selected from the group consisting of 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 domain. The amino acid sequences of such receptors (eg, human receptors) and their transmembrane domains, as well as suitable nucleotide sequences for encoding and expressing such transmembrane domains, are known and can be readily obtained from databases such as UniProt, NCBI Protein, GenBank, and the like.

[0063] As outlined above, the CCR of the present disclosure comprises a first subunit and a second subunit, which comprise a first cytokine receptor intracellular signaling domain and a second cytokine receptor intracellular signaling domain (ICD), respectively. According to some embodiments, the first cytokine receptor ICD, the second cytokine receptor ICD, or both comprise IL-2RβICD, IL-3RαICD, IL-4R ICD, IL-5RαICD, IL-6RαICD, IL-7R ICD, IL-9R ICD, IL-10RαICD, IL-10RβICD, IL-11RαICD, IL-12Rβ1ICD, IL-12Rβ2ICD, IL-13Rα1ICD, IL-13Rα2ICD, IL-20RαICD, IL-20Rβ, IL-21R ICD, IL-22Rα1, IL-22Rα2, IL-23R ICD, IL-27RαICD, IL-28R ICD, IL-31RαICD, TGFβR1 ICD, TGFβR2 ICD, TGFβR3 ICD, IFNAR1 ICD, ICD, IFNAR2 ICD, IFNGR1 ICD, IFNGR2 ICD, IFNLR ICD, CSF2RB ICD, gp130 ICD, CD9 ICD, OSMR ICD, CSF-1R ICD, or any combination thereof.

[0064] In certain embodiments, the first cytokine receptor ICD is an IL-22Rα1ICD, and the second cytokine receptor ICD is an IL-10RβICD. Such CCRs are used, for example, when it is desired that cells exhibit constitutive IL-22 signaling 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-12Rβ1ICD. For example, when it is desired that cells exhibit constitutive IL-23 signaling or regulatable IL-23 signaling in the absence of IL-23, such CCRs are used.

[0065] According to some embodiments, the first cytokine receptor ICD comprises an ICD from a common cytokine receptor γ chain family. γc is used 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 γ chain family is IL-2RβICD, IL-4R ICD, IL-7R ICD, IL-9R ICD or IL-21R ICD. When the first cytokine receptor ICD comprises an ICD from a common cytokine receptor γ chain family, in some embodiments, the second cytokine receptor ICD is a common γ chain (γc) ICD.

[0066] In certain embodiments, the first subunit, the second subunit or both comprise a joint sequence between the domains of the subunit. Such subunits may include one, two, three, four or five or more joints. In a specific embodiment, the length of the joint is about 1 to about 25 amino acids, about 5 to about 20 amino acids or about 10 to about 20 amino acids or the amino acid of any intermediate length. In some embodiments, the length of the joint 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. The limiting examples of the joints that can be incorporated between the domains of the first subunit and / or the second subunit include serine-glycine joints, disulfide joints (e.g., to enhance the association between the first subunit and the second subunit), etc., and include any joint sequence provided in the following table 1.

[0067] According to some embodiments, the first subunit, the second subunit or both include a transition domain arranged between a transmembrane domain and ICD. As will be understood by the benefit of the present disclosure, compared with the subunit without a transition domain, such a transition domain can be incorporated into the first subunit and / or the second subunit to enhance the expression of CCR, as shown in the following experimental section. For example, in some embodiments, the subunit comprising γc ICD can be engineered so that the transition domain is arranged between the transmembrane domain and γc ICD. Suitable transition domains include but are not limited to transition domains comprising near membrane (JM) regions of cell surface receptors. In some embodiments, the JM region of CD8α or IL-15Rα is used as a transition domain between a transmembrane domain and ICD, such as γc ICD. A non-limiting example of a CD8α transition domain (sometimes referred to as a transition peptide in this article) is a domain comprising an amino acid sequence NHRNRRRVCKCPRPVV (SEQ ID NO: 54). A non-limiting example of an IL-15Rα transition domain is a domain comprising the amino acid sequence KSRQTPP (SEQ ID NO: 55). Variants of such domains are also provided by the present disclosure.

[0068] 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 can be configured to allow the polycistronic expression of the first subunit and the second subunit. That is, two or more (e.g., each) proteins encoded by a single nucleic acid can be used as independent protein expression from the same promoter. In certain embodiments, a single nucleic acid includes a ribosomal skipping site to allow the polycistronic expression of two or more (e.g., each) protein coding regions. The non-limiting example of a suitable ribosomal skipping site that can be incorporated into such a single nucleic acid is the P2A ribosomal 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.

[0069] According to some embodiments, the first subunit, the second subunit or both comprise a protease cleavage site disposed between the transmembrane domain and the ICD. The term "cleavage site" refers to a key (e.g., a scissile bond) cut by an agent (e.g., a protease). The cleavage site of a protease is included in a specific amino acid sequence recognized by a protease during proteolytic cleavage, and may include surrounding amino acids (e.g., one to six amino acids) on either side of the scissile bond, which are required to bind to the active site of the protease and to identify as a substrate. In some embodiments, the cleavage site is provided as a cleavable joint, wherein a "cleavable joint" refers to a joint comprising a protease cleavage site. A cleavable joint is generally cleavable under physiological conditions.

[0070] 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 potyviral family proteases. Potyviral 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 mottle 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:

[0071] GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKS MSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPEEPFQPVKEATQLMN(SEQ ID NO:59)

[0072] 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 sequence, and / or functional (proteolytic) fragments thereof, such as fragments having a length of 100 to 125, 125 to 150, 150 to 175, 175 to 200, 200 to 225, or 225 to 235 amino acids.

[0073] According to some embodiments, the viral protease cleavage site is for HCV protease. In certain embodiments, the viral protease cleavage site is for the viral protease derived from HCV nonstructural protein 3 (NS3). NS3 is composed of N-terminal serine protease domain and C-terminal helicase domain. "Derived from HCV NS3" means that the protease is the proteolytic activity variant of the cleavage site of the serine protease domain of HCV NS3 or its serine protease domain that can cut HCV NS3. The protease domain of NS3 forms a heterodimer with the HCV nonstructural protein 4A (NS4A) that activates proteolytic activity. The protease derived from HCV NS3 can include the whole NS3 protein or its proteolytic activity fragment, and can further include a cofactor polypeptide, such as a cofactor polypeptide derived from HCV nonstructural protein 4A (NS4A), for example, an activated NS4A district. NS3 protease is highly selective and can be inhibited by many non-toxic cell-permeable drugs 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.

[0074] Exemplary proteases derived from HCV NS3

[0075] APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTD(SEQ ID NO:60)

[0076] APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIMSTATQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDGRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTD(SEQ ID NO:61)

[0077] APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTD(SEQ ID NO:62)

[0078] In some embodiments, the protease comprises one of the sequences shown above, 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 with one of such sequences, and / or functional (proteolytic) fragments thereof, such as fragments having a length of 100 to 185, 120 to 185, 140 to 185, 160 to 185, 170 to 185, 180 to 185, 182 to 185, or 184 to 185 amino acids.

[0079] NS3 protease cleavage sites can include four junctions between nonstructural (NS) proteins of HCV polyproteins that are usually cleaved by NS3 protease during HCV infection, including NS3 / NS4A, NS4A / NS4B, NS4B / NS5A and NS5A / NS5B junction cleavage sites. For a description of representative sequences of NS3 protease and its 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.

[0080] In some embodiments, protease is derived from HCV NS3 and is engineered to include one or more amino acid replacements relative to the HCV NS3 protease amino acid sequence shown above. For example, protease can include a replacement at the position corresponding to position 54 of the following amino acid sequence: APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTD (SEQ ID NO:60). In some embodiments, this type of replacement is a replacement of threonine to alanine.

[0081] NS3 nucleic acid and protein sequences can be derived from HCV, including any isolate of HCV having any genotype (e.g., genotype 1-7) or subtype. Many NS3 nucleic acid and protein sequences are known and described in, for example, USSN 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, e.g., NCBI entries: Accession Nos. 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, all of which 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 of these sequences or proteolytic fragments thereof can be employed.

[0082] 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. Many 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, NCBI entries: Accession Nos. NP_751925, YP_001491554, GU945462, HQ822054, FJ932208, FJ932207, FJ932205, and FJ932199; all of these sequences (as entered prior 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 of these sequences or proteolytic fragments thereof may be employed.

[0083] HCV polyprotein nucleic acid and protein sequences can be derived from HCV, including any isolate of HCV with any genotype (e.g., genotype 1-7) or hypotype. 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, e.g., NCBI entry: 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 (as entered before 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 of these sequences or proteolytic fragments thereof may be employed.

[0084] 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.

[0085] In certain embodiments, the protease cleavage site is a human protease cleavage site. Non-limiting examples of human protease cleavage sites include human kallikrein (KLK) protease, human enterokinase protease, human thrombin, human matrix metalloproteinase (MMP), human urokinase-type plasminogen activator receptor (uPAR), human plasmin (human plasmin) or human cathepsin cleavage sites. According to some embodiments, the protease cleavage site is a cleavage site of human kallikrein (KLK) protease, 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 of these sequences or proteolytic fragments thereof can be employed.

[0086] In certain embodiments, the protease cleavage site is a protease cleavage site for a human protease selected from the group consisting of acrosin protease (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), azurin-like metalloendopeptidase (ASTL), ATP23 metallopeptidase and ATP synthase assembly factor homolog (ATP23), ataxin 3 (ATXN3), ataxin-like protein 3 (ATXN3L), azurocidin 1 (AZ U1), β-secretase 1 (BACE1), β-secretase 2 (BACE2), bone morphogenetic protein 1 (BMP1), BRCA1 / BRCA2 containing complex subunit 3 (BRCC3), calpain 14 (CAPN14), calpain 3 (CAPN3), cysteine ​​protease recruitment domain family member 8 (CARD8), cysteine ​​protease 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), 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 multicystin 3 (CHMP3), CLN5 intracellular transporter (CLN5), chymotrypsin-like 1 (CMA1), collagen subfamily member 11 (COLEC11), COP9 signaling body subunit 5 (COPS5), corin serine peptidase (CORIN), carboxypeptidase A4 (CPA4), carboxypeptidase yolk-like (CPVL), cystatin SN (CST1), cystatin 1 1 (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-induced 1 homolog 2 (DDI2), DAP3-binding cell death enhancer 1 (DELE1), complement factor (DF), dickkopfWNT 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 inner membrane peptidase subunit 2 (IMMP2L), jumonji domain-containing protein 7 (JMJD7), Josephin domain-containing protein 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 (KLK8), kallikrein-related peptidase 9 (KLK9), kallikrein pseudogene 1 (KLKP1), lipocalin 2 (LCN2), legumin (LGMN), leishmaniasis leishmanolysin-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 aspartate peptidase (NAPSA), neurolytic protein (NLN), NLR family CARD domain-containing protein 4 (NLRC4), NLR family pyrin domain-containing protein (domain protein 1 (NLRP1), puromycin-sensitive aminopeptidase (NPEPPS), opiorphin propeptide (OPRPN), OTU deubiquitinase, ubiquitin aldehyde-binding protein 2 (OTUB2), poly (ADP-ribose) polymerase family member 9 (PARP9), proprotein convertase subtilisin / kexin type 1 (PCSK1), proprotein convertase subtilisin / kexin type 1 inhibitor (PCSK1N), proprotein convertase subtilisin / kexin type 2 (PCSK2), proprotein convertase subtilisin / kexin type 4 (PCSK4), proprotein proprotein convertase subtilisin / kexin type 5 (PCSK5), proprotein convertase subtilisin / kexin type 6 (PCSK6), 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-induced kinase 1 (PINK1), prolyl endopeptidase-like (PREPL), parkin RBRE3 ubiquitin protein ligase (PRKN), serine protease gene family (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), protease 3 (PRTN3), presenilin 2 (PSEN2), protein containing PYD and CARD domains (PYCARD), retinoic acid receptor response protein 1 (RAR RES1), RING finger and FYVE-like domain-containing E3 ubiquitin protein ligase (RFFL), Rhomboid-like protein 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), synaptosome-associated protein 25 (SNAP25), secreted phosphoprotein 2 (SPP2), small proline-rich protein 3 (SPRR3), spleen-associated tyrosine kinase (SYK), transcription factor EB (T FEB), transglutaminase 2 (TGM2), Toll-like receptor adaptor molecule 1 (TICAM1), tubulointerstitial nephritis antigen-like 1 (TINAGL1), transmembrane serine protease 11D (TMPRSS11D), 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 α / β1 (TPSAB1), tryptase β2 (TPSB2), tryptase δ 1 (TPSD1), tryptase gamma 1 (TPSG1), tryptase pseudogene 2 (TPSP2), tyrosyl-protein sulfotransferase 1 (TPST1), tyrosyl-protein sulfotransferase 2 (TPST2), tyrosyl-protein sulfotransferase 2 pseudogene 1 (TPST2P1), thyrotropin-releasing hormone-degrading enzyme (TRHDE), thyroid hormone receptor interacting factor 4 (TRIP4), ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), ubiquitin-specific peptidase 27X-linked (USP27X), tubulin carboxypeptidase (vasohibin) 2 (VASH2), valosin-containing protein (VCP), and WAP tetradisulfide core domain 1 (WFDC1).

[0087] In some embodiments, the protease is highly selective for the cleavage site. In addition, the protease activity may be capable of being inhibited by known small molecule inhibitors that are cell permeable and non-toxic to the cells or individuals under study or treatment. For a discussion of proteases, see, e.g., VYH Hook, Proteolytic and cellular mechanisms in prohormone and proprotein processing, RG Landes Company, Austin, Tex., USA (1998); NM Hooper et al., Biochem. J. 321:265-279 (1997); Z. Werb, Cell 91:439-442 (1997); TG Wolfsberg et al., J. Cell Biol. 131:275-278 (1995); T. Berg et al., J. Biochem. 307:313-326 (1995); MJ Smyth and JA Trapani, Immunology Today. Today, 16:202-206 (1995); RV Talanian et al., J. Biol. Chem. 272:9677-9682 (1997); and NAThornberry et al., J. Biol. Chem. 272:17907-17911 (1997), the disclosures of which are incorporated herein by reference in their entirety. In some embodiments, the protease employed is a sequence-specific non-human protease for which FDA-approved drug inhibitors can be used.

[0088] Therefore, in some embodiments, one or more nucleic acids further encode protease, wherein the protease cleavage site is a cleavage site for protease. In a configuration (sometimes referred to as "trans" configuration in this article, wherein protease is expressed as a single polypeptide relative to the first subunit and the second subunit), one or more nucleic acid encodings comprise a fusion rotein of protease and a transmembrane domain. In other cases, protease can be expressed as a part of a fusion rotein with the first subunit, the second subunit or both, sometimes referred to as "cis" configuration in this article.

[0089] The proteolytic activity of the protease can be adjustable. In some embodiments, the proteolytic activity of the protease can be regulated via a cell permeable small molecule. For example, such a cell permeable small molecule can be an inhibitor of the protease. Such inhibitors are known and available. When the protease is derived from HCV NS3, in some cases, the inhibitor of the protease is selected from asunaprevir (ASV), danoprevir (DPV), simeprevir (SPV), grazoprevir (GPV), glecaprevir, voxiprevir and any combination thereof. When the protease is HIV protease, in some cases, the inhibitor of the protease 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 SARS-CoV-2 protease, in some cases, the inhibitor of the protease is namatevir (Paxlovid). When the protease is human renin protease, in some cases, the inhibitor of the protease is aliskiren.

[0090] The first subunit and / or the second subunit may include any additional domains and functions as needed. For example, in certain embodiments, the first subunit, the second subunit or both include extracellular protein tags. Such tags are used, for example, to assess the use of cells expressing CCR on the cell surface. In some cases, the extracellular protein tag is an N-terminal protein tag. Non-limiting exemplary tags that can be incorporated into the first subunit and / or the second subunit include FLAG tags, HA tags, etc. Exemplary amino acid sequences of such tags are provided in Table 1 below.

[0091] In certain embodiments, the first subunit and the second subunit are engineered to each comprise an extracellular cysteine ​​residue, wherein the 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. For example, a disulfide linker sequence (e.g., CGG ) can be incorporated into the extracellular domain of each of the first and second subunits to stabilize their association.

[0092] Aspects of the present disclosure further include chimeric cytokine receptors (CCRs) encoded by one or more nucleic acids of the present disclosure.

[0093] The amino acid sequences of exemplary CCR components are provided in Table 1 below. For each sequence, the domains are listed in the left column as ordered from N-terminus to C-terminus. The sequences in the right column indicate the domains by alternating underlining.

[0094] Table 1 - Amino acid sequences of exemplary CCR components

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109] The present disclosure provides each polypeptide provided in Table 1 and each separate domain therein, and nucleic acids encoding such polypeptides and separate domains. Cells comprising such polypeptides and nucleic acids are also provided. As will be appreciated, the present disclosure also provides variants of any polypeptide and separate domains therein, wherein in some cases, the variant polypeptide or its domain 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 with a parent / reference sequence or its functional fragment, wherein the variant retains the functionality of the parent / reference sequence (e.g., dimerization ability, intracellular signaling activity, protease activity, protease cleavage ability, transition domain, etc.).

[0110] For example, in certain embodiments, variants of such polypeptides with 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 categories. Amino acid substitutions can be introduced into the polypeptide of interest, and the product is screened for desired activity (e.g., retained / improved cell surface expression, dimerization, signal transduction, adjustability, etc.).

[0111] Table 2 – Amino Acid Substitutions

[0112]

[0113]

[0114] Amino acids can be grouped based on common side chain properties:

[0115] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0116] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0117] (3) Acidic: Asp, Glu;

[0118] (4) Basic: His, Lys, Arg;

[0119] (5) Residues that affect chain orientation: Gly, Pro;

[0120] (6) Aromatic: Trp, Tyr, Phe.

[0121] Non-conservative substitutions will entail exchanging a member of one of these classes for another.

[0122] Exemplary nucleotide sequences encoding the various domains and polypeptides shown in Table 1 are provided in SEQ ID NOs: 31 to 53. The present disclosure also encompasses nucleic acids encoding polypeptides or domains thereof that differ from the nucleotide sequences in SEQ ID NOs: 31 to 53 due to the degeneracy of the genetic code.

[0123] 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 them. In some cases, there is provided a nucleic acid encoding a membrane-associated polypeptide, the membrane-associated polypeptide comprising a transmembrane domain and a cytokine receptor ICD, wherein the polypeptide does not include an extracellular cytokine binding domain. Such polypeptides are based in part on unexpected findings herein, that is, compared with CAR T cells lacking a single cytokine chain, a single cytokine chain (that is, not paired with a second cytokine chain) can enhance the anti-tumor efficacy of CAR T cells (see, e.g., Example 11). In certain embodiments, cytokine receptor ICD is IL-2RβICD, IL-7R ICD, IL-9R ICD or a common γ chain (γc) ICD. According to some embodiments, cytokine receptor ICD is IL-2RβICD. In some cases, the polypeptide includes a heterodimerization domain, such as an extracellular heterodimerization domain. In certain embodiments, the polypeptide includes a protease cleavage site arranged between a transmembrane domain and ICD.

[0124] Expression constructs, cells and compositions

[0125] Expression constructs

[0126] Aspects of the present disclosure further include one or more expression constructs, which include any one of one or more nucleic acids of the present disclosure. As used herein, "expression construct" is a circular or linear polynucleotide (a polymer consisting of naturally occurring and / or non-naturally occurring nucleotides), which includes a first subunit, a second subunit or a region of both CCR encoding the present disclosure, which region is operably connected to a suitable promoter (e.g., a constitutive promoter or an inducible promoter). In some embodiments, the 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, the expression of the first subunit, the second subunit or both can be controlled by one or more endogenous regulatory elements (e.g., promoters, enhancers, etc.) at or near the genomic locus, and the expression construct is inserted into the genomic locus.

[0127] In certain embodiments, the promoter can be a single shared promoter between each protein coding region of the expression construct, or at least one protein coding region can be operably connected to a promoter that is not shared with any other protein coding region of the expression construct. The expression construct can be configured to allow the polycistronic expression of two or more (e.g., each) protein coding regions. That is, two or more (e.g., each) proteins encoded by the expression construct can be expressed from the same promoter as a single protein. In certain embodiments, the expression construct includes a ribosomal skipping site to allow the polycistronic expression of two or more (e.g., each) protein coding regions. The non-limiting example of a suitable ribosomal skipping site that can be incorporated into the expression construct is the P2A ribosomal skipping site from porcine Teschovirus.

[0128] Expression constructs (e.g., vectors) can be suitable for replication and integration in prokaryotes, eukaryotes, or both. Expression constructs can include functionally appropriate directional transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of nucleic acids encoding the first subunit, the second subunit, or both. Expression constructs optionally include a universal expression cassette containing at least one independent terminator sequence, sequences that allow the expression cassette to replicate in both eukaryotes and prokaryotes (e.g., as found in shuttle vectors), and selection markers for both prokaryotic and eukaryotic systems.

[0129] In order to obtain high levels of expression of the cloned nucleic acid, an expression construct is usually constructed that generally contains a strong promoter to direct transcription, a ribosome binding site for translation initiation, and a transcription / translation terminator, each functionally oriented to each other and the protein coding sequence. Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator regions of the E. coli tryptophan biosynthetic pathway, i.e., the phage lambda (P L ) and L-arabinose (araBAD) operon. It is also useful to include a selection marker in the DNA vector transformed in Escherichia 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 can be obtained using, for example, Escherichia coli, Bacillus sp. and Salmonella. Escherichia coli systems can also be used. Transducing cells with nucleic acids (e.g., expression constructs) can involve, for example, incubating lipid microparticles containing nucleic acids with cells, or incubating viral vectors containing nucleic acids with cells within the host range of the vector.

[0130] In certain embodiments, after one or more expression constructs are delivered to the cell, one or more expression constructs are additional (e.g., extrachromosomal), wherein "episomal" or "episomal" means a polynucleotide that is independent of the cell chromosome DNA replication. The non-limiting example of an additional body that can be adopted is a plasmid.

[0131] According to some embodiments, after one or more expression constructs are delivered to the cell, one or more expression constructs are integrated into the genome of the cell. In certain embodiments, one or more expression constructs are suitable for site-specific integration into the genome. For example, the expression construct can be suitable for site-specific integration into the genome, wherein the site-specific integration inactivates the target gene in the genome of the cell. For example, site-specific integration can knock out the target gene by knocking in the expression construct. Any suitable method for site-specific gene editing and functional integration can be used. The functional integration of the expression construct can be achieved by various means, including by using an integration vector, including viruses and non-viral vectors. In some cases, retroviral vectors, such as lentiviral vectors, can be used. In some cases, non-retroviral integration vectors can be used. The integration vector can be in contact with the cell at a suitable concentration (or infection multiplicity) in a suitable transduction medium, and lasts for a suitable time so that the vector infects the target cell, thereby promoting the functional integration of the expression construct. Non-limiting examples of useful viral vectors include retroviral vectors, lentiviral vectors, adenovirus (Ad) vectors, adeno-associated virus (AAV) vectors, hybrid Ad-AAV vector systems, etc.

[0132] Strategies for site-specific integration that can be used for the methods of the present disclosure include strategies using homologous recombination, non-homologous end joining (NHEJ), etc. Such strategies can use non-naturally occurring or engineered nucleases, including but not limited to zinc finger 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 homologous templates to repair damaged DNA chains. Due to its homology-based mechanism, HR has been used as a tool for site-specific engineering genomes. Gene targeting by HR requires the use of two homologous arms flanking the transgene / target site of interest. HR efficiency can be increased by introducing DSB at the target site using a specific rare-cutting endonuclease. The discovery of this phenomenon has promoted the development of methods for generating site-specific DSBs in the genomes of different species. In the past decade, a variety of chimeric enzymes, i.e., ZFN, meganuclease and TALEN, have been designed for this purpose. ZFN is a modular chimeric protein containing a ZF-based DNA binding domain (DBD) and a Fok1 nuclease domain. DBD is usually composed of three ZF domains each with 3 base pair specificities; the Fok1 nuclease domain provides DNA nicking activity, which is targeted by two flanking ZFNs. Due to the modular nature of DBD, any site in the genome can be targeted. Except that DBD is derived from a transcription activator-like effector (TALE), TALEN is similar to ZFN. TALE DBD is modular, and it is composed of 34 residues repeated, and its DNA specificity is determined by the number and order of repetitions. Each repeat is bound to a single nucleotide in the target sequence by only two residues.

[0133] cell

[0134] 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 cell is a prokaryotic cell (e.g., bacteria), a yeast cell, an insect (e.g., fruit fly) cell, an amphibian (e.g., frog, e.g., African clawed frog) cell, a plant cell, etc. According to some embodiments, the cell is a mammalian cell. Mammalian cells of interest include human cells, rodent cells, etc.

[0135] In some embodiments, the cell is an immune cell. 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 cell is a T cell. When the immune cell includes a T cell, the T cell may include a naive T cell (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, αβT cells (T αβ ), γδT cells (T γδ ) or any combination thereof.

[0136] According to some embodiments, the cell is a stem cell, for example, a mammalian (e.g., human) stem cell. For example, the cell population may include embryonic stem (ES) cells, adult stem cells, hematopoietic stem cells (HSC), induced pluripotent stem cells (iPSC), mesenchymal stem cells (MSC), neural stem cells (NSC), or any combination thereof.

[0137] Methods 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 where the one or more nucleic acids or one or more expression constructs are delivered to the cells of the cell population. The method may further include selecting cells that exhibit cell surface expression of a CCR.

[0138] The contacting step can include contacting the cell population with the one or more nucleic acids or the one or more expression constructs, for example, by combining the cells with the one or more nucleic acids or the one or more expression constructs in a single mixture under conditions suitable for delivering (e.g., transfecting, transducing, etc.) each of the one or more nucleic acids or the one or more expression constructs into the cells of the cell population.

[0139] A variety of suitable methods and conditions for delivering nucleic acid and expression constructs to cells are known. According to some embodiments, delivery is carried out by microinjection, transfection, lipofection, heat shock, electroporation, transduction, gene gun, DEAE-dextran mediated transfer, etc. In certain embodiments, one or more nucleic acids or one or more expression constructs are introduced into the cells of the cell group by AAV transduction. The AAV vector can include the ITR from AAV2, and the serotype from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10. According to some embodiments, the AAV vector includes the ITR from AAV2 and the 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. The lentiviral vector backbone can be derived from HIV-1, HIV-2, Mae Di-Visna 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). The lentiviral vector can be an integration competent or integrase defective lentiviral vector (TDLV). In one embodiment, an IDLV vector comprising an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is employed. Non-limiting exemplary methods for preparing retroviral expression constructs and transducing cells with such constructs are provided in the experimental section below.

[0140] In certain embodiments, the cells of the present disclosure (e.g., T cells, NK cells, etc.) are genetically modified to express receptors (e.g., recombinant receptors) on their surfaces. That is, cells can be engineered to co-express CCR and additional receptors, 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, tactile receptors (Tango receptors), ChaCha receptors, generalized extracellular molecule sensors (GEMS) receptors, growth factor receptors, switch receptors, adhesion molecules, integrins, inhibitory receptors, stimulatory receptors, receptors containing immunoreceptor tyrosine-based activation motifs (ITAMs), receptors containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs), hormone receptors, receptor tyrosine kinases, immune receptors (such as CD28, CD80, ICOS, CTLA4, PD1, PD-L1, BTLA, HVEM, CD27, 4-1BB, 4-1BBL, OX40, OX4 OL, DR3, GITR, CD30, SLAM, CD2, 2B4, TIM1, TIM2, TIM3, TIGIT, CD226, CD160, LAG3, LAIR1, B7-1, B7-H1 and B7-H3), 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 α / β receptor, interferon γ 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 (such as tumor necrosis factor receptor 2 (1B), tumor necrosis factor receptor 1), lymphotoxin β receptor, OX40, CD4 0, 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-induced TNFR-related receptor, TROY, death receptor 6, death receptor 3, ectodysplasin A2 receptor, chemokine receptors (such as CCR1, CCR2, CCR3, CCR4, CCR5,

[00136] The invention relates to proteins that are involved in the regulation of the growth factor receptor (GFAP) and / or EGFR (EGFR) family. The proteins that are involved in the regulation of the growth factor receptor (GFAP) and / or EGFR family are preferably proteins that are involved in the regulation of the growth factor receptor (GFAP) and / or EGFR family. The proteins that are involved in the regulation of the growth factor receptor (GFAP) and / or EGFR family are preferably proteins that are involved in the regulation of the growth factor receptor (GFAP) and / or EGFR family. In some embodiments, such receptors are immune cell receptors selected from the group consisting of 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.

[0141] 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.

[0142] As described above, according to some embodiments, the cells of the present disclosure (e.g., human T cells) can be engineered to express CAR in addition to CCR. The extracellular binding domain of CAR can include 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 a non-limiting example, the single-chain antibody is a single-chain variable fragment (scFv). In some embodiments, the extracellular binding domain of CAR is a single-chain form (e.g., scFv form) of an antibody approved for use as a therapeutic antibody by the U.S. Food and Drug Administration and / or the European Medicines Agency (EMA). Non-limiting examples of single chain antibodies that can be employed when the protein of interest is a CAR include single chain forms (e.g., scFv forms) of the following: Adecatumumab, Ascrinvacumab, Cixutumumab, Conatumumab, Daratumumab, Drozitumab, Duligotumab, Durvalumab, valumab), Dusigitumab, Enfortumab, Enoticumab, Figitumumab, Ganitumab, Glembatumumab, Intetumumab, Ipilimumab, Iratumumab, Icrucumab, Lexatumumab, Lucatumumab, Mapatumumab, Narnatumab, Necitumumab, Nesvacumab, Ofatumumab, Olaratumab, Panitumumab, Patritumab, Pritutumumab mumab), Radretumab, Ramucirumab, Rilotumumab, Robatumumab, Seribantumab, Tarextumab, Teprotumumab, Tovetumab, Vantictumab, Vesencumab,Votumumab, Zalutumumab, Flanvotumab, Altumomab, Anatumomab, Arcitumomab, Bectumomab, Blinatumomab, Detumomab, Ibritumomab, Minretumomab, Mitumomab, Moxetumomab, Naptumomab Naptumomab, Nofetumomab, Pemtumomab, Pintumomab, Racotumomab, Satumomab, Solitomab, Taplitumomab, Tenatumomab, Tositumomab, Tremelimumab, Abagovomab, Igovomab, Oregovomab b), Capromab, Edrecolomab, Nacolomab, Amatuximab, Bavituximab, Brentuximab, Cetuximab, Derlotuximab, Dinutuximab, Ensituximab, Futuximab, Girentuximab, Indatuximab, I Isatuximab, Margetuximab, Rituximab, Siltuximab, Ublituximab, Ecoromeximab, Abituzumab, Alemtuzumab, Bevacizumab, Bivatuzumab, Brontictuzumab, Cantuzumab, Cantuzumab,Citatuzumab, Clivatuzumab, Dacetuzumab, Demcizumab, Dalotuzumab, Denintuzumab, Elotuzumab, Emactuzumab, Emibetuzumab, Enoblituzumab, Etaracizumab, Farletuzumab, Ficlatuzumab clatuzumab), Gemtuzumab, Imgatuzumab, Inotuzumab, Labetuzumab, Lifastuzumab, Lintuzumab, Lorvotuzumab, Lumretuzumab, Matuzumab, Milatuzumab, Nimotuzumab, Obinutuzumab, Ocatuzumab caratuzumab), Otlertuzumab, Onartuzumab, Oportuzumab, Parsatuzumab, Pertuzumab, Pinatuzumab, Polatuzumab, Sibrotuzumab, Simtuzumab, Tacatuzumab, Tigatuzumab, Trastuzumab, Sibrotuzumab, Simtuzumab, Tacatuzumab, Tigatuzumab, Trastuzumab, Tucotuzumab, Vandortuzumab, Vanucizumab, Veltuzumab, Vorsetuzumab, Sofituzumab, Catumaxomab, Ertumaxomab, Depatuxizumab, Ontuxizumab, Blontuvetmab, Tamtuvetmab, or antigen-binding variants thereof.

[0143] Additional receptors (e.g., CAR) may include one or more linker sequences between various domains. A "variable region linker sequence" is an amino acid sequence that connects the heavy chain variable region to the light chain variable region and provides a spacer function compatible with the interaction of the two sub-binding domains, so that the resulting polypeptide retains the same specific binding affinity for the target molecule as an antibody containing the same light chain and heavy chain variable regions. A non-limiting example of a variable region linker sequence is a glycine-serine linker, such as (G 4 S) 3 (SEQ ID NO:63) joint. In certain embodiments, the joint separates one or more heavy chain or light chain variable domains, hinge domains, transmembrane domains, costimulatory domains, and / or primary signaling domains. In a specific embodiment, the receptor (e.g., CAR) includes one, two, three, four, or five or more joints. In a specific embodiment, the length of the joint 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 intermediate length of amino acids. In some embodiments, the length of the joint 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.

[0144] In some embodiments, the antigen binding domain of a receptor (e.g., CAR) is followed by one or more spacer domains, which remove the antigen binding domain from the cell surface expressing the receptor to achieve appropriate cell / cell contact, antigen binding and / or activation. The spacer domain (and any other spacer domains, joints and / or analogs described herein) can be derived from natural, synthetic, semi-synthetic or recombinant sources. In certain embodiments, the spacer domain is a part of an immunoglobulin, including but not limited to one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain may include a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region amino acid sequence. In some embodiments, the spacer domain includes CH2 and / or CH3 of IgG1, IgG4 or IgD. The illustrative spacer domains suitable for receptors described herein (e.g., CAR) include hinge regions such as CD8α and CD4 derived from the extracellular region of type 1 membrane proteins, which may be wild-type hinge regions from these molecules or variants thereof. In certain embodiments, the hinge domain includes 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 an IgG4 hinge.

[0145] "Transmembrane domain" (Tm domain) is a part of a receptor (e.g., CAR) that fuses an extracellular binding portion and an intracellular signaling domain, and anchors the receptor to the plasma membrane of a cell (e.g., a T cell, such as Treg). The Tm domain can be derived from a natural, synthetic, semisynthetic, or recombinant source. In some embodiments, the Tm domain is derived from an α or β chain (e.g., at least including a transmembrane region or a functional portion thereof) 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.

[0146] In one embodiment, the receptor (e.g., CAR) includes a Tm domain derived from CD28. In certain embodiments, the receptor includes a Tm domain derived from CD28 and a short oligomer or polypeptide linker (e.g., a length of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids), which connects the Tm domain of the receptor and the intracellular signaling domain. For example, a glycine-serine linker can be used as such a linker.

[0147] The "intracellular signaling" domain of a receptor (e.g., CAR) refers to a portion of a receptor that is involved in transducing a signal bound to a target molecule / antigen into the interior of a cell to initiate a cell function. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transduces a signal and instructs the cell to perform a specific function. Insofar as a truncated portion of an intracellular signaling domain is used, such a truncated portion can be used to replace the full-length intracellular signaling domain as long as it transduces a signal. The term intracellular signaling domain means any truncated portion of an intracellular signaling domain that is sufficient to transduce a signal.

[0148] The signal generated by the T cell receptor (TCR) alone is not enough to fully activate T cells, and secondary or costimulatory signals are also required. Therefore, T cell activation is mediated by two different types of intracellular signaling domains: a primary signaling domain that initiates antigen-dependent primary activation through TCR (e.g., TCR / CD3 complex) and a costimulatory signaling domain that provides secondary or costimulatory signals in an antigen-independent manner. Therefore, a receptor (e.g., CAR) expressed by a genetically modified cell may include an intracellular signaling domain, which includes one or more (e.g., 1, 2 or more) "costimulatory signaling domains" and "primary signaling domains".

[0149] The primary signaling domain regulates the primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner. The primary signaling domain that acts in a stimulatory manner may include a signaling motif known as an immunoreceptor tyrosine-based activation motif (or "ITAM"). Non-limiting examples of ITAM-containing primary signaling domains suitable for 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 includes a CD3ζ primary signaling domain and one or more costimulatory signaling domains. The intracellular main signaling domain and the costimulatory signaling domain are operably connected to the carboxyl terminus of the transmembrane domain.

[0150] In some embodiments, receptor (for example, CAR) includes one or more costimulatory signal domains, to enhance the efficacy and amplification of immune effector cells (for example, T cells) expressing receptors. As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule or its active fragment. Suitable for exemplary costimulatory molecules for receptors expected in a particular embodiment 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) includes one or more co-stimulatory signaling domains selected from the group consisting of 4-1BB (CD137), CD28, and CD134, and a CD3ζ primary signaling domain.

[0151] The receptor (e.g., CAR) expressed by the cells genetically modified according to the methods of the present disclosure may include any variety of suitable domains, including but not limited to a leader sequence; a hinge, spacer and / or linker domain; a transmembrane domain; a co-stimulatory domain; a signaling domain (e.g., a CD3ζ domain); a ribosomal jumping element; a restriction enzyme sequence; a reporter protein domain, etc.

[0152] 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 that 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, delta-like canonical incision ligand 3 (DLL3), endothelin receptor type B (EDNRB), ephrin A4 (EFNA4), epidermal growth factor receptor (EGFR), EGFRvIII, extracellular nucleotide 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 nontransfer B (GPNMB), guanylate cyclase 2C (GUCY2C), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), integrin α, lysosomal associated membrane protein 1 (LAMP-1), Lewis Y, LIV-1, leucine-rich repeat-containing protein 15 (LRRC15), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), sodium-dependent phosphate transporter 2B (NaPi2b), nectin-4, NMB, NOTCH3, p-cadherin (p-CAD), 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-1 (TIM-1), Tn antigen, trophoblast cell surface antigen (TROP-2), Wilms tumor 1 (WT1), and VEGF-A.

[0153] In certain embodiments, the cells of the present disclosure are genetically modified to express antibodies. The term "antibody" (also used interchangeably with "immunoglobulin") covers antibodies of any isotype (e.g., IgG (e.g., IgG1, IgG2, IgG3 or IgG4), IgE, IgD, IgA, IgM, etc.), intact antibodies (e.g., antibodies composed of tetramers, which in turn are composed of two dimers of heavy and light chain polypeptides); single-chain antibodies (e.g., scFv); fragments of antibodies that retain specific binding to an antigen (e.g., fragments of intact or single-chain antibodies), including but not limited to 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.

[0154] Immunoglobulin polypeptides include kappa and lambda light chains as well as alpha and gamma (IgG 1 IgG 2 IgG 3 IgG 4 ), δ, ε and μ heavy chains or equivalents in other species. A full-length immunoglobulin "light chain" (usually about 25 kDa or about 214 amino acids) is contained in NH 2 A full-length immunoglobulin "heavy chain" (about 150 kDa or about 446 amino acids) similarly comprises a variable region (about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (about 330 amino acids).

[0155] The variable region of the immunoglobulin light or heavy chain (V L and V H ) consists of a "framework" region (FR) interrupted by three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs"). The extent of the framework regions and CDRs has been defined (see E. Kabat et al., Sequences of proteins of immunological interest, 4th ed., U.S. Department of Health and Human Services, Public Health Services, Bethesda, MD (1987); and Lefranc et al., IMGT, (the international ImMunoGeneTicsinformation ), "Nucl. Acids Res.", 2005, 33, D593-D597). The sequences of the framework regions of different light chains or heavy chains are relatively conserved among species. The framework region of an antibody, i.e., the combined framework region of the light chain and the heavy chain, is used to locate and align CDRs. CDRs are primarily responsible for binding to the epitope of an antigen. Unless otherwise indicated, all CDRs and frameworks provided by the present disclosure are defined according to Kabat above.

[0156] Therefore, "antibodies" encompass proteins having one or more polypeptides that may be genetically coded, for example, by immunoglobulin genes or fragments of immunoglobulin genes. Recognized immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the classes of immunoglobulins: IgG, IgM, IgA, IgD, and IgE, respectively. In some embodiments, antibodies of the present disclosure are IgG antibodies, for example IgG1 antibodies, such as human IgG1 antibodies. In some embodiments, cells express antibodies comprising human Fc domains.

[0157] It is known that the typical immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two pairs of identical polypeptide chains, each pair of polypeptide chains having one "light chain" (about 25 kD) and one "heavy chain" (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The term variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains, respectively.

[0158] According to some embodiments, the cells of the present disclosure have not been genetically modified to express an engineered receptor other than a CCR.

[0159] As will be appreciated with the benefit of this disclosure, cells of the present disclosure (e.g., T cells) can be expanded during the therapeutic cell manufacturing process. "Expanding" or "expanded" means culturing cells under conditions of cell proliferation. Suitable conditions can vary, for example, depending on the type of cells being expanded (e.g., the type of T cells). Such conditions can include culturing cells in a suitable container (e.g., a cell culture plate or wells thereof, a box, tube, bottle, or bag suitable for an automated therapeutic cell manufacturing system, such as a closed automated therapeutic cell manufacturing system, such as the CliniMACS by Miltenyi Biotec). System, Cytiva Cell Expansion System, Wilson Wolf Cell expansion system, from Terumo Cell expansion system, Lonza system, etc.), in a suitable culture medium (e.g., cell culture medium such as RPMI, DMEM, IMDM, MEM, DMEM / F-12, etc.), at a suitable temperature (e.g., 32° C. to 42° C., such as 37° C.) and pH (e.g., pH 7.0-7.7, such as pH 7.4), in an atmosphere with a suitable percentage of CO 2 Cells (e.g., T cells) are cultured in an environment with an ambient temperature of 50° C. (e.g., 3% to 10%, such as 5%).

[0160] Methods for activating and expanding cells for therapy (e.g., therapeutic T cells, etc.) are known in the art and are described, for example, in U.S. Patent Nos. 6,905,874, 6,867,041, and 6,797,514; and PCT Publication No. WO 2012 / 079000, the contents of which are hereby incorporated by reference in their entirety. In the example of T cells, such methods can include contacting PBMCs or isolated T cells with stimulators and co-stimulators such as anti-CD3 antibodies and anti-CD28 antibodies (usually attached to beads or other surfaces) in a culture medium with appropriate cytokines (such as IL-2). Anti-CD3 and anti-CD28 antibodies attached to the same beads are used as "surrogate" antigen presenting cells (APCs). An example is System, a CD3 / CD28 activator / stimulator system for physiological activation of human T cells. In other embodiments, feeder cells and appropriate antibodies and cytokines are used to activate and stimulate T cell proliferation using methods such as those described in U.S. Pat. Nos. 6,040,177 and 5,827,642 and PCT Publication No. WO 2012 / 129514 (the contents of which are hereby incorporated by reference in their entirety).

[0161] 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 from Miltenyi Biotec System, Cytiva Cell expansion system, Wilson Wolf's G- Cell expansion system, from Terumo Cell expansion system, Lonza Systems, etc. Detailed guidelines and protocols for producing therapeutic cells on such systems are available from the suppliers of such systems.

[0162] Composition

[0163] The present disclosure also provides compositions. According to some embodiments, provided are compositions comprising any cell or cells of the present disclosure, one or more nucleic acids, one or more expression constructs, and / or cells or progeny thereof.

[0164] Such compositions can 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 can be an aqueous liquid medium, such as water, a buffer solution, etc. One or more additives, such as salts (e.g., NaCl, MgCl2, KCl, MgSO4), 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.), solubilizing agents, detergents (e.g., nonionic detergents, such as Tween-20 etc.), ribonuclease inhibitors, glycerol, chelating agents, etc. can be present in such compositions. In certain embodiments, the liquid medium is a cell culture medium. Non-limiting examples of cell culture media include Minimum Essential Medium, DMEM, a-MEM, RPMI medium, Clicks, F-12, X-Vivo 15, X-Vivo 20, Optimizer, and the like.

[0165] In certain embodiments, a composition comprising a cell of the present disclosure is provided, wherein the composition is suitable for application to a subject, such as a human subject. Such compositions may include cells and a pharmaceutically acceptable carrier. Pharmaceutical compositions generally include a therapeutically effective amount of cells. "Therapeutically effective amount" means the number of cells sufficient to produce the desired result, such as the amount sufficient to achieve a beneficial or desired treatment (including prevention) result (such as the reduction of the symptoms of a disease (e.g., cancer) or illness associated with, for example, a target cell or its colony (e.g., cancer cell) compared to a control). An effective amount may be administered in one or more administrations. The therapeutically effective amount may vary according to factors such as individual disease state, age, sex, and body weight, and the ability of cells to elicit the desired response in an individual. The therapeutically effective amount is also the amount in which the beneficial effect of the treatment exceeds any toxic or harmful effect of the cell. The term "therapeutically effective amount" includes an amount effective for "treating" a subject (e.g., a human subject). When indicating a therapeutic amount, the precise amount of the composition to be administered in a particular embodiment may be determined by a physician according to the instructions and taking into account the individual differences in the patient's (individual's) age, body weight, tumor size, degree of infection or metastasis, and symptoms. In some embodiments, the pharmaceutical composition of the present disclosure comprises 1×10 6 Up to 5×10 10Cells of the present disclosure.

[0166] The cells of the present disclosure can be incorporated into a variety of preparations for therapeutic administration. For example, the cells of the present disclosure can be formulated into a pharmaceutical composition by combining with an appropriate pharmaceutically acceptable excipient or diluent. Preparations of cells suitable for administration to a patient (e.g., suitable for human administration) are typically sterile and may further be free of detectable pyrogens or other contaminants that prohibit administration to a patient according to the selected route of administration.

[0167] The cells can be formulated for parenteral (eg, intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration or any other suitable route of administration.

[0168] Aqueous preparations of cells can be prepared in pH buffered solutions, e.g., pH ranges from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers suitable for pH within this range include phosphate buffers, histidine buffers, citrate buffers, succinate buffers, acetate buffers, and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, for example, on the desired tonicity of the buffer and the preparation.

[0169] Tension agents can be included in the formulation to adjust the tension of the formulation. Exemplary tension agents include sodium chloride, potassium chloride, glycerol and any component from the group of amino acids, sugars and combinations thereof. In some embodiments, the aqueous formulation is isotonic, but hypertonic or hypotonic solutions may also be suitable. The term "isotonic" means a solution with the same tension as some other solution compared thereto, such as a physiological saline solution or serum. The tension agent can be used in an amount of about 5mM to about 350mM, for example, in an amount of 100mM to 350mM.

[0170] Surfactants can also be added to the formulation to reduce aggregation and / or minimize the formation of particles in the formulation 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 (Poloxamers, Pluronic) and sodium lauryl sulfate (SDS). An example of a suitable polyoxyethylene sorbitan-fatty acid ester is polysorbate 20 (known under the trademark Tween 20 TM Polysorbate 80 (sold under the trademark Tween 80 TM Examples of suitable polyethylene-polypropylene copolymers are marketed under the name F68 or Poloxamer 188 TMExamples of suitable polyoxyethylene alkyl ethers are those sold under the trademark Brij TM Exemplary concentrations of surfactants may range from about 0.001% to about 1% w / v.

[0171] In some embodiments, the composition comprises a cell of the present disclosure and one or more of the agents identified above (e.g., surfactants, buffers, stabilizers, tonicity agents) and is substantially 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, the preservative is included in the formulation, e.g., at a concentration ranging from about 0.001 to about 2% (w / v).

[0172] Reagent test kit

[0173] The present disclosure also provides a kit. In certain embodiments, a kit is provided, which includes any one of one or more nucleic acids of the present disclosure, or any one of one or more expression constructs. In certain embodiments, the kit of the present disclosure includes a transfection / transduction reagent that can be used to introduce any one of one or more nucleic acids or one or more expression constructs into a cell of interest, such as an immune cell (e.g., a T cell) or other cell of interest.

[0174] The components of the kit may be present in separate containers, or multiple components may be present in a single container. For example, one or more nucleic acids, or any one of one or more expression constructs, may be provided in separate containers or in the same container. Suitable containers include single tubes (e.g., vials), one or more wells of plates (e.g., 96-well plates, 384-well plates, etc.), etc.

[0175] The kit of the present disclosure may further include instructions for contacting the cell population with one or more nucleic acids or any one of the one or more expression constructs under conditions where any one of the one or more nucleic acids or any one of the one or more expression constructs is delivered to the cells of the cell population. The kit of the present disclosure may further include instructions for selecting cells that exhibit cell surface expression of CCR.

[0176] The instructions for use of a test kit can be recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastics, etc. Therefore, the instructions can be present in the test kit as a package insert, in the label of a container of a test kit or its components (i.e., relevant to packaging or subpackaging). In other embodiments, the instructions exist as an electronic storage data file present in a suitable computer-readable storage medium, and the computer-readable storage medium is, for example, a portable flash drive, a DVD, a CD-ROM, a floppy disk, etc. In other embodiments, the actual instructions are not present in the test kit, but provide, for example, a method for obtaining instructions from a remote source via the Internet. The example of this embodiment is a test kit comprising a website, in which instructions can be viewed and / or instructions can be downloaded from the website. As with the instructions, the method for obtaining instructions is recorded on a suitable substrate.

[0177] Treatment

[0178] Aspects of the present disclosure further include methods of treatment. As shown herein, when expressed on cells, CCR of the present disclosure is a strong driving force for cell proliferation in vitro and in vivo, and compared with control cells lacking CCR, enhanced in vivo anti-tumor activity is provided. Therefore, CCR and cells expressing it are used for cell therapy, and the non-limiting examples of the cell therapy 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, etc.

[0179] According to some embodiments, there is provided a method for 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 cells of the present disclosure. The cell comprises any one of one or more expression constructs of the present disclosure, such that the cell expresses a CCR comprising a first subunit and a second subunit encoded by one or more nucleic acids. The cell may be further modified to express any additional receptor described herein, such as CAR, recombinant TCR, etc.

[0180] "Cell-based therapy" or "cell therapy" refers to the transfer of autologous or allogeneic cellular material into 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 expressing recombinant T cell receptors (TCRs)), therapy comprising administering T cells that do not express recombinant receptors other than CCRs, CAR NK cell therapy, etc.

[0181] In certain embodiments, the signaling of the chimeric cytokine receptor is regulatable. For example, in some embodiments: the first subunit, the second subunit, or both comprise a protease cleavage site disposed between the transmembrane domain and the ICD; the one or more nucleic acids further encode a protease, wherein the protease cleavage site is a cleavage site for a protease; and the proteolytic activity of the protease is regulatable.

[0182] 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 can further include administering an effective amount of the cell permeable small molecule to the subject when signal transduction through the CCR is required. In certain embodiments, such methods further include stopping the administration of the cell permeable small molecule when signal transduction through the CCR is no longer required.

[0183] When the cell-based therapy can be regulated using a cell-permeable protease inhibitor, the protease inhibitor is selected based on the protease employed in the regulatable system. For example, in some embodiments, when the protease is derived from HCV NS3, the inhibitor of the protease can be asunavir (ASV), danoprevir (DPV), simeprevir (SPV), grazoprevir (GPV), glecaprevir, voxiprevir, or any combination thereof. Also for example, when the protease is HIV protease, the inhibitor of the protease can be atazanavir (Reyataz), darunavir (Prezista), fosamprevir (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 another example, when the protease is SARS-CoV-2 protease, the inhibitor of the protease can be Paxlovid. As another example, when the protease is human renin protease, the inhibitor of the protease can be Aliskiren.

[0184] Therapeutic cells can be autologous / autologous ("self") or non-autologous ("non-self", e.g., allogeneic, isogenic, or xenogeneic). "Autologous" as used herein refers to cells obtained from a subject to which the therapeutic cells are subsequently administered. "Allogeneic" as used herein refers to cells obtained from a donor other than the subject to which 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 a primate. In some embodiments, the cells are obtained from humans.

[0185] Any of the cell-based therapeutic methods disclosed herein can be used to treat a variety of conditions in a subject. In certain embodiments, the subject suffers from cancer. The terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth / proliferation. The method can be used to treat a variety of cancers. In certain embodiments, cancer includes solid tumors. Examples of solid tumors that can be treated by the methods disclosed herein include carcinomas, lymphomas, blastomas, and sarcomas. In certain embodiments, cancer includes hematological malignancies, non-limiting examples of which include leukemias, lymphomas, or multiple myeloma.

[0186] 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, liver cancer, breast cancer, colon cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, various types of head and neck cancer, etc. In certain embodiments, the individual suffers from a cancer selected from the group consisting of solid tumors, 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 head and neck cancer, squamous cell carcinoma, 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 aspects, the individual suffers from a cancer selected from the group consisting of melanoma, Hodgkin's lymphoma, renal cell carcinoma (RCC), bladder cancer, non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC).

[0187] Treatment means at least improving one or more symptoms associated with a subject's condition (e.g., cancer), wherein improvement is used in a broad sense to refer to at least reducing the magnitude of a parameter (e.g., symptom) associated with the treated condition. Thus, treatment also includes situations in which a condition (e.g., cancer) or at least one or more symptoms associated therewith are completely suppressed (e.g., prevented from occurring) or stopped (e.g., terminated), such that the subject no longer suffers from the condition, or at least no longer suffers from the symptoms characterizing the condition.

[0188] The following examples are offered by way of illustration and not by way of limitation.

[0189] experiment

[0190] Example 1 - Chimeric Cytokine Receptor (CCR) that Signals in the Absence of Its Cognate Cytokine Production

[0191] In this embodiment, the production of a chimeric cytokine receptor (CCR) is described, which is capable of signaling in the absence of its cognate cytokine. CCR includes a first subunit and a second subunit, the first subunit comprising a first heterodimerization domain and a first cytokine receptor intracellular signaling domain (ICD), and the second subunit comprising a second heterodimerization domain homologous to the first heterodimerization domain and a second cytokine receptor ICD. As a proof of concept, in this embodiment, CCR uses: a leucine zipper as the first heterodimerization domain and the second heterodimerization domain; a first ICD, which is IL-2RB or IL-21R; and a second ICD as a common γ chain (γC) ICD.

[0192] exist Figure 2 A schematic diagram of a chimeric cytokine receptor (CCR) system is shown, which allows constitutive signaling of receptors in the absence of their cognate cytokines. The receptor 1 of CCR is composed of a first dimerization domain (such as a leucine zipper fused to a transmembrane domain and a first intracellular domain (ICD 1)). The receptor 2 of CCR is composed of a second dimerization domain homologous to the first dimerization domain (such as a second leucine zipper fused to a transmembrane domain and a second intracellular domain (ICD 2)). The association of two dimerization domains results in close proximity of two intracellular signaling domains and triggers the signaling of the receptor complex. Cysteine ​​residues are optionally included in the extracellular domain to stabilize the association of the receptor complex via disulfide bonds. Detection tags are optionally included to facilitate detection of cell surface expression of receptor components. Figure 2 B shows an exemplary signaling domain of a chimeric cytokine receptor based on the gamma chain cytokine receptor.

[0193] Figure 3It is a series of flow charts showing the cell surface expression of the CCR receptor components of various common γ chain receptors.The receptor components are composed of extracellular detection modules (HA or Flag tags), leucine zipper domains, CD8α hinges and transmembrane domains and intracellular signal transduction domains (IL-2RB, IL-21R or γC).As can be seen from the data, the receptor (with or without GGSGS joints) containing γC intracellular domains shows poor cell surface expression.The T cells of the untransduced Mock adding or not adding cytokines are used as the negative control of FLAG and HA tags.

[0194] exist Figure 4 A schematic diagram of the chimeric cytokine receptor (CCR) system is shown in A. To improve expression of CCR receptors containing a common gamma chain intracellular domain, the transition between the transmembrane domain and the intracellular domain was engineered. Figure 4 B is a list of various CCR constructs that contain a common γ chain intracellular domain and a transition peptide between the transmembrane domain and the ICD domain. The transition peptide is derived from the membrane-proximal region of CD8α or IL-15Rα. HTM refers to the hinge and transmembrane domain. Figure 4 C shows a series of flow charts of IL-2R CCR, which demonstrates the cell surface expression of HA tag and phosphorylated STAT5 (pSTAT5), a cytokine signaling molecule downstream of IL-2R signaling. As can be seen from the data, the inclusion of transition peptides in the juxtamembrane domain improves the cell surface expression of the common γ chain CCR receptor component and allows the induction of high levels of IL-2 signaling in the absence of cytokines. Mock untransduced T cells exposed to exogenously added IL-2 are used as a positive control for pSTAT5.

[0195] exist Figure 5 The display is shown in Figure 4 Figure 2 shows the proliferation of primary human T cells transduced with various CCRs shown in Figure 2. As can be seen from the data, in the absence of added IL-2, CCR drives the proliferation of T cells. Mock untransduced T cells with added IL-2 were used as a positive control. T cells transduced with only one component of the CCR (CCR IL-2RB alone) did not proliferate in the absence of IL-2. CCR drives T cell proliferation to varying degrees, and the magnitude of proliferation varies with Figure 4 The intensity of pSTAT5 signaling is correlated with that shown in .

[0196] Example 2 - Development of a regulatable CCR system to address in vivo toxicity observed with constitutive CCRs

[0197] In this example it is shown that the constitutive CCR described in Example 1 exhibits toxicity in vivo. However, here the development of a regulatable CCR system that addresses such toxicity is demonstrated.

[0198] Figure 6 A is a series of bioluminescent images showing the expansion of primary human T cells transduced with HER2 chimeric antigen receptor (CAR) and IL-2 or IL-21CCR in NSG mice bearing 143B osteosarcoma tumors. As can be seen from the data, IL-2CCR is a strong driver of T cell proliferation in vivo. Figure 6 B shows the Figure 6 Quantification of T cell expansion from the bioluminescence images shown in A. As can be seen from the data, IL-2CCR is a strong driver of T cell proliferation in vivo. Figure 6 C is a graph showing tumor size of mice implanted with 143B osteosarcoma cells and treated with the indicated T cells. Figure 6 D is a graph of the survival curves of mice treated with the indicated T cells. Mice treated with CCR-IL-2 rapidly succumbed to treatment-related toxicity due to uncontrolled proliferation of T cells. Thus, constitutive IL-2CCR is a strong driver of proliferation in vivo but causes lethal toxicity in NSG mice.

[0199] Figure 7 It is a schematic diagram of the chimeric cytokine receptor (CCR) system regulated by the drug developed in the present embodiment. The protease cleavage site is integrated between the transmembrane domain and the intracellular signal transduction domain of CCR. Due to the proteolytic activity of the protease at the cleavage site, the co-expression of the protease specific to the cleavage site causes the cleavage of CCR, thereby causing the inactivation of CCR (receptor closure). Adding a protease inhibitor (drug) suppresses this cleavage event, and makes CCR in an open state (receptor opening). Therefore, the signal transduction of CCR becomes dependent on the presence of medicine. Regulated CCR can be circumvented by regulating the proliferation of cells to fall into the treatment window to be associated with constitutive CCR toxicity.

[0200] exist Figure 8 The following shows how to use Figure 7A series of flow charts of primary human T cells transduced with IL-2R CCR in a regulated form as described in , which show the cell surface expression of HA / FLAG tags and phosphorylated STAT5 (pSTAT5), which is a cytokine signaling molecule downstream of IL-2R signaling. Regulated CCR is constructed by integrating a 33 amino acid linker containing HCV NS3 protease cleavage between the transmembrane and intracellular domains of CCR. HCV NS3 protease is co-expressed in cells to allow receptor cleavage in the absence of protease inhibitors (3 μM grazoprevir, drug). As can be seen from the data, a 33 amino acid linker containing HCV NS3 cleavage site eliminates CCR signaling in the presence or absence of drugs, and is not the optimal configuration for regulated CCR signaling. Mock untransduced T cells exposed to exogenously added IL-2 are used as positive controls for pSTAT5.

[0201] exist Fig. 9 It was demonstrated that insertion of a linker containing a cleavage site upstream of the γ-chain ICD allowed drug-regulated CCR signaling. Fig. 9 A is a list showing amino acid linkers comprising the HCV NS3 protease cleavage site inserted at various positions within the gamma chain ICD. Fig. 9 B is used as Fig. 9 A series of flow charts of primary human T cells transduced with IL-2R CCR in a regulated form shown in A, which demonstrates cell surface expression of HA / FLAG tags and phosphorylated STAT5 (pSTAT5). Regulated CCR is constructed by integrating a 19 amino acid linker containing HCV NS3 protease cleavage between the transmembrane and intracellular domains of the γ chain CCR component. HCV NS3 protease is co-expressed in the cell to allow receptor cleavage in the absence of protease inhibitors (3 μM grazoprevir, drug). As can be seen from the data, the inclusion of a 19 amino acid linker containing an HCV NS3 cleavage site at position 1 allows drug-dependent signaling of CCR. Mock untransduced T cells exposed to exogenously added IL-2 are used as a positive control for pSTAT5.

[0202] exist Fig.10A series of flow charts of primary human T cells transduced with regulated forms of IL-2R CCR are shown in Figure 2, which show phosphorylated STAT5 (pSTAT5). Regulated CCR is constructed by integrating 19 amino acid linkers containing HCV NS3 protease cleavage between the transmembrane and intracellular domains of the γ chain CCR component, the IL-2RB CCR component, or both. HCVNS3 protease is co-expressed in the cell to allow receptor cleavage in the absence of protease inhibitors (drugs). HCV4a4B or 4b5A cleavage sites are used as HCV cleavage sites. "RB" represents a CCR component containing IL-2RB ICD. "gc" represents a CCR component containing a common γ chain ICD. "Off" and "On" represent the absence or presence of protease inhibitors (3 μM Grazoprevir), respectively. As can be seen from the data, several configurations allow control of drug regulation of CCR signaling. Mock untransduced T cells exposed to exogenously added IL-2 are used as positive controls for pSTAT5.

[0203] exist Fig.11 Pharmacological control of in vitro cell expansion is demonstrated in . Fig.11 A is a diagram showing the Fig.10 A series of graphs showing the in vitro proliferation of primary human T cells transduced with various CCRs shown in and grown in the presence (Reg CCR On) or in the absence (Reg CCR Off) of a protease inhibitor (3 μM Grazoprevir). Original (OG) constitutive IL-2 CCR and untransduced T cells (Mock) served as positive and negative controls, respectively. Fig.11 A graph showing the percentage survival 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) is shown in B. As can be seen from the data, several configurations allowed drug-regulated control T cells to survive and proliferate.

[0204] Example 3 - CCR enhances anti-tumor activity in vivo

[0205] In this example it is demonstrated that CCR enhances anti-tumor activity in vivo, including subsequent rechallenge.

[0206] Fig.12A is a schematic diagram showing the construct used in this experiment.By fusing anti-CD22 scFv with CD8a hinge, CD8a transmembrane domain, 41BB intracellular domain and CD3z signaling domain, CD22 targeting CAR (CD22 CAR) is constructed.Const CCR (Const CCR) is constructed using the intracellular domain of IL-2RB and a common γ chain, and is expressed in a bicistronic vector.By integrating 4b5a HCV cleavage site between transmembrane domain and common γ chain intracellular domain, drug-regulated CCR (Reg CCR) is constructed.HCV NS3 protease is co-expressed in a tricistronic vector.

[0207] exist Fig.12 A series of bioluminescent images tracking the growth of Nalm6 leukemia expressing firefly luciferase are shown in B. On day 0, 1x10^6Nalm6 was infused to NSG mice, and then treated with the indicated CAR-T cells. Four days later, 4X10^6 CAR-T or control cells were implanted by tail vein injection. On the 17th day, mice were re-stimulated with 4.6x10^6Nalm6 tumor cells. Mice in the "+GPV" group were also implanted with an osmotic drug pump (Azlet model 2002) containing 54mg / mL grazoprevir and 0.6mg / mL ritonavir. These mice were additionally administered 50mg / kg grazoprevir and 25mg / kg ritonavir by oral gavage 1-2 times a day. Mock untransduced T cells were used as negative controls.

[0208] Fig.12 C is shown Fig.12 Quantification of the bioluminescence images shown in B. As can be seen from the data, mice that received CD22 CAR-T cells co-transduced with regulated IL-2CCR (Reg CCR) and administered grazoprevir were able to maintain long-term tumor control, while control CD22 CAR-T cells lacking regulated CCR died from tumor growth. Mice that received CD22 CAR-T cells co-expressing constitutive IL-2CCR (Const CCR) died due to treatment-related toxicity.

[0209] exist Fig.13 In the study, we demonstrated that regulated CCRs can expand CAR-T cells in vivo without toxicity. Fig.13A shows a schematic diagram of the construct used in this experiment.By fusing anti-CD22 scFv with CD8a hinge, CD8a transmembrane domain, 41BB intracellular domain and CD3z signaling domain, CD22 targeting CAR (CD22CAR) is constructed.Const CCR (Const CCR) is constructed using the intracellular domain of IL-2RB and a common γ chain and is expressed in a bicistronic vector.Drug-regulated CCR (Reg CCR) is constructed by integrating the 4b5a HCV cleavage site between the transmembrane domain and the common γ chain intracellular domain.HCV NS3 protease is co-expressed in a tricistronic vector.

[0210] Fig.13 B is the tracking of T cells expressing Antares luciferase in Fig.12 A series of bioluminescent images of the growth in mice shown in B. On day 0, NSG mice were infused with 1x10^6Nalm6 and then treated with the indicated CAR-T cells. Four days later, 4X10^6 CAR-T or control cells were implanted by tail vein injection. On the 17th day, mice were re-stimulated with 4.6x10^6Nalm6 tumor cells. Mice in the "+GPV" group were also implanted with an osmotic drug pump containing 54mg / mL grazoprevir and 0.6mg / mL ritonavir (Azlet model 2002). These mice were additionally administered 50mg / kg grazoprevir and 25mg / kg ritonavir by oral gavage 1-2 times a day. Mock untransduced T cells were used as negative controls.

[0211] exist Fig.13 C shows the display Fig.13 Quantification of the bioluminescent images shown in B. As can be seen from the data, mice that received CD22 CAR-T cells co-transduced with regulated IL-2CCR (Reg CCR) and administered grazoprevir expanded and persisted to a greater extent than control CD22 CAR-T cells lacking regulated CCR. Mice that received CD22 CAR-T cells co-expressing constitutive IL-2CCR (Const CCR) had rapid, uncontrolled expansion of CAR-T cells, which led to fatal treatment-related toxicity.

[0212] exist Fig.14 Here we show that CAR-T cells expressing drug-regulated IL-2CCR from two separate vectors had enhanced expansion in vivo in the presence of grazoprevir. Fig.14A is a schematic diagram showing the constructs used in this experiment. The two chains of the regulated IL-2CCR are expressed on separate vectors, with the IL-2RB component co-expressed with the CD22CAR bicistronic. The common γ chain component containing the 4b5A HCV cleavage site is co-expressed with the HCV NS3 protease on a separate vector.

[0213] exist Fig.14 A series of bioluminescent images tracking the growth of T cells expressing Antares luciferase are shown in B. On day 0, NSG mice were infused with 1x10^6Nalm6 and then treated with the indicated CAR-T cells. Four days later, 4X10^6 CAR-Ts were implanted by tail vein injection. On day 17, mice were re-stimulated with 4.6x10^6Nalm6 tumor cells. Mice in the "+GPV" group were also implanted with osmotic drug pumps containing 54mg / mL grazoprevir and 0.6mg / mL ritonavir (Azletmodel 2002). These mice were additionally administered 50mg / kg grazoprevir and 25mg / kg ritonavir by oral gavage 1-2 times a day.

[0214] Fig.14 C is shown Fig.14 Graph of quantification of the bioluminescence images shown in B. As can be seen from the data, mice that received CD22 CAR-T cells co-expressing drug-regulated IL-2CCR (Reg CCR) and were administered grazoprevir (+GPV) expanded to a higher extent in vivo compared to mice that did not receive GPV.

[0215] exist Fig.15 In the figure, it is shown that the leucine zipper domain enhances the STAT5 signaling in IL-2CCR. A flow chart is shown, which shows the phosphorylation level of STAT5 from primary human T cells transduced with IL-2CCR containing (leucine zipper IL-2CCR) or without (CD8a H / Tm IL-2CCR) leucine zipper components. Mock untransduced T cells exposed to exogenously added IL-2 are used as positive controls for pSTAT5, while those T cells not exposed to IL-2 are used as negative controls. As can be seen from the data, the inclusion of leucine zipper components in IL-2CCR leads to higher levels of phosphorylated STAT5.

[0216] exist Fig.16In the figure, the expression of IL-21CCR in primary human T cells was shown to cause the high phosphorylation level of STAT3. The flow chart of IL-21CCR is shown, and it shows the phosphorylation (pSTAT3) of STAT3, and pSTAT3 is the cytokine signal transduction molecule in the downstream of IL-21R signal transduction. The T cell that the Mock that is exposed to the IL-21 of exogenous addition is not transduced is used as the positive control of pSTAT3, and those T cells that are not exposed to IL-21 are used as negative control. As can be seen from the data, the expression of IL-21CCR in primary human T cells causes the high phosphorylation level of STAT3.

[0217] Example 4 - IL-7R and IL-9R CCR

[0218] In this example the development of additional types of CCRs is described, in particular CCRs based on the IL-7R and IL-9R signaling domains (ICDs).

[0219] exist Fig.17 Flowchart 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 is shown in . "On" and "Off" represent cell culture in the presence or absence of 3 μM grazoprevir (HCV NS3 protease inhibitor). The receptor regulated by HCV NS3 protease contains the HCV 4b5a cleavage site between the transmembrane domain and the γ chain ICD. These data show the constitutive signaling of IL-2R, IL-9R and IL-7R CCR, as determined by high levels of pSTAT5 and / or pSTAT3. In contrast, the chimeric receptors Reg.IL-2R and Reg.IL-9R regulated by HCV NS3 protease only express high levels of pSTAT5 and / or pSTAT3 when exposed to the HCV NS3 protease inhibitor grazoprevir. Reg.IL-7R failed to induce pSTAT5 and / or pSTAT3 signaling even in the presence of grazoprevir, indicating that this specific HCV NS3 protease-regulated chimeric receptor embodiment is incompatible with the IL-7R ICD.Phosphoflow analysis was performed 48 hours after removal of IL-2 from the culture medium.

[0220] Fig.18 Data are presented showing that constitutive IL-2, IL-9, and IL-7 CCR induce lethal toxicity in mice due to unregulated growth of CAR-T cells. Fig.18A is a series of bioluminescent images tracking the growth of T cells expressing Antares luciferase. On day 0, NSG mice were infused with 1x10^6Nalm6. Four days later, 3X10^6 indicated CAR+CCR T cells were implanted by tail vein injection. On day 11, mice were re-stimulated with 5x10^6 Nalm6 tumor cells. Fig.18 B is quantification Fig.18 A graph of bioluminescence from the image. Fig.18 C is Fig.17 Survival curves of mice shown in A. As can be seen from the data, mice receiving constitutive IL2CCR, IL-9CCR, and IL-7CCR experienced lethal toxicity due to the unregulated growth of CAR-T cells.

[0221] exist Fig.19 Data demonstrating controlled growth of CAR-T cells expressing protease-regulated CCRs are shown in . Fig.19 A is a series of bioluminescent images tracking the growth of T cells expressing Antares luciferase. On day 0, NSG mice were infused with 1x10^6Nalm6. Four days later, 3X10^6 indicated CAR+CCR T cells were implanted by tail vein injection. On day 11, the mice were re-stimulated with 5x10^6 Nalm6 tumor cells. Mice in the "on" group were also implanted with an osmotic drug pump (Azlet model2002) containing 54mg / mL grazoprevir and 0.6mg / mL ritonavir. These mice were additionally administered 50mg / kg grazoprevir and 25mg / kg ritonavir by oral gavage once a day. One component of the regulated CCR is delivered on a CD22.BBz CAR vector (i.e., 22-IL-2RB or 22-IL-9R), while a second vector delivers an adjustable γ chain (Reg gc) component and HCV NS3 protease, such as Fig.13 As shown in A. As can be seen from the data, GPV can drive the growth of T cells expressing regulatable CCRs. Even in the absence of GPV, the regulatable IL-9 CCR showed significant growth, which may be due to the signaling ability of the IL-9R ICD even in the absence of the cognate γ chain ICD. Fig. 20 As schematically shown in FIG, a further iteration of the regulated IL-9CCR can employ dual cleavage of the IL-9CRICD in addition to cleavage of the γ chain ICD to reduce this “leakage” activity.

[0222] Example 5 - Regulatable CCR with a protease fused to one of the CCR subunits

[0223] In this example the development of a regulatable CCR comprising a protease fused to one of the CCR subunits is described.

[0224] Fig.21 A is a schematic diagram of a Reg CCR with a protease directly fused to the ICD2 system. The protease cleavage site is integrated between the transmembrane domain and the intracellular signaling domain 2 (ICD2). The protease is integrated between the c-terminal of one of the chains / subunits of ICD2 and CCR. Due to the proteolytic activity of the protease at the cleavage site, the expression of the protease as a part of the subunit causes the cleavage of CCR, thereby causing the inactivation of CCR (receptor closure). Adding a protease inhibitor (drug) suppresses this cleavage event and makes CCR in an open state (receptor opening). Therefore, the signal transduction of CCR becomes dependent on the presence of drugs. It is expected that adjustable CCR avoids the toxicity associated with constitutive CCR by regulating the proliferation of cells to fall into the treatment window.

[0225] exist Fig.21 A flow chart showing staining of primary human T cells expressing HCV NS3 protease-regulated IL2CCR T cells is shown in B. "+GPV" and "-GPV" indicate cell culture in the presence or absence of 3 μM grazoprevir (HCV NS3 protease inhibitor), respectively. The HCV NS3 protease-regulated receptor contains the HCV 4b5a cleavage site between the transmembrane domain and the γ chain ICD. The HCV NS3 protease is delivered to the γ chain ICD as a direct fusion via a short linker (cis-protease short L), delivered to the γ chain ICD as a direct fusion via a long linker (cis-protease long L), or in the initial approach, delivered via HCV NS3 protease delivered on a separate transmembrane protein (e.g. Figure 7 These data suggest that the HCV NS3 protease-regulated chimeric receptor can be regulated with 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). Phosphorylation flow analysis was performed 48 hours after removal of IL-2 from the culture medium.

[0226] Example 6 - T cells co-expressing CD22 CAR and CCR exhibited Similar levels of CAR expression

[0227] In this example, it is demonstrated that T cells co-expressing CD22 CAR and CCR exhibit similar levels of CAR expression compared to T cells expressing CD22 CAR alone.

[0228] Data in Fig. 22Provided in. A flow chart showing the surface expression levels of CD22.BBzCAR from primary human T cells transduced with various CCRs is shown. The data show that all configurations result in similar levels of surface CAR expression. "Constit." and "Reg." refer to constitutive CCR and regulatable CCR, respectively.

[0229] Example 7 - CCR enhances the proliferation and anti-tumor efficacy of CAR T cells

[0230] NSG mice were inoculated with Nalm6 leukemia and then treated with CD22.BBzCAR-T cells engineered with various IL2 CCR constructs ( Fig.23 ). GPV was administered to 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 CCR lacking ICD (ΔICD) were used as controls. A: Quantification of tumor progression, B: Quantification of T cell expansion.

[0231] 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 ( Fig.24 ). Mock untransduced T cells and conventional CD22.BBz CAR-T cells were used as controls. A: Quantification of tumor progression, B: Quantification of T cell expansion.

[0232] The data suggest that CCR enhances the proliferation and anti-tumor efficacy of CAR T cells.

[0233] Example 8 - In vitro drug control of cell expansion of CD22.BBz CAR-T cells

[0234] In this example, drug control of cell expansion of CAR-T cells was evaluated. CD22.BBz CAR-T cells were cultured in medium lacking IL2. Fig.25 As shown in , CD22 CAR-T cells with IL2 CCR controlled by GPV had enhanced proliferation in vitro, and a significant >40-fold expansion of Reg IL2 CCR CAR-T cells was observed in the presence of GPV compared to cells grown in the absence of GPV.

[0235] Example 9 - Regulatable expression of CCR when cultured in medium lacking exogenous cytokines HER2CAR-T cells express high levels of pSTAT5

[0236] In this example, in vitro drug control of pSTAT5 levels in HER2.BBz CAR-T cells is demonstrated. HER2.BBz CAR-T cells were cultured in a medium lacking IL2. Fig.26As shown in , HER2 CAR-T cells engineered with various constitutive CCRs and regulatable CCRs expressed high levels of pSTAT5 when cultured in medium lacking exogenous cytokines.

[0237] Example 11 - CAR T cells lacking the membrane-associated IL2RB ICD show increased expression of the common γ-chain ICD in the absence of CAR T cells expressing membrane-associated IL2RB ICD exhibit enhanced anti-tumor efficacy

[0238] In this example, the anti-tumor efficacy of CAR T cells expressing membrane-associated IL2RB ICD in the absence of a common γ-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 were used as controls. The data show quantification of tumor progression ( Fig. 27 ). The data unexpectedly demonstrated that CAR T cells expressing a membrane-associated IL2RB ICD in the absence of a common γ-chain ICD exhibited enhanced anti-tumor efficacy compared to CAR T cells lacking a membrane-associated IL2RB ICD.

[0239] Example 12 - In vitro expression and in vivo activity of CAR-T cells engineered with non-γ chain CCRs

[0240] In this example, the in vitro expression and in vivo activity of CAR-T cells engineered with non-γ chain CCRs (IL18 CCR in this example as a proof of concept) were evaluated. Fig.28 A is a flow chart showing cell surface expression of CCRs with IL18RA and IL18RB ICDs. Fig.28 As shown in B, NSG mice were inoculated with Nalm6 leukemia and then treated with CD22.BBz CAR-T cells engineered with regulated IL18CCR constructs or constitutive IL18CCR constructs. For the regulated IL18CCR group, GPV was administered to mice every day (+GPV) or not at all (-GPV). Mock untransduced T cells and CD22.BBz CAR-T cells engineered with CCR (ΔICD) lacking ICD were used as controls. Data show quantification of tumor progression.

[0241] method

[0242] Construction of retroviral plasmid vector

[0243] DNA sequences were synthesized as oligonucleotides or gBlocks (Integrated DNA Technologies) In-Fusion cloning (TakaraBio) was used to clone DNA sequences into MSGV1 retroviral vectors. The products of the In-Fusion reaction were transformed into chemically competent cells (Stellar Cell, Takara Bio) using a heat shock method. The resulting colonies were sequence verified using Sanger sequencing. Bacterial cultures were grown in LB-ampicillin medium for 16 hours. Subsequently, plasmid DNA was extracted from bacterial cultures using a miniprep kit (QIAprep Spin Miniprep Kit, Qiagen).

[0244] Isolation of primary human T cells from blood donors

[0245] 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.

[0246] Retroviral production

[0247] 293GP packaging cells and RD114 envelope plasmids were used to prepare retroviral supernatant RD114. Briefly, 11 μg of RD114 and 22 μg of the corresponding MSGV1 transfer plasmid were mixed in Lipofectamine 2000 reagent (Thermo Fisher) and used to transfect 293GP cells grown to approximately 80% confluence on poly-D-lysine cell culture dishes (Corning). 293GP cells were cultured at 37°C in 5% CO 2 The cells were cultured in 293GP medium (DMEM, 10% FBS, 2 mM L-glutamine, 10 mM HEPES, and 100 μg / mL streptomycin, 100 U / mL penicillin, Gibco) in an environment. The medium was replenished every 24 hours. After 48 hours and 72 hours after transfection, the retroviral supernatant was harvested, centrifuged to remove dead cells and debris, and then stored at -80°C until further use.

[0248] Retroviral transduction of T cells

[0249] On day 0, primary human T cells were thawed and activated using anti-CD3 / CD28 human T-ExpanderDynabeads (Thermo Fisher) at a 3:1 bead to cell ratio. Two days later, the retroviral supernatant was spun onto a 12-well or 24-well cell culture plate that had been pre-coated with RetroNectin (Takara Bio) according to the manufacturer's instructions. Approximately 0.1 mL to 1 mL of retroviral supernatant diluted in DMEM was added to the retronectin plate and centrifuged at 3200 RPM, 32°C for about two hours. The supernatant was then aspirated from the wells. Subsequently, 0.25-0.5x10 6 T cells prepared at a concentration of 10 cells / mL were added to 1 mL of T cell culture medium composed 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 gently rotated at 1200 RPM for 2 min and then incubated at 37°C, 5% CO 2 Incubate overnight at 4 °C. Repeat the transduction process the next day. On day 4, remove the Dynabeads by magnetic separation. 6 Cells are cultured at a concentration of 10 cells / mL and expanded for up to several weeks.

[0250] In vitro cell proliferation assay

[0251] To determine whether CCR-transduced T cells can 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, IL15, etc.) Cell number and viability were determined using a trypan blue exclusion viability test and a Cellometer Auto 1000 cell counter (Nexcelom Biosciences).

[0252] Antibodies and flow cytometry

[0253] The following antibody was used to stain the cells: Brilliant Violet 421 TM Anti-DYKDDDDK tag antibody (Biolegend), anti-HA.11 epitope tag antibody (BioLegend), Alexa 647 mouse anti-Stat5 (pY694) (BD) and PE mouse anti-Stat3 (pY705) clone 4 / P-STAT3 (BD). Recombinant CD22-Fc fluorescently labeled with DyLight 650 micro-antibody labeling kit (Thermo Fisher) was used for CAR detection. Cells were stained according to the instructions of the antibody manufacturer. Flow cytometry samples were run using a BD Fortessa instrument. Data were analyzed using FlowJo software (Tree Star). Intracellular phosphorylation flow cytometry (phospho-flow cytometry) was performed using a methanol-modified Life Technologies Fix&perm kit (Thermo Fisher Scientific) to assess the phosphorylation levels of STAT3 and STAT5.

[0254] Preparation and administration of grazoprevir

[0255] For in vitro experiments, grazoprevir (Acme Bioscience, custom synthesis) was reconstituted to 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% PEG 300 (Rigaku) ​​by incubation in a 37°C water bath and vortexing. Similarly, ritonavir (ACROS Organic), used as a pharmacokinetic enhancer, was 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) PEG 300, 5% (w / v) sucrose (Sigma), and 5% (v / v) DPBS (Gibco)) and delivered to mice by oral gavage in a total volume of 100 μL per dose using a 20 gauge 30 mm gavage tube (Instech).

[0256] Nalm6 leukemia mouse model

[0257] 1 × 10 NSG cells were implanted into 6- to 10-week-old male or female NSG mice. 6 Nalm6 leukemia cells were transduced with firefly luciferase to monitor tumor progression. Four days later, 4X10^6 CAR-T or control cells were implanted via tail vein injection. T cells were transduced with Antares luciferase to allow in vivo monitoring. On day 17, mice were re-challenged with 4.6x10^6 Nalm6 tumor cells.

[0258] 143B Osteosarcoma Mouse Model

[0259] Male or female NSG mice aged 6 to 10 weeks were implanted with 1x10 6 143B cells were injected into the tibia periosteum. Four days later, mice were treated with 3X10^6 human T cells expressing the indicated transgenes via tail vein injection. Caliper measurements were used to monitor tumor progression. T cells were transduced with Antares luciferase to allow in vivo monitoring.

[0260] In vivo bioluminescence imaging

[0261] Firefly luciferase assay is used to monitor tumor progression. 200 μ L of 15 μ g / Ml D-luciferin (D-luciferin) is applied to mice to detect tumor cells expressing firefly luciferase. Antares luciferase assay is used to monitor T cell expansion. Nano-Glo substrate (Promega, diluted in DPBS) diluted 1:40 of 200 μ L is applied to mice by intraperitoneal injection to detect T cells expressing Antares luciferase. Images are collected using IVIS imaging system using 30 seconds exposure and medium binning for 5 min after injection. If the pixel is saturated in the image, other images are obtained using automatic exposure settings. Living Image software is used to calculate the total flux around the region of interest around each mouse body. Unsaturated images are used to quantify BLI images. The BLI images in the same experiment are set to the same scale.

[0262] Therefore, the above only illustrates the principle of the present disclosure.It should be understood that those skilled in the art will be able to design various arrangements, which, although not explicitly described or shown in this article, embody the principle of the present invention and are included in its spirit and scope.In addition, all examples and conditional language narrated in this article are mainly intended to help readers understand the principles of the present invention and the concepts contributed by the inventor to promote this area, and should be interpreted as not being limited to such specific examples and conditions of narration.In addition, all statements of the principles, aspects and embodiments of the present invention and their specific examples are narrated herein and are intended to cover both their structural equivalents and functional equivalents.In addition, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e. any element of the performance of the same function developed, regardless of the 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 said chimeric cytokine receptor, wherein: The first subunit comprises a first heterodimerization domain and a first cytokine receptor intracellular signaling domain (ICD); and The second subunit comprises a second heterodimerization domain homologous to the first heterodimerization domain and a second cytokine receptor ICD.

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

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

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

6. One or more nucleic acids according to claim 5, wherein the heterologous transmembrane domains of the first subunit and the second subunit 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. According to one or more nucleic acids described in any one of claims 1 to 4, wherein the first subunit and the second subunit comprise a transmembrane domain independently selected from the following: 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 domain.

8. One or more nucleic acids according to any one of claims 1 to 7, wherein the first cytokine receptor ICD, the second cytokine receptor ICD, or both comprise IL-2RβICD, IL-3RαICD, IL-4R ICD, IL-5RαICD, IL-6RαICD, IL-7RICD, IL-9RICD, IL-10RαICD, IL-10RβICD, IL-11RαICD, IL-12Rβ1ICD, IL-12Rβ2ICD, IL-13Rα1ICD, IL-13Rα2ICD, IL-20RαICD, IL-20Rβ, IL-21RICD, IL-22Rα1, IL-22Rα2, IL-23RICD, IL-27RαICD, IL-28RAICD, IL-31RαICD, TGFβR1 ICD, TGFβR2 ICD, TGFβR3 ICD, IFNAR1 ICD, IFNAR2 ICD, IFNGR1 ICD, IFNGR2 ICD, IFNLRICD, CSF2RB ICD, gp130 ICD, CD9 ICD, OSMR ICD, CSF-1R ICD, or any combination thereof.

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

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

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

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. One or more nucleic acids according to claim 12, wherein the transition domain comprises the juxtamembrane (JM) region of a cell surface receptor.

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

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

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

18. The one or more nucleic acids of 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. 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 disposed between the transmembrane domain and the ICD.

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

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. One or more nucleic acids according to 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 can be cleaved by the viral protease.

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

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

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

26. One or more nucleic acids according to 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. 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, wherein the protease cleavage site is a cleavage site for the protease.

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

30. The one or more nucleic acids of claim 27, wherein the one or more nucleic acids encode: a protease fused to the first subunit; a protease fused to the second subunit; or A protease fused to the first subunit and a protease fused to the second subunit.

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

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

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

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

35. The one or more nucleic acids of claim 33, wherein the protease is HIV protease, and wherein 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. The one or more nucleic acids of claim 33, wherein the protease is SARS-CoV-2 protease, and wherein the inhibitor of the protease is Paxlovid.

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

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

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

41. according to one or more nucleic acids described in any one of claims 1 to 40, wherein the first subunit and the second subunit each comprise an extracellular cysteine ​​residue, wherein when expressed on the surface of a cell, 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.

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

43. One or more expression constructs comprising one or more nucleic acids according to 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 the polypeptide does not comprise an extracellular cytokine binding domain.

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

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

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

49. A nucleic acid according to claim 47 or 48, wherein the heterodimerization domain is as defined in claim 3 or 4.

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

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 according to any one of claims 44 to 51.

53. An expression construct comprising the nucleic acid according to 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. A cell comprising one or more expression constructs according to claim 43.

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

58. The cell of claim 57, wherein the cell does not express a cytokine receptor subunit comprising a 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. The cell of claim 60, wherein the immune cell is a T cell.

62. A cell according to any one of claims 54 to 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 tactile receptor, a ChaCha receptor, a generalized extracellular molecule sensor (GEMS) receptor, a cytokine receptor, a chemokine receptor, a switch receptor, an adhesion molecule, an integrin, an inhibitory receptor, a stimulatory receptor, a receptor containing an immunoreceptor tyrosine-based activation motif (ITAM), or a receptor containing an immunoreceptor tyrosine-based inhibitory motif (ITIM).

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

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

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 a composition according to claim 64, wherein the cell expresses: a chimeric cytokine receptor comprising a first subunit and a second subunit encoded by the one or more nucleic acids; or a membrane-associated polypeptide encoded by the nucleic acid.

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

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

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

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

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

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

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

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

74. The method of claim 70 or claim 71, wherein the protease is HIV protease, and wherein 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. The method of claim 70 or claim 71, wherein the protease is SARS-CoV-2 protease, and wherein the inhibitor of the protease is Paxlovid.

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

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

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

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

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

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

Images