Constitutive Cytokine Receptors

Abstract

Recombinant proteins are provided that include an exodomain derived at least in part from the extracellular region of EPOR, the exodomain including a dimerization domain that allows the recombinant protein to dimerize with a second protein and provide a signal to the T cell in the absence of a signal inducer molecule. Also provided are nucleic acid molecules encoding such recombinant proteins, recombinant constructs, vectors, and cells containing the nucleic acid molecules, methods of making such cells, and their uses for therapeutic purposes.

Description

[Technical field]

[0001] Field The present disclosure and invention relates to recombinant proteins and their use in adoptive cell therapy (ACT). In particular, recombinant proteins can provide desired constitutive signals, such as constitutive STAT-mediated signals, to cells expressing the proteins. The signals can confer desired effects or properties to cells, such as increased function, activity, vitality, or survival (e.g., persistence in transplanted host subjects). Also provided are nucleic acid molecules encoding such recombinant proteins, recombinant constructs, vectors, and cells containing the nucleic acid molecules, methods for making such cells, and their use for therapeutic purposes.

[0002] background Adoptive cell therapy (ACT), which involves the administration of functional immune cells to a subject, has become an established and evolving immunotherapeutic approach for a variety of medical conditions, including malignant and infectious diseases, among others. Initially, tumor-infiltrating lymphocytes were shown to be effective in the treatment of metastatic melanoma, and subsequently, redirected T cells or NK cells expressing chimeric antigen receptors (CARs) or xenogeneic T cell receptors (TCRs) to target various cellular target molecules have been developed and adopted for clinical use. Initial approaches used immune cells with cytotoxic properties, e.g., cytotoxic T cells or NK cells, to target and kill unwanted or harmful cells in the body, but more recently, regulatory T cells (Tregs) have been developed for ACT. Tregs have an immunosuppressive function. Tregs act to control cytopathic immune responses and are essential for the maintenance of immune tolerance. The suppressive properties of Tregs can be exploited for therapeutic purposes, e.g., to ameliorate and / or prevent immune-mediated organ damage in inflammatory disorders, autoimmune diseases, transplants, etc.

[0003] To be useful in ACT, the transplanted or administered cells must survive and persist in the recipient (the subject of the ACT treatment) in a functional state for a period of time sufficient to exert a useful therapeutic effect. Furthermore, the cells must be generated (e.g., engineered), cultured, and expanded in vitro to prepare sufficient numbers of cells for therapeutic use.

[0004] The growth factor interleukin-2 (IL-2) is essential for the homeostasis (development, proliferation, and survival) of immune cells, especially Tregs, as well as for their suppressive function and phenotypic stability. Activated conventional T cells (Tcon) are the major source of IL-2 in vivo. On the other hand, Tregs cannot produce IL-2 and rely on paracrine access to IL-2 produced by Tcon in the microenvironment.

[0005] The acquisition of IL-2 has a significant impact on the therapeutic efficacy of Tregs expanded in vitro and transplanted into patients. This is because 1) in vitro expansion protocols generally require high concentrations of IL-2, making Tregs highly dependent on this cytokine, 2) immunosuppressant administration often results in reduced IL-2 concentrations in patients, and 3) IL-2 is often only accessible to the inflamed tissue microenvironment. Liver transplantation presents a particularly challenging challenge, given that IL-2 levels are known to be reduced in inflamed livers, further exacerbated by chronic use of calcineurin inhibitors, which substantially reduces the ability of Tcon to produce IL-2. The administration of low doses of exogenous IL-2 reverses calcineurin inhibitor-induced Treg dysfunction and promotes Treg accumulation in the liver. However, the therapeutic use of low doses of IL-2 raises concerns about the risk of concomitant activation of Tcon, which may promote tissue damage.

[0006] WO2020 / 044055 describes an approach to avoid the need for exogenous IL-2 administration. In this case, Treg cells are engineered to express a modified CAR that can provide a productive IL-2 signal to the cell upon binding to a target antigen. In other words, the endodomain, the intracellular signaling domain of the CAR, contains sequences or domains derived from the IL receptor, which allow it to transmit an "IL-2 signal" in the absence of endogenous IL-2 and without the need for IL-2 binding. IL-2 transmits signals through the transcription factor STAT5 (signal transducer and activator of transcription 5). STAT5 is phosphorylated in the active state by the kinases JAK1 and / or JAK2, which are normally activated upon binding of interleukins (e.g., IL-2) to the receptor. Thus, the CAR in WO2020 / 044055 contains an endodomain that contains a STAT5 association motif and a JAK1- and / or JAK2-binding motif.

[0007] Similarly, other immune cells for ACT, such as cytotoxic T cells and other T effector cells, including CAR-T cells, may require or benefit from being provided with additional signaling capabilities to enhance survival or persistence of function. Thus, the need for additional, and more specifically engineered, signaling, whether to enhance the survival or persistence of cells for ACT or to improve their functional activity or therapeutic efficacy, is not limited to Treg cells.

[0008] While WO2020 / 04405 provided important advances, the field of ACT continues to need new and improved approaches, particularly more universally applicable approaches that avoid or reduce the need to develop targeted, engineered CARs.

[0009] Erythropoietin (EPO) is a glycoprotein made by the fetal liver or, in adults, by perivascular stromal fibroblasts in the kidney. In vivo, EPO production is primarily stimulated by hypoxia, which leads to stabilization of the hypoxia-inducible factor (HIF)-1α transcription factor and subsequent transcription of EPO. EPO induces erythropoiesis by binding to the erythropoietin receptor (EPOR) on erythroid precursor cells, but is also expressed in non-hematopoietic tissues. EPOR is a 508 amino acid transmembrane receptor that contains an extracellular domain containing a WSXWS motif, one transmembrane hydrophobic region, and a cytoplasmic domain. Upon binding to EPO, EPOR can form homodimers and signal through activation of JAK2, MAPK, and PI3 kinases and phosphorylation of STAT5. It has also been suggested in the literature that EPOR may be capable of heterodimerization with CD131.

[0010] WO2019 / 169290 describes an approach for inducibly introducing a signal into a cell using a chimeric receptor that contains a dimerization domain capable of dimerization in the presence of a drug. Some constructs disclosed in WO2019 / 169290 contained a domain derived from EPOR or a domain derived from IL2RB, but signaling was only induced in the presence of a ligand. Inducibility allows for control of signaling in cells and may be useful in certain disease states, but under conditions where the ligand is absent or present at low levels, signaling from such constructs may be reduced, resulting in a lack of cell persistence. Thus, there is a need to develop cytokine receptors that can provide a constitutive signal to cells, useful under conditions where inducibility is not possible and / or is undesirable.

[0011] overview The recombinant protein identified by the inventors is capable of homodimerization in the absence of ligand and of providing a constitutive signal to cells expressing the recombinant protein. In particular, the recombinant protein has an exodomain derived from a modified extracellular domain of a cytokine receptor, which modification promotes homodimerization and constitutive signaling in the absence of a natural ligand for the cytokine receptor. The recombinant protein may in particular further comprise an endodomain and can be used in some cell types to provide a consistent signal, such as a STAT signal, that allows cells to survive in environments where cytokine receptor ligands are or may be limited, particularly in the context of ACT. In such environments, the use of inducible proteins to provide survival signals to cells may not be appropriate, and the present invention has particular utility here and addresses this particular problem.

[0012] In particular, the recombinant protein identified for use by the inventors may include an exodomain derived from the extracellular region of EPOR, but may further include modifications that allow constitutive signaling through the receptor in the absence of EPO. The extracellular region may also retain the ability to bind EPO when EPO is present in the environment, potentially increasing signaling through the modified cytokine receptor. Thus, advantageously, the recombinant protein may be capable of dimerizing to provide a constitutive signal to the cell, and may also be capable of binding to environmental ligands when available, providing the potential for enhanced signaling. However, constitutive signaling in the absence of ligand is sufficient to provide cells expressing the recombinant protein with the desired functional / survival advantage. In one embodiment, the recombinant protein may include a modified human extracellular region of EPOR with an amino acid substitution of residue 154 to cysteine, which promotes homodimerization of the monomeric recombinant protein chain and constitutive signaling in the absence of EPO.

[0013] Although a recombinant protein for use in the present invention may provide any signal to a cell expressing the recombinant protein, it is particularly envisaged that the recombinant protein may be utilized, particularly in the context of cell therapy, to allow cells to survive or persist after administration to a subject, providing the best chance for a treatment to be effective in a diseased subject. In particular, signal transduction via the recombinant protein involves tyrosine kinase activity and protein phosphorylation, more particularly involving the phosphorylation and activity of Janus kinases (JAK), such as activation of the JAK-STAT signal transduction pathway involving JAK1 and / or JAK2. Thus, in particular, the endodomain of the recombinant protein described herein may be derived or partially derived from the same protein as the exodomain (EPOR, which contains a JAK2 binding motif and a STAT5 association motif), or may be derived from one or more different proteins (the recombinant protein may be a chimeric recombinant protein), which may allow the same or similar signal transduction as EPOR. Or, it may allow the provision of a different cellular signal than the natural EPOR receptor. Advantageously, the inventors further confirmed that when the endodomain of a recombinant protein is derived from EPOR, it is possible to utilize a modified endodomain with reduced binding ability to SHP1, which when activated can inhibit phosphorylation of JAK2, as well as having the ability to affect other endogenous receptors, such as IL2R. The inventors further confirmed that when the endodomain of a recombinant protein is derived from EPOR, inserting an amino acid sequence, such as a cytoplasmic tail, at the C-terminus of the portion of said endodomain derived from the endodomain of EPOR can increase or stabilize the cell surface expression of the recombinant protein and / or increase its sensitivity to EPO.

[0014] The recombinant proteins described herein are particularly useful in T cells that may be required to persist in a subject for a period of time after administration, and more specifically in Tregs that require STAT5 signaling to survive (usually via endogenous IL2R) and are entirely dependent on the supply of exogenous ligands to induce such signaling. Given the constitutive signaling capacity of the receptor, Tregs' requirement for constant STAT5 signaling, and the lack of naturally available ligands (e.g., IL2) in certain disease states (e.g., type I diabetes), the use of recombinant proteins in Tregs is particularly advantageous. The inventors have advantageously shown that Tregs expressing the recombinant proteins described herein maintain their normal phenotype and suppressive function, so that cellular stability can be preserved.

[0015] Thus, the invention provides a T cell comprising a recombinant protein, the recombinant protein comprising an exodomain derived at least in part from the extracellular region of EPOR, the exodomain comprising a dimerization domain that enables the recombinant protein to dimerize with a second protein and provide a signal to the T cell in the absence of a signal inducer molecule.

[0016] The dimerization domain may be any dimerization domain that allows the recombinant molecule to bind to a second protein in the absence of a signal inducer molecule and transmit a signal to the cell. Thus, the dimerization domain allows for the provision of a constitutive signal to the cell. The dimerization domain may be outside (i.e., at a different location in the exodomain than) the portion of the exodomain derived from the extracellular region of the EPOR, or may in particular be included within the portion of the exodomain derived from the extracellular region of the EPOR.

[0017] Thus, in this regard, the invention provides a T cell comprising a recombinant protein, the recombinant protein comprising an exodomain derived at least in part from the extracellular region of EPOR, the portion comprising a modification to the extracellular region of EPOR that enables the recombinant protein to dimerize with a second protein in the absence of a signal inducer molecule and provide a signal to the T cell. Thus, a recombinant protein present in a T cell may be expressed within the T cell from a nucleic acid molecule transduced into the cell or a precursor cell, or may be expressed from an endogenous nucleic acid sequence that has been modified using gene editing techniques.

[0018] In certain embodiments, the recombinant protein comprises an exodomain that allows disulfide bond dimerization of the recombinant protein with a second protein. Thus, the recombinant protein comprises at least one dimerization domain that specifically allows disulfide bond dimerization.

[0019] It will be further understood by those skilled in the art that dimerization may be homodimerization, in which a recombinant protein binds to another recombinant protein as described herein, or heterodimerization, in which a recombinant protein dimerizes with a different protein. It will be understood that for heterodimerization to occur, the second protein must contain a cognate dimerization domain that can bind to the dimerization domain present in the recombinant protein. It is typically the dimerization of the recombinant protein that allows the production of a signal to the T cell. In a particular embodiment of the present invention, the dimerization is homodimerization, and in this respect, the present invention provides a T cell comprising a recombinant protein, the recombinant protein comprising an exodomain derived at least in part from the extracellular region of EPOR, the exodomain comprising a dimerization domain that allows the recombinant protein to homodimerize in the absence of a signal inducer molecule and provide a signal to the T cell. In particular, as described above, at least a portion of the exodomain derived from the extracellular region of EPOR may contain modifications to the extracellular region of EPOR that enable homodimerization of the recombinant protein in the absence of a signal inducer and the provision of a signal to the T cell.

[0020] Dimerization of the recombinant proteins occurs spontaneously in the presence of a second protein and does not require the presence of a signal inducer molecule, such as erythropoietin, and more particularly does not require the presence of any signal inducer molecule (including neither a ligand nor a dimerization inducer molecule).

[0021] In this respect, the present invention further provides a T cell comprising a recombinant protein, the recombinant protein comprising an exodomain derived at least in part from the extracellular region of EPOR, the exodomain comprising a dimerization domain that allows the recombinant protein to dimerize with a second protein and provide a signal to the T cell in the absence of EPO. In particular, as described above, at least a portion of the exodomain derived from the extracellular region of EPOR may comprise a modification relative to the extracellular region of EPOR that allows the recombinant protein to homodimerize in the absence of EPO and provide a signal to the T cell.

[0022] In particular, the present invention provides a T cell comprising a recombinant protein, the recombinant protein comprising an exodomain derived at least in part from the extracellular region of EPOR, the exodomain comprising a dimerization domain that allows the recombinant protein to homodimerize and provide a signal to the T cell in the absence of EPO. In particular, as described above, at least a portion of the exodomain derived from the extracellular region of EPOR may comprise a modification to the extracellular region of EPOR that allows the recombinant protein to homodimerize and provide a signal to the T cell in the absence of EPO.

[0023] The portion of the exodomain derived from the extracellular region of the EPOR may comprise the full length of the extracellular region of the EPOR, or a partial or variant sequence thereof. In particular, the EPOR extracellular region sequence present may comprise at least one modification that allows the recombinant protein to dimerize and provide constitutive signaling in the absence of a signal inducer molecule, particularly EPO. Thus, the at least one modification may be any modification that results in constitutive signaling of the recombinant protein. The EPOR extracellular region sequence present in the recombinant protein may comprise other modifications in addition to the at least one modification that allows the recombinant protein to dimerize and provide constitutive signaling in the absence of a signal inducer molecule, particularly EPO. In particular, the portion of the exodomain derived from the extracellular region of the EPOR may be derived from SEQ ID NO: 1, more particularly from amino acids 1 to 250 of SEQ ID NO: 1 (SEQ ID NO: 5). The at least one modification may be a mutation in the amino acid sequence of the extracellular domain of EPOR that allows dimerization, in particular disulfide bond dimerization, and / or may be the insertion of a sequence that allows dimerization in the absence of a signal inducer molecule, such as a leucine zipper. More specifically, the recombinant protein comprises an arginine to cysteine ​​modification at position 154 of SEQ ID NO:5 (R154C). In a most specific embodiment, the exodomain may comprise or consist of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.

[0024] Thus, in another embodiment, the present invention provides a T cell comprising a recombinant protein, the recombinant protein comprising an exodomain comprising the extracellular region of EPOR or a portion or variant thereof modified to enable dimerization of the recombinant protein with a second protein in the absence of a signal inducer molecule, particularly EPO, and provide a signal in the T cell.

[0025] In one embodiment, the portion or variant of the extracellular domain of EPOR may retain the function of the extracellular domain of EPOR and thus be capable of binding to EPO. In this way, it may be possible to increase the signal provided to the cell in the presence of EPO, as described above. Alternatively, or in addition, such a portion or variant may have at least 70%, 80%, or 90% identity to the extracellular domain of EPOR. In certain embodiments, the variants and portions may include at least one modification that provides a dimerization domain, which allows the recombinant protein to dimerize with a second protein and provide a signal to the expressing cell in the absence of a signal inducer molecule, such as EPO.

[0026] In a further embodiment, the present invention provides a T cell comprising a recombinant protein comprising an exodomain comprising SEQ ID NO:2 or SEQ ID NO:4, or an exodomain having at least 90% identity to SEQ ID NO:2 or SEQ ID NO:4 and comprising a cysteine ​​at position 130 of SEQ ID NO:2 or a cysteine ​​at position 154 of SEQ ID NO:4, wherein the recombinant protein is capable of dimerizing with a second protein in the absence of a signal inducer molecule, in particular EPO, and is capable of providing a signal in the T cell.

[0027] The exodomain of a recombinant protein may contain, in addition to the portion derived from the extracellular region of EPOR, additional heterologous domains or regions not involved in dimerization (i.e., non-dimerizing domains or regions not present in wild-type EPOR), such as a suicide motif or tag, and thus the exodomain may not consist solely of the portion derived from the extracellular region of EPOR.

[0028] As mentioned above, the exodomain of the recombinant protein described herein comprises at least one dimerization domain (e.g., at least two, three, four, or five) that allows dimerization and constitutive signaling in the absence of a signal inducer molecule. In particular, the dimerization domain can be introduced into a portion of the exodomain derived from the extracellular region of EPOR, for example, by adding one or more modifications (e.g., at least two, three, four, or five modifications) to the amino acid sequence. Thus, a signal inducer molecule (e.g., EPO) is not required for signaling to occur in a cell comprising the recombinant protein described herein. In particular, the recombinant protein is capable of dimerizing and providing a constitutive signal in the absence of EPO, the natural ligand of EPOR. Thus, although not required for constitutive signaling, the exodomain of the recombinant protein described herein may comprise one or more dimerization domains that allow inducible dimerization of the recombinant protein with a second protein. Such "inducible dimerization domains" require the presence of a dimerization inducer molecule in order to dimerize. In one embodiment, the presence of at least one inducible dimerization domain can allow for enhanced or increased signaling through the recombinant protein, as described in detail below. Typically, the one or more inducible dimerization domains can be heterologous to the EPOR (i.e., not present in the wild-type EPOR). Those skilled in the art will understand that the one or more inducible dimerization domains can be included anywhere in the recombinant protein, including within any endodomain or exodomain, but in particular within the exodomain, e.g., within a portion of the exodomain derived from the extracellular region of the EPOR, or within the exodomain but not within a portion derived from the extracellular region of the EPOR, e.g., as a separate domain or region.

[0029] The recombinant protein of the invention may comprise a transmembrane domain to anchor the exodomain in the cell membrane. The transmembrane domain may be derived from any protein having a transmembrane domain, including, for example, EPOR, IL2RB, CD28, CD8, etc. In one embodiment, the transmembrane domain may be associated with the transmembrane domain of another protein, for example, the transmembrane domain may be derived from a TREM protein capable of associating with DAP10 / 12.

[0030] The signal that can be provided by recombinant protein can be a signal that improves or increases the functional properties or activity of cells. Thus, the function or effect of cells can be increased, and the function or effect can be a function or effect in vitro or in vivo, i.e. during the generation or proliferation of cells in the process of preparing for ACT or after the cells are administered to a subject. This can be, for example, cell survival, cell persistence, cell function persistence, vitality, functional effect (e.g., immunosuppressive effect or cytotoxic effect), cell phenotype, including memory phenotype, proliferation ability, and / or therapeutic efficacy of cells. The increase can be observed in cells that contain recombinant protein compared to cells that do not contain recombinant protein. The above-mentioned signals are particularly constitutive signals.

[0031] In certain embodiments, the signal is a pro-survival signal, which helps the cells to survive and maintain their ability to function during and after culture, and helps the cells to persist and maintain their functional ability after being administered to a subject during the course of treatment. This may also be called a persistence signal. Thus, the recombinant protein can be expressed in cells to confer inducible pro-survival signaling ability to the cells. This protein is particularly useful in cells prepared for use in ACT therapy, and may be expressed in such cells together with an antigen receptor, such as a TCR or CAR or any chimeric receptor. Thus, this protein is useful in engineering cells for ACT.

[0032] In one embodiment, the signal is a STAT-mediated signal (e.g., a STAT3-mediated signal or a STAT5-mediated signal), more specifically, a STAT5-mediated signal, which can normally be induced in cells by an interleukin such as IL-2 or by EPO.

[0033] The recombinant protein of the present invention may in particular comprise an endodomain or may associate with a further signaling protein comprising an endodomain to provide a signal to a cell. Thus, the endodomain may be part of the recombinant protein described herein or may be comprised within a signaling protein that associates with the recombinant protein, for example, via the respective transmembrane domain, where, for example, the transmembrane domain of the recombinant protein may be derived from a myeloid receptor and the transmembrane domain of the signaling protein may be derived from DAP10 or DAP12.

[0034] In certain embodiments, the endodomain comprises at least one JAK1-binding motif and / or JAK2-binding motif and at least one STAT-association motif (e.g., STAT3-association motif and / or STAT5-association motif). In particular, the endodomain may comprise at least one JAK2-binding motif and at least one STAT5-association motif, or at least one JAK1-binding motif and at least one STAT5-association motif. The endodomain may comprise further domains, for example, at least one JAK3-binding motif.

[0035] The present invention further provides a T cell comprising a nucleic acid molecule (also referred to as a polynucleotide) comprising a base sequence encoding a recombinant protein as defined herein.

[0036] The nucleic acid molecule may be in the form of a construct, more specifically in the form of a recombinant construct comprising the nucleic acid molecule and one or more other base sequences (base sequences of interest). For example, the construct may comprise, together with the nucleic acid molecule, regulatory sequences (e.g., expression control sequences) and / or sequences encoding another functional protein (more generally, a protein of interest) (e.g., a receptor such as a CAR or TCR). If the endodomain is present in an additional signaling protein, the base sequences encoding the recombinant protein and the base sequences encoding the signaling protein may be provided in the same construct. Alternatively, separate nucleic acid molecules or constructs may be provided for the recombinant protein and the signaling protein. The construct may comprise one or more co-expression sequences linking the nucleic acid molecule with one or more other coding base sequences.

[0037] The nucleic acid molecules or constructs defined herein may be contained within a vector. If separate recombinant proteins and other expressed proteins (e.g., functional proteins or signal transduction proteins) are encoded by separate molecules or constructs, they may each be contained in a separate vector. Thus, there may be a set of vectors, each of which contains a sequence encoding a separate desired protein.

[0038] The vector may be a viral vector or a non-viral vector. In one embodiment, the vector may comprise the nucleic acid molecule defined herein and an additional base sequence that codes for a protein of interest, in particular a receptor such as a CAR or TCR.

[0039] The cells (T cells) express the recombinant protein on their cell membrane.

[0040] The present invention also provides a cell population, such as a T cell population, comprising a cell as defined herein.

[0041] In one embodiment, the cell is a T cell, including CD4+ T cell or CD8+ T cell or any precursor thereof.Accordingly, the cell may be a stem cell, more specifically a hematopoietic stem cell (HSC) or a pluripotent stem cell (PSC), such as an induced pluripotent stem cell (iPSC), i.e. a cell before differentiation or conversion into a T cell.In particular, the T cell may be a Treg cell.The cell may be a primary cell or may be derived from a cell line.

[0042] The invention further provides a method of preparing a T cell as defined herein (i.e. a cell containing a recombinant protein or a cell containing a nucleic acid molecule encoding a recombinant protein), comprising introducing into the T cell (e.g. transducing or transducing into the cell) a nucleic acid molecule, construct or vector as defined herein. The method may comprise expressing the recombinant protein in the cell. This may include, for example, culturing the cell.

[0043] Such methods may further include a preceding step of isolating, enriching, preparing, or generating cells for use in the methods.Furthermore, the isolation or enrichment or generation of cells may be performed after the nucleic acid molecule introduction step.For example, the nucleic acid molecule may be introduced into precursor or progenitor cells, such as stem cells, and then the cells may be induced or transformed into T cells.For example, iPSC cells may be differentiated into Treg cells or other T cells, and Tcon cells may be converted into Treg cells.

[0044] This embodiment may also include a method of preparing a recombinant protein as defined herein, comprising introducing a nucleic acid molecule, construct or vector as defined herein into a T cell, expressing the chimeric protein by the cell, and, optionally, detecting and / or recovering the chimeric protein.

[0045] The present invention further provides a method for preparing a T cell as defined herein, comprising genetically engineering an endogenous nucleic acid encoding EPOR to include a modification such that the expressed EPOR includes an extracellular domain that homodimerizes in the absence of EPO, thus providing a signal to the T cell. In particular, the method may be performed using T cell precursors, such as iPSCs, prior to differentiation into T cells, and more particularly, the EPOR expressed from the genetically engineered endogenous nucleic acid includes an R to C amino acid substitution at position 154.

[0046] The present invention further provides a method of promoting the survival or persistence of a cell, comprising introducing into a cell a nucleic acid molecule, construct or vector as defined herein, or genetically manipulating an endogenous nucleic acid as described above.

[0047] This embodiment may include the additional step of administering the cells defined herein to a subject, and optionally administering a signal inducer molecule (e.g., EPO, if it is desired to increase constitutive signaling) to the subject. Any inducer can be administered before, during, or after administration of the cells. Thus, in this embodiment, the method can be performed in vivo. Alternatively, the method can be performed in vitro / ex vivo.

[0048] Viewed from another aspect, the present invention provides the use of a recombinant molecule described herein to promote the survival or persistence of a cell expressing the recombinant molecule.

[0049] As mentioned above, recombinant proteins can advantageously be expressed in cells in a therapeutic context. The cells can be unmodified cells, in the sense that they have not been further genetically engineered for therapeutic use, such as T cells isolated from a subject or cells derived from such isolated cells (although of course the cells are modified by the method to express recombinant proteins), but typically the cells are cells that have been further modified or engineered to express additional molecules (i.e. additional proteins), in particular receptors such as CAR or TCR.

[0050] Thus, the present invention further provides a method for preparing a cell for use in adoptive cell transfer therapy (ACT), comprising providing the cell with a recombinant protein as defined herein. More specifically, the method may comprise introducing a nucleic acid molecule, construct, or vector as defined herein into the cell. The method may also comprise introducing a separate nucleic acid molecule, construct, or vector into the cell, for example a nucleic acid molecule, construct, or vector comprising a sequence encoding a separate (e.g., second) protein, or therapeutic protein, in particular a receptor, for example a CAR or TCR.

[0051] Furthermore, the present invention provides a pharmaceutical composition comprising a cell, a cell population, or a vector comprising a nucleic acid molecule encoding a recombinant protein as defined herein, together with at least one pharma- ceutically acceptable carrier or excipient.In one embodiment, the cell or vector comprises an additional base sequence encoding an additional protein, particularly a chimeric protein, or a receptor, such as a CAR or TCR.In another embodiment, the cell comprises a separate nucleic acid molecule, construct, or vector comprising a base sequence encoding an additional protein, particularly a chimeric protein, or a receptor, such as a CAR or TCR.

[0052] Furthermore, the present invention provides a cell or cell population comprising a nucleic acid molecule encoding a recombinant protein as defined herein, or a pharmaceutical composition as defined herein, or a vector comprising a nucleic acid molecule encoding a recombinant protein as defined herein, for use in a method of therapy. In particular, said cell, cell population, or pharmaceutical composition comprising said cell or cell population may be for ACT. Said vector or pharmaceutical composition comprising said vector may be for gene therapy. Said ACT or gene therapy may be for treating or preventing any condition that responds to ACT or gene therapy, in particular immunotherapy by ACT or gene therapy.

[0053] The present invention further provides a cell, cell population, or vector comprising a nucleic acid molecule encoding a recombinant protein as defined herein, or a pharmaceutical composition as defined herein, for use in the treatment or prevention of cancer, or an infectious, neurodegenerative, inflammatory, autoimmune, or allergic disease, or any condition associated with an unwanted or harmful immune response. In particular, when the cell is a Treg or other immunosuppressive cell, the cell may be used to induce immunosuppression (i.e., to suppress unwanted or harmful immune responses) to ameliorate and / or prevent immune-mediated organ damage, for example, in inflammatory, autoimmune, or allergic disorders or conditions, and in transplantation.

[0054] This aspect also provides a method of adoptive cell transfer therapy comprising administering to a subject in need of said therapy a cell or cell population comprising a nucleic acid molecule encoding a recombinant protein as defined herein, or a pharmaceutical composition as defined herein, in particular an effective amount of said cell, cell population, or pharmaceutical composition.

[0055] Also provided is a method for treating or preventing cancer, or an infectious, neurodegenerative, inflammatory, autoimmune or allergic disease, or a condition associated with an unwanted or harmful immune response, comprising administering to a subject in need thereof a cell, a cell population or a vector comprising a nucleic acid molecule encoding a recombinant protein as defined herein, or a pharmaceutical composition as defined herein, in particular an effective amount of said cell, cell population, vector or pharmaceutical composition.

[0056] Further provided is the use of a cell, a population of cells, or a vector comprising a nucleic acid molecule encoding a recombinant protein as defined herein in the manufacture of a medicament for use in the treatment or prevention of cancer, or an infectious, neurodegenerative, inflammatory, autoimmune, or allergic disease, or a condition associated with an unwanted or deleterious immune response.

[0057] In some embodiments of such therapeutic aspects, the use may be in inducing tolerance to transplantation; in treating and / or preventing cellular and / or humoral transplant rejection; in treating and / or preventing graft-versus-host disease (GvHD), autoimmune disease, or allergic disease; for promoting tissue repair and / or tissue regeneration; or for ameliorating inflammation. In particular, in such embodiments, the cells may be Treg cells.

[0058] In the various therapeutic aspects described above, the cells, cell populations, vectors or pharmaceutical compositions may be for use in combination or with a signal inducer molecule, particularly EPO.

[0059] Thus, a further aspect provides a combination product comprising (a) a cell, a cell population, or a vector comprising a nucleic acid molecule encoding a recombinant protein as defined herein, or a pharmaceutical composition as defined herein, and (b) a signal inducer molecule, in particular EPO, for use in a therapy, in particular an ACT therapy or gene therapy. The therapy may be any of the therapies defined above and further described herein.

[0060] Each component (a) and (b) of the above combination product may be for separate, sequential or simultaneous use. [Brief description of the drawings]

[0061] [Figure 1] Figure 1 shows wild-type mouse EPOR showing homodimerization of the EPOR chain in the presence of EPO. When the receptor is activated by EPO, JAK2 can associate with the endodomain of the receptor, resulting in phosphorylation of Y343 and recruitment and dimerization of STAT5. SHP1 can directly inhibit JAK2, providing a negative feedback loop for the control of EPOR-mediated signaling. [Figure 2A] Figure 2 shows various recombinant proteins described herein. Figure 2A shows a recombinant protein that includes an exodomain from EPOR modified to cysteine ​​at position 129 (mouse sequence numbering), a transmembrane domain of EPOR, and an endodomain of EPOR that includes Y343 (mouse sequence numbering). This recombinant protein can homodimerize and provide a constitutive signal to cells via STAT5. [Figure 2BC]Figure 2B shows a recombinant protein comprising an exodomain from EPOR modified to cysteine ​​at position 129 (mouse sequence numbering), a transmembrane domain from EPOR, and a truncated endodomain from EPOR that removes the binding site for SHP1, thereby eliminating the negative feedback of SHP1 on JAK2 and enhancing STAT5 signaling. Figure 2C shows a recombinant protein comprising an exodomain from EPOR modified to cysteine ​​at position 129 (mouse sequence numbering), a transmembrane domain from EPOR, and an endodomain that comprises a truncated endodomain from IL2RB that retains the JAK1 binding motif and the STAT5 association motif (with Y510). [Figure 3-1] Figure 3 shows the constructs tested in Example 5. The exodomain derived from the EPOR exodomain allowed the identification of cells expressing the protein using the EPOR antibody. [Figure 3-2] Figure 3 continued [Figure 4A] Figure 4A shows FACS plots showing the transduction efficiency of fresh Tregs transduced with various constructs, as measured using HLA-A2 dextramer and EPOR expression. [Figure 4B] Figure 4B shows FACS plots showing the transduction efficiency of frozen Tregs transduced with various constructs, as measured using HLA-A2 dextramer and EPOR expression. [Diagram 5] FIG. 5 shows the pSTAT5 MFI for transduced and non-transduced fractions of cells with and without EPO treatment. [Figure 6A] FIG. 6A shows the percentage of transduced cells (as determined by the presence of A2 CAR) in the cell population with and without IL2 over 6 days. [Figure 6B] FIG. 6B shows FoxP3 expression in transduced (A2 Dex+) and non-transduced (A2 Dex-) fractions of cells with and without IL2 over a 6 day period. [Figure 6C]FIG. 6C shows the fold expansion of Tregs transduced with either the 658 or 786 constructs over 5 and 7 days. [Figure 7-1] FIG. 7 shows the expression of various Treg markers in transduced and non-transduced fractions of cells as well as mock-transduced cells. [Figure 7-2] Figure 7 continued [Figure 7-3] Figure 7 continued [Figure 8-1] FIG. 8 shows the percentage suppression of Tregs against the proliferation of T effector cells using different stimuli, aCD3 / CD28 beads, HLA-A2− B cells, and HLA-A2+ B cells. [Figure 8-2] Figure 8 continued [Figure 9] FIG. 9 shows the percentage of killed target cells, indicating the cytotoxicity of Tregs transduced with the different constructs against target cells. [Figure 10] FIG. 10 shows the levels of various intracellular cytokines in mock Tregs and Tregs transduced with the 658 or 786 constructs (both transduced and non-transduced cell fractions).

[0062] Detailed Description The subject of the products, methods and uses herein is a recombinant protein that can be used to promote the functionality or survival or indeed any property of the cell in which it is expressed.Thus, this protein is useful for adoptive cell transfer, aids in the preparation of cells for ACT, and / or helps to keep cells alive and functional after transfer into a subject.The therapeutic effect of cells can be improved.

[0063] The recombinant proteins described herein are based on the presence of an exodomain derived at least in part from the extracellular region of EPOR, which contains a dimerization domain (e.g., at least one dimerization domain) that allows the recombinant protein to dimerize with another protein (a second protein) and provide a signal to a cell containing the recombinant protein in the absence of a signal inducer molecule, in particular EPO. The recombinant proteins described herein can spontaneously dimerize with a second protein (e.g., another recombinant protein) and provide a constitutive signal to a cell in which the recombinant protein is expressed. In particular, the signal can be transmitted to the cell via an endodomain, which in certain embodiments may be included within the recombinant molecule or, in other embodiments, may be provided on a separate protein ("signaling protein").

[0064] Thus, the present invention is based on the ability of recombinant molecules to provide constitutive signals to cells. In particular, the dimerization of the recombinant proteins described herein activates the signal transduction pathway mediated by JAK kinase activity, particularly JAK1 or JAK2 activity, in particular the JAK1-STAT or JAK2-STAT signal transduction pathway. In this way, the recombinant proteins can mimic the signal transduction induced by the activation of natural cytokine receptors, such as EPOR or interleukin (e.g., IL-2) receptors. By "mimic", we mean that the signal transduction cascade activated by the recombinant proteins of the present disclosure is similar to the signal transduction cascade activated by natural cytokine receptors, while the magnitude of activation induced by the recombinant proteins of the present disclosure may differ from that of natural cytokine receptors.

[0065] As described herein, a recombinant protein includes an exodomain. In this specification, "exodomain" refers to a portion or part of a recombinant protein that can be found outside the cell when expressed as a membrane-bound protein. Thus, typically, an exodomain refers to a portion of a protein that is found outside the cell, not in the cell membrane or in the cytoplasm. Those skilled in the art will understand that recombinant protein expression first occurs inside the cell, and in the process, the entire protein is found inside the cell. Furthermore, recombinant proteins can be periodically internalized and circulated (e.g., in response to binding of a ligand such as EPO). However, in the normal form after expression, recombinant proteins are typically membrane-bound proteins, and the exodomain as a part of the protein can be found outside the cell membrane and the cell. The terms "exodomain", "extracellular domain", and "extracellular region" are used interchangeably herein.

[0066] The exodomain of the recombinant protein comprises a sequence derived from the extracellular region of EPOR. For example, the exodomain of the recombinant protein may consist of a sequence derived from the extracellular region of EPOR. "Derived from" in this specification means that the exodomain of the recombinant protein comprises a sequence having at least 70% identity to the extracellular region of EPOR, more specifically, a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the extracellular region of EPOR. The sequence derived from the extracellular region of EPOR contained within the exodomain of the recombinant protein may comprise at least one modification (e.g., at least one amino acid substitution, insertion, deletion, or translocation) with respect to the wild-type extracellular region of EPOR. In this regard, in one embodiment, the exodomain of the recombinant protein may not comprise the wild-type extracellular region of EPOR, for example, as shown in SEQ ID NO: 3 or SEQ ID NO: 5. Thus, the EPOR extracellular domain sequence contained within the exodomain may contain one modification, or may contain two or more modifications, for example, at least two, three, four, five, ten, fifteen, or twenty modifications. In particular, at least one of the modifications may result in the introduction of a dimerization domain, which may result in the ability of the recombinant protein to dimerize, in particular homodimerize, and provide a signal to a cell expressing the recombinant protein in the absence of any signal inducer molecule (in particular in the absence of EPO). The EPOR extracellular domain sequence contained within the exodomain may also or alternatively contain modifications that do not result in the introduction of a dimerization domain, and thus do not result in the ability of the recombinant protein to dimerize and / or signal in the absence of any signal inducer molecule. However, if the dimerization domain is provided elsewhere in the exodomain (i.e., not within the portion derived from the extracellular domain of the EPOR), the EPOR extracellular region sequence within the exodomain may comprise or consist of the wild-type sequence, or may contain one or more modifications that do not result in the introduction of a dimerization domain.

[0067] EPOR is a receptor that binds erythropoietin (EPO) in its native conformation, and EPO binding induces dimerization and the JAK2 / STAT5 signaling cascade. Thus, wild-type EPOR provides an inducible signal, rather than a constitutive signal, to cells expressing the receptor. Wild-type human EPOR comprises 508 amino acids, including a signal peptide (SEQ ID NO:6, consisting of amino acids 1-24 of SEQ ID NO:1), an extracellular region (SEQ ID NO:3 (without signal peptide), consisting of amino acid residues 25-250 of SEQ ID NO:1), a transmembrane domain (SEQ ID NO:7, consisting of amino acid residues 251-273 of SEQ ID NO:1), and a cytoplasmic domain (SEQ ID NO:8, consisting of amino acid residues 274-508 of SEQ ID NO:1). The human full-length wild-type sequence of EPOR is shown in SEQ ID NO:1.

[0068] As used herein, the term "extracellular region of EPOR" refers to the region of EPOR that is typically found extracellularly, i.e., outside the cell. In particular, when considering human EPOR or mouse EPOR, the "extracellular region of EPOR" may refer to the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5 (for humans) or the sequence shown in SEQ ID NO: 11 (for mice). Thus, the extracellular region of EPOR may include or be free of a signal peptide.

[0069] As mentioned above, the exodomain of the recombinant protein described herein comprises a sequence derived from the extracellular region of EPOR, which sequence may comprise one or more modifications. In particular, at least one of the modifications may enable the recombinant protein to dimerize with a second protein in the absence of a signal inducer molecule and provide a signal to a cell containing the recombinant protein (more specifically, the recombinant protein may have an increased ability to dimerize with a second protein in the absence of a signal inducer molecule (particularly EPO) compared to wild-type EPOR in the absence of a signal inducer molecule (particularly EPO) and / or an increased ability to provide a signal to a cell containing the recombinant protein compared to wild-type EPOR in the absence of a signal inducer molecule (particularly EPO)). Thus, the recombinant protein may have an increased ability to dimerize and / or provide a signal in the absence of a signal inducer molecule (e.g., EPO) compared to wild-type EPOR by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. Increased dimerization and / or signaling can be determined as further described below.

[0070] Typically, one or more modifications that result in the above-mentioned function (signal transduction in the absence of a signal inducer molecule, i.e., constitutive signal transduction) can be made to the extracellular region of the EPOR in the present invention. Such one or more modifications can enable the recombinant protein to dimerize with a second protein based on cognate dimerization domains that associate, e.g., bind or interact in some way, when in close proximity. Thus, the modification of the extracellular region of the EPOR can introduce a dimerization domain that allows dimerization in the absence of a signal inducer molecule.

[0071] In a particular embodiment, the one or more modifications may allow the recombinant protein to dimerize with a second protein via a disulfide bond (disulfide bond dimerization), and more specifically, may allow disulfide bond homodimerization. Typically, to achieve this effect, one or more non-naturally occurring cysteine ​​residues can be inserted or substituted into the extracellular region of EPOR. In this regard, the sequence contained within the exodomain derived from the extracellular region of EPOR may have an amino acid modification at position 130 of SEQ ID NO:3, position 154 of SEQ ID NO:5, or position 129 of SEQ ID NO:11. More specifically, the modification may be an amino acid substitution from arginine to cysteine, and may therefore be referred to as R130C, R154C, or R129C in SEQ ID NO:3, SEQ ID NO:5 (or SEQ ID NO:1), or SEQ ID NO:11 (or SEQ ID NO:9), respectively. Those skilled in the art will understand that this embodiment, in particular, allows disulfide bonds between recombinant protein chains, resulting in homodimerization of the recombinant protein. Thus, the exodomain of the recombinant protein may comprise or consist of the amino acid sequence of SEQ ID NO:3 with an R to C amino acid substitution at position 130, SEQ ID NO:5 with an R to C amino acid substitution at position 154, or SEQ ID NO:11 with an R to C amino acid substitution at position 129.

[0072] Viewed alternatively, the exodomain may comprise or consist of the sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:10.

[0073] As used herein, the term "dimerization domain" refers to a first domain capable of binding to a second domain, and these first and second domains may be the same (homodimerization domain) or different (heterodimerization domain). The dimerization domain of the present invention may also be referred to as a constitutive dimerization domain, since dimerization occurs with its cognate partner in the absence of a signal inducer molecule (e.g., a dimerization inducer molecule). As described above, the dimerization domain may be present within any portion of the exodomain, for example, within a portion derived from the extracellular region of the EPOR, or elsewhere within the exodomain. Thus, the dimerization domain may be formed by modifications made to the extracellular region of the EPOR as described above, or may be based on other constitutive dimerization systems known in the art, which may be located within sequences derived from the extracellular region of the EPOR, at the N-terminus or C-terminus of the extracellular region of the EPOR, or elsewhere within the exodomain, as described below.

[0074] In this regard, particular mention may be made of leucine zippers, which are widely known and described in the art. Leucine zipper domains are a type of protein-protein interaction domain commonly found in transcription factors, characterized by an a-helix of leucine residues spaced at regular intervals. Thus, in one embodiment, the dimerization domains herein are or contain leucine zipper sequences. Depending on the leucine zipper sequence used, they can be used for heterodimerization or homodimerization. Leucine zipper domains derived from Fos or Jun protein molecules are described in Patel et al., 1996, J. Biol. Chem. 271(8), 30386-30391 and Stuhlmann-Laeisz et al., 2006, Mol. Biol. Cell 17, 2986-2995. A representative leucine zipper sequence based on human c-Jun is shown in SEQ ID NO:20 (which may contain GG at the N-terminus), and the Fos leucine zipper sequence is shown in SEQ ID NO:21.

[0075] Heterodimerization domains containing the Jun leucine zipper and the Fos leucine zipper, respectively, may be used, or homodimerization domains containing the Jun leucine zipper may be used.

[0076] Other leucine zipper dimerization domains known in the art include those based on ZIP proteins, a class of transcription factors. ZIP domains are regions of alpha-helices that contain leucines arranged to form a leucine zipper motif. ZIP domains can interact with leucines on other ZIP domains to reversibly bind (i.e., dimerize) the alpha-helices. Thus, the dimerization domains herein can include bZIP leucine zipper domains or aZIP leucine zipper domains. For example, the heterodimerization domains can be or include BZIP (RR) domains that heterodimerize with AZIP (EE) domains. Leucine zippers are examples of coiled-coil structural protein motifs that can be used to create dimerization domains. Heterodimerization domains based on bZIP and synthetic coiled-coil peptides are described in Reinke et al. 2010, J. Am. Chem. Soc. 132(17), 6025-6031, any of which can be used. For example, suitable leucine zipper domains include SYNZIP1-SYNZIP48. Other examples of leucine zipper domains include BATF, ATF4, ATF3, BACH1, JUND, NFE2L3, and HEPTAD. The sequence of the BZip (RR) leucine zipper domain is shown in SEQ ID NO: 22. The sequence of the AZip (EE) leucine zipper domain is shown in SEQ ID NO: 23.

[0077] In some embodiments, suitable leucine zipper domain pairs have a dissociation constant (Kd) of 1000 nM or less, eg, 100 nM or less, 10 nM or less, or 1 nM or less.

[0078] Further examples of dimerization domain pairs include PSD95-Dlgl-zo-1 (PDZ) domain, or streptavidin domain and streptavidin-binding protein (SBP) domain.Other dimerization domains can be obtained or derived from other proteins that are known to interact or bind with each other.For example, one heterodimerization domain pair can include CD80 and PDL-1.

[0079] Further examples of homodimerization domains include the Fc region of immunoglobulin G. The Fc region has been widely used in fusion proteins, for example to provide a dimerization domain, and various fragments and variants of the Fc region capable of dimerization have been described in the literature, for example a fragment lacking the first five amino acids of the Fc region.

[0080] As mentioned above, the extracellular domain sequence from EPOR contained within the exodomain may contain additional modifications that may or may not contribute to the ability of the recombinant molecule to dimerize and signal in the absence of a signal inducer molecule. In particular, the inventors envision that variants or fragments of the extracellular domain of EPOR may be utilized in the present invention, so long as the recombinant protein has the desired ability to spontaneously dimerize (i.e., dimerize in the absence of a signal inducer molecule when co-located with a second protein capable of dimerization) and provide a signal to a cell containing the recombinant protein (and the second protein, if different from the recombinant protein). As indicated above, in particular, such variants or fragments / portions may have at least 70% sequence identity to the wild-type extracellular domain of EPOR (e.g., SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 11). Thus, for example, further modified extracellular regions of EPORs, including additional amino acid substitutions, deletions, additions, or translocations, for example, at least 1, 2, 3, 4, or 5 amino acid substitutions, deletions, additions, or translocations, can be used in the present invention. In this regard, the exodomain may comprise or consist of a variant or fragment of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:10 (e.g., a variant or fragment having at least 60%, 70%, 80%, 90%, or 95% identity to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:10), provided that the recombinant protein has the desired function of dimerizing with a second protein in the absence of a signal inducer molecule and providing a signal to a cell expressing the recombinant protein. It will be understood that a recombinant protein comprising such a variant or fragment may have the same or similar ability to dimerize with a second protein and / or provide a signal as a recombinant protein comprising an exodomain comprising SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:10, or may have an altered ability to dimerize with a second protein.In particular, a recombinant protein comprising said variant or fragment may have a dimerization and / or signaling capability of at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 120%, 150%, 170%, 190%, or 200% of that of a recombinant protein comprising an exodomain comprising SEQ ID NO: 2. Dimerization can be detected and measured as further described below.

[0081] More specifically, the exodomain may comprise or consist of a variant or fragment of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:10 (e.g., a variant or fragment having at least 60%, 70%, 80%, 90%, or 95% identity to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:10), with the proviso that a variant or fragment of SEQ ID NO:2 comprises R130C, a variant or fragment of SEQ ID NO:4 comprises R154C, and a variant or fragment of SEQ ID NO:10 comprises R129C. Furthermore, said variant or fragment should confer to the recombinant protein the ability to dimerize with a second protein in the absence of a signal inducer molecule and provide a signal to a cell containing the recombinant protein.

[0082] Modifications to the extracellular domain of EPOR reported in the art include T114A, S115A, S116A, F117A, F117L, F117W, F117Y, V118A, L120A, E121A, R165A, M174A, S176A, H177A, and R179A. Any one or more of these modifications may be present with the exodomain sequence derived from the extracellular domain of EPOR in the present invention.

[0083] Although preferred in some embodiments, it is not essential that the sequence derived from the extracellular region of EPOR contained with the exodomain of the recombinant protein retains the ability to bind EPO. Thus, modifications to the EPO binding site of the extracellular region of EPOR to reduce (e.g., reduce by more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90%) or eliminate the ability to bind EPO are within the scope of the present invention. Alternatively, as mentioned above, in one embodiment, it may be desirable for the exodomain (and thus the extracellular region of EPOR) to be capable of binding EPO to provide an additional signal to the cell beyond the constitutive signal provided in light of the modifications made to that region. The present invention further encompasses providing an extracellular region of EPOR in which the EPO binding site has been specifically modified to enhance binding to EPO compared to the unmodified wild-type EPOR extracellular region. In this regard, modification of the EPO binding site may make it possible to increase the affinity of EPO for the binding site by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. The EPO binding site is found at position 117 of SEQ ID NO:1 or SEQ ID NO:5, and therefore modifications of this position are encompassed by the present invention.

[0084] The exodomain may further comprise other heterologous domains or tags in addition to sequences derived from the extracellular region of EPOR and / or in addition to any constitutive dimerization domain. Thus, in this specification, reference to an exodomain derived at least in part from the extracellular region of EPOR means that the exodomain comprises amino acid sequences derived from the extracellular region of EPOR, but additional heterologous sequences (i.e., sequences not present in wild-type EPOR) may be present. For example, the exodomain may further comprise a suicide moiety to allow induction of cell death that may be necessary or desirable, such as during the occurrence of an adverse event during or after administration of a cell therapy. An example of a possible suicide moiety is the CD20 epitope, in which case cell death can be induced by administration of rituximab. WO2013 / 15339 describes CD20 epitopes that can be used in this way. Other domains that can be used include tags that can be used to identify and select cells expressing the recombinant molecule, such as Strep tags or myc tags.

[0085] Furthermore, in some cases, it may be desirable to include one or more additional inducible dimerization domains in the exodomain of the recombinant molecule to allow for possible enhancement of signaling (e.g., at least 10%, 20%, 30%, 40%, or 50% increase in signaling compared to a recombinant molecule having the same amino acid sequence but not including one or more additional inducible dimerization domains). In another view, the presence of one or more additional inducible dimerization domains may allow for a greater amount of dimerization between a recombinant protein defined herein and a second protein (e.g., at least 10%, 20%, 30%, 40%, or 50% increase) compared to a recombinant protein not including an additional inducible dimerization domain. The additional inducible dimerization domains may allow for dimerization by any means. For example, dimerization may be a direct or indirect association, and does not mean that the two dimerization domains are directly bound or linked, although this is not excluded. Typically, each inducible dimerization domain may bind to a dimerization inducer molecule. Those skilled in the art will understand that if a dimerization inducer molecule is required for dimerization between any additional inducible dimerization domains, then increased signaling or increased percentage of dimers obtained may only occur in the presence of the dimerization inducer molecule. Such additional inducible dimerization domains may be present in addition to the presence of an EPO binding site in the sequence derived from the extracellular region of EPOR, or may be present to replace the loss of such an EPO binding site.

[0086] Chemically induced dimerization systems using a variety of dimerization inducer molecules and different protein domains for dimerization are known in the art and are further described below.

[0087] The concept of chemically induced dimerization mediated by small molecule inducers has been known for many years and has been used as a tool to control the dimerization of proteins of interest fused to inducer binding domains. Such systems have been described for a variety of applications in cell biology, for example to bring proteins into close proximity to investigate signal transduction pathways and other biological mechanisms, in medicine to degrade or inactivate pathogenic proteins, and in gene and cell therapy. A typical chemical dimerization inducer (CID), or dimerization inducer as the term is used herein, has the characteristic that it can interact or bind with two proteins or protein domains, one on each side of the molecule. Thus, a dimerization inducer has two binding sites or binding faces (or more generally, interaction sites). In the case of heterodimerization, the dimerization inducer can interact or bind with two different proteins or dimerization domains. In the case of homodimerization, the dimerization inducer can interact or bind with two copies or molecules of the same dimerization domain. The original system was based on the macrolides FK506 and rapamycin. These macrolides can bind to and induce heterodimerization of a variety of different proteins or protein domains, including FK506-binding protein (FKBP), the FKBP-rapamycin domain (FRB) of mTOR, calcineurin, and cyclophilin, which can be used in different combinations to realize heterodimerization domain pairs and CID combinations. Such systems may also include the use of cyclosporine, which binds to calcineurin or cyclophilin. Other CID heterodimerization systems based on different molecules have since been developed and are described in the literature. In addition, homodimerization systems based on FK506 derivatives capable of binding to two FKBP molecules have been developed; that is, homodimerization systems based on symmetric or dimer inducers containing two binding sites for the same dimerization domain.

[0088] In one embodiment, the dimerization inducer molecule is rapamycin or its analogs, and the dimerization domain is the protein domain that binds to it.Rapamycin and rapamycin analogs induce heterodimerization by generating an interface between the FRB domain of mTOR and FK506 binding protein (FKBP). This association allows FKBP to block access to the mTOR active site and inhibit its function.Although mTOR is a very large protein, the exact small segment of mTOR that is required for interaction with rapamycin is known and can be utilized.

[0089] The macrolides rapamycin and FK506 act by inducing heterodimerization of cellular proteins. Each drug binds with high affinity to the FKBP12 protein, forming a drug-protein complex that then binds to and inactivates mTOR / FRAP and calcineurin, respectively. The FKBP-rapamycin binding (FRB) domain of mTOR has been defined and applied as an isolated protein moiety of 89 amino acids that can be fused to a protein of interest. Rapamycin can then induce the access of the FRB fusion to FKBP12 or a protein fused to FKBP12.

[0090] The terms "FRB" and "FKBP" include variants thereof. Such variants may include amino acid sequences having one or more amino acid modifications (e.g., substitutions, additions, and / or deletions) relative to the native sequence. The term "FKBP" includes FKBP12.

[0091] Rapamycin has several properties of an ideal inducible dimerizer: it has high affinity (KD<1nM) for FRB when bound to FKBP and high specificity for the FRB domain of mTOR. Rapamycin is an effective therapeutic immunosuppressant with favorable pharmacokinetic and pharmacodynamic profiles in mammals. Pharmacological analogs of rapamycin, such as everolimus, temsirolimus, and deforolimus, with different pharmacokinetic and dynamic properties (Benjamin et al, Nature Reviews, Drug Discovery, 2011), may also be used depending on the clinical environment.

[0092] To prevent rapamycin from binding to and inactivating endogenous mTOR, the surface of rapamycin that contacts FRB can be modified: compensatory mutations in the FRB domain to form a "bumped" rapamycin-accommodating surface restore dimerization interactions only with the FRB mutant and not with the endogenous mTOR protein.

[0093] Bayle et al. (Chem Bio; 2006; 13; 99-107) describes various rapamycin analogs, or "rapalogs," with their corresponding modified FRB-binding domains. For example, Bayle et al. (2006) describe the rapalogs C-20-methyllyrlrapamycin (MaRap), C16(S)-butylsulfonamiderapamycin (C16-BS-Rap), and C16-(S)-7-methylindolerapamycin (AP21976 / C16-AiRap) in combination with their respective complementary binding domains, as shown in Figure 2. Other rapamycins / rapalogs include sirolimus and tacrolimus (FK506).

[0094] Thus, in such embodiments, the inducible dimerization domain in a recombinant protein described herein may comprise FKBP and a second protein may comprise the cognate dimerization domain FRB, or vice versa. FKBP / FRB may have or comprise a sequence as set forth in any one of SEQ ID NO: 15 to SEQ ID NO: 19 or a variant thereof.

[0095] As used interchangeably herein, a "signal inducer molecule" or a "signal inducer molecule" refers to a molecule that can induce signal transduction through a protein or receptor that contains an exodomain, a transmembrane domain, and an endodomain to a cell that contains such a protein or receptor. A signal inducer molecule can induce any kind of signal, and a person skilled in the art will understand that the induced signal depends on the endodomain present in the protein or receptor. Typically, a signal inducer molecule binds to a protein or receptor to induce a signal. In one embodiment, a signal inducer can bind to a protein or receptor to induce dimerization or multimerization, resulting in the transmission of a signal through the endodomain of the protein or receptor. In this embodiment, the signal inducer molecule can be a dimerization inducer molecule. However, it is also possible for a signal inducer to bind to a receptor or protein and induce a conformational change, which results in the production of a signal that is translocated by the endodomain of the protein or receptor. In this respect, the signal inducer molecule may bind to an already dimerized receptor or protein (e.g., no signal or weak signal) to generate a signal through the endodomain in the cell. In the present invention, the recombinant protein can provide a signal to the cell in the absence of such a signal inducer molecule. Thus, the recombinant protein does not require the presence of a signal inducer molecule to provide a signal to the cell (and / or to dimerize with a second protein). In particular, the signal inducer molecule may be EPO.

[0096] "EPO" herein refers to erythropoietin and includes the amino acid sequence of SEQ ID NO: 14. EPO typically binds to EPOR at amino acid position 117 of SEQ ID NO: 1. As detailed above, the recombinant proteins of the present invention do not require the presence of EPO to produce a signal to cells containing the recombinant proteins.

[0097] A "dimerization inducer molecule" as used herein may form an interface between two inducible dimerization domains (e.g., in a recombinant protein and in a second protein) to bind them as a dimer, or may allow chemical crosslinking between two inducible dimerization domains (e.g., in a recombinant protein and in a second protein) to allow the formation of a dimer. As described multiple times herein, a recombinant protein can dimerize with a second protein in the absence of a signal inducer molecule, including in the absence of a dimerization inducer molecule, and provide a signal in a cell containing the recombinant protein. However, it is not excluded that one or more other inducible dimerization domains may be present in the recombinant molecule. Dimerization inducer molecules include, for example, rapamycin, as described above.

[0098] Dimerization of the recombinant protein described herein with a second protein (due to the modifications described herein and / or due to the presence of one or more inducible dimerization domains) can be determined or measured by non-denaturing SDS PAGE or dynamic light scattering techniques well known in the art. Dimerization can therefore generally be detected by the difference in size between the monomeric and dimeric forms of a protein (in the present invention, the recombinant protein). The ability of a recombinant protein to dimerize (particularly to homodimerize) with a second protein can therefore be measured (or determined) by detecting the presence of a dimer of the recombinant protein in a cell or cell population. The recombinant protein described herein has the ability to dimerize with a second protein in the absence of a signal inducer molecule, particularly EPO. This dimerization ability can therefore be determined by detecting the dimeric form of the recombinant protein when expressed in a cell (either alone in the case of homodimerization or together with a different second protein in the case of heterodimerization). Typically, the recombinant protein of the present invention can dimerize in the absence of signal inducer molecule to the same or similar extent as wild-type EPOR dimerizes in the presence of EPO. Alternatively, the recombinant protein can have an altered ability to dimerize with a second protein compared to wild-type EPOR in the presence of EPO. Thus, altered dimerization ability can refer to an increase or decrease in the relative amount of recombinant protein in dimeric or monomeric form in a cell or cell population compared to a certain standard, for example, compared to wild-type EPOR in the presence of EPO, or compared to SEQ ID NO: 2 for variants or fragments of SEQ ID NO: 2 (e.g., an increase or decrease of at least 40%, 50%, 60%, 70%, 80%, 90%, 95% of recombinant protein in dimeric form). One of skill in the art will understand that even if dimerization / signaling of a recombinant protein in the absence of EPO is reduced (e.g., at least 10%, 20%, 30%, 40%, or 50% reduced) compared to wild-type EPOR in the presence of EPO, a sufficient signal is still transmitted to the cell to provide a desired result (e.g., persistence) and may be preferred in certain embodiments.

[0099] Dimerization with a "second protein" as used herein means that the recombinant protein can form a dimer with the second protein (i.e., a monomer of the recombinant protein can associate with a monomer of the second protein to form a dimer (comprising one copy of each monomer)). In one embodiment, the second protein is a recombinant protein, and thus in this case the recombinant protein is capable of homodimerization, e.g., dimerization with itself, in which case the dimer comprises two recombinant protein monomers. Thus, in this embodiment, it is sufficient for the recombinant protein to be expressed in the cell for dimerization to occur (expression of a different protein is not required for dimerization), and as mentioned above, no signal inducer molecule is required. Dimerization can be detected as described above.

[0100] Alternatively, or in addition, a recombinant protein as defined herein may be capable of heterodimerizing with a different second protein that is not a recombinant protein of the invention. Thus, the second protein may comprise a domain or moiety different from the recombinant protein. However, the second protein must be capable of dimerizing with the recombinant protein and providing a signal in the absence of a signal inducer molecule. Thus, the second protein must comprise a dimerization domain cognate to the dimerization domain of the recombinant protein that is capable of association and signal transduction in the absence of a signal inducer molecule. For example, in one embodiment where the recombinant protein comprises an exodomain comprising a modified extracellular region of EPOR, the modification comprises the insertion or substitution of an amino acid residue to a cysteine ​​(for the purpose of disulfide bond dimerization), the second protein must comprise a cognate cysteine ​​residue in an appropriate position to allow dimerization in the absence of a signal inducer molecule. Alternatively (or additionally), if the recombinant protein contains an exodomain with a leucine zipper, the second protein may contain a cognate leucine zipper to allow dimerization in the absence of a signal inducer molecule. For example, the recombinant protein may contain a Jun leucine zipper or a Fos leucine zipper, and the second protein may contain a Jun leucine zipper (or vice versa). Those skilled in the art will understand that the recombinant proteins described herein are capable of homodimerization and heterodimerization. Thus, a cell expressing a recombinant protein and a different second protein may contain a homodimer of the recombinant protein as well as a heterodimer of the recombinant protein and the different second protein.

[0101] Thus, in one embodiment, the second protein may be a variant or part of the recombinant molecule, in particular the second protein may comprise an exodomain that is a variant or part of the exodomain of the recombinant molecule (e.g., may have at least 70%, 80%, 90%, or 95% sequence identity thereto). Alternatively, the second protein may comprise an endodomain that is a variant or part of the endodomain of the recombinant molecule (e.g., may have at least 70%, 80%, 90%, or 95% sequence identity thereto). The second protein must be capable of binding to the recombinant protein in the absence of the signal inducer molecule. In particular, in the case of recombinant proteins comprising an additional inducible dimerization domain, e.g. an inducible heterodimerization domain, the additional inducible dimerization domain present in the recombinant protein may be different from the additional inducible dimerization domain (e.g., FRB / FKBP or a functional variant thereof) present in the second protein (in this embodiment, the recombinant protein may further comprise an FRB and the second protein may comprise an FKBP, or vice versa).

[0102] Additionally or alternatively, the recombinant protein and the second protein may comprise different tags, for example, the recombinant protein may comprise a strep tag and the second protein may comprise a myc tag, or vice versa. Those skilled in the art will understand that the second protein may typically be a recombinant protein, and the present invention may further require expressing a second recombinant protein together with the recombinant protein of the present invention in the cell. Thus, the present invention further allows for the introduction into the cell of a second nucleic acid molecule comprising a base sequence encoding the second protein, for example together with a nucleic acid comprising a base sequence encoding a recombinant protein as defined herein.

[0103] Variants of any amino acid sequence presented herein may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the reference sequence (i.e., the reference sequence number specified herein), unless otherwise stated. In particular, such variants retain the desired or required properties of the parent molecule from which they are derived, i.e., the reference sequence. Thus, variant sequences may have the stated sequence identity (%), provided that the variant sequence provides an effective dimerization system and signal.

[0104] The term "derivative" or "variant" as used interchangeably herein in relation to the protein or polypeptide of the present invention includes any substitution, mutation, modification, replacement, deletion, and / or addition of one (or more) amino acid residues from or to a sequence, provided that the resulting protein or polypeptide retains the desired function. For example, if the derivative or variant is an endodomain, the desired function may be the ability of the domain to signal (e.g., activate or inactivate a downstream molecule), and if the derivative or variant is a dimerization domain, the desired function is interaction (directly or indirectly) with the cognate dimerization domain. In another view, the variant or derivative referred to herein is typically a functional variant or functional derivative. For example, the variant or derivative may have at least at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the function compared to the corresponding reference sequence. The variants or derivatives may have a similar or the same level of function as compared to the corresponding reference sequence, or may have a higher level of function than the corresponding reference sequence (e.g., at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% increased).

[0105] Typically, the amino acid substitutions may consist of, for example, one, two or three to ten or twenty substitutions, provided that the modified sequence thereby retains the desired activity or potency. Amino acid substitutions may include the use of non-naturally occurring analogues. For example, the variant or derivative may have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the activity or potency of the corresponding reference sequence. The variant or derivative may have a similar or the same level of activity or potency as the corresponding reference sequence, or may have a higher level of activity or potency (e.g., at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher) than the corresponding reference sequence.

[0106] Proteins or peptides may also have deletions, insertions, or substitutions of amino acid residues that produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made based on the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues, so long as the intrinsic function is preserved. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0107] Conservative substitutions may be made, for example according to Table 1 below. [Table 1]

[0108] The derivative may be a homologue. The term "homologue" as used herein means an entity having a certain homology with a wild-type amino acid sequence and a wild-type nucleotide sequence. The term "homology" can be equated with "identity".

[0109] A homologous or variant sequence may comprise an amino acid sequence that may have at least 80%, 85%, or 90%, preferably at least 95%, 96%, 97%, 98%, or 99% identity to the subject sequence. Typically, the variant may comprise the same active site, etc. as the subject amino acid sequence. Although homology may also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), in the context of this specification it is preferred to express homology in terms of sequence identity.

[0110] Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percentage homology or identity between two or more sequences. Percentage homology or sequence identity can be calculated over a contiguous sequence, i.e., one sequence is aligned with the other and each amino acid in one sequence is directly compared, residue by residue, with the corresponding amino acid in the other sequence. This is called an "ungapped" alignment. Typically, such ungapped alignments are only performed over a relatively short number of residues.

[0111] This is a very simple and consistent method, but it does not take into account that in a pair of sequences that are identical except for, for example, one insertion or deletion, the insertion or deletion in the base sequence may cause the subsequent codon to be disaligned, i.e., potentially resulting in a large decrease in % homology when a global alignment is performed. As a result, most sequence comparison methods are designed to create an optimal alignment that takes into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to maximize local homology.

[0112] However, these more complex methods assign a "gap penalty" to each gap that occurs during the alignment, such that for the same number of identical amino acids, a sequence alignment with as few gaps as possible (reflecting a high similarity between the two compared sequences) achieves a higher score than one with many gaps. An "affine gap cost" is usually used, which imposes a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties naturally produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalty to be modified. However, it is preferred to use the default values ​​when using such software for sequence comparison. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0113] Calculation of maximum percentage homology / sequence identity therefore first requires the creation of an optimal alignment, taking into account gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387). Examples of other software capable of performing sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid-Ch.18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and the GENEWORKS comparison tool suite. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. Another tool called BLAST2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174:247-50; FEMS Microbiol. Lett. (1999) 177:187-8).

[0114] Although the final percentage homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​commonly used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. One example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs typically use either the public default values ​​or a custom symbol comparison table if supplied (see user manual for further details). However, for some applications, it is preferred to use the default values ​​in the GCG package, and for other software, a default matrix such as BLOSUM62. Preferably, the percentage identity is determined over the entire reference and / or query sequence.

[0115] Once the software has produced an optimal alignment, it is possible to calculate percentage homology, preferably percentage sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0116] A "fragment" typically refers to a selected region of a polypeptide or polynucleotide that is of interest in terms of function, e.g., is functional or encodes a functional fragment. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is a portion (or part) of a full-length polypeptide or polynucleotide.

[0117] Such variants, derivatives, and fragments may be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where an insertion is made, a synthetic DNA may be made that encodes the insert, with 5' and 3' flanking regions corresponding to the naturally occurring sequence on either side of the insertion position. The flanking regions contain appropriate restriction enzyme sites corresponding to sites in the naturally occurring sequence, so that the sequence may be cleaved with the appropriate enzyme (or enzymes) and the synthetic DNA ligated to the cleavage site. The DNA is then expressed in accordance with the invention to produce the encoded protein. These methods are merely illustrative of the many standard techniques well known in the art for DNA sequence manipulation, and other known techniques may also be used.

[0118] The recombinant proteins described herein in particular typically comprise a transmembrane domain that anchors the exodomain of the recombinant protein to the cell membrane. The transmembrane domain may be derived from any protein having a transmembrane domain, such as a type I transmembrane protein, a type II transmembrane protein, or a type III transmembrane protein.

[0119] The transmembrane domain of the chimeric protein may also comprise an artificial hydrophobic sequence. Further transmembrane domains will be apparent to those skilled in the art. The TM domain may be selected, for example, from any of those typically used in recombinant transmembrane proteins. Examples of transmembrane (TM) regions that may be used are shown below. 1)CD28 TM region (Pule et al, Mol Ther, 2005, Nov; 12(5):933-41; Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426-35; Casucci et al, Blood, 2013, Nov 14;122(20):3461-72.);2)OX40 TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41);3)41BB TM region (Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426-35);4)CD3 zetaTM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41; Savoldo B, Blood, 2009, Jun 18;113(25):6392-402.);5) CD8a TM region (Maher et al, Nat Biotechnol, 2002, Jan;20(1):70-5.; Imai C, Leukemia, 2004, Apr;18(4):676-84; Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426-35; Milone et al, Mol Ther, 2009, Aug;17(8):1453-64.). Other transmembrane domains that can be used include those derived from CD4, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, or CD154. The transmembrane domain may also be derived from IL2RB or EPOR.

[0120] By way of example, the transmembrane domain may be derived from the CD28 transmembrane domain and may comprise or consist of the amino acid sequence shown as SEQ ID NO: 34, or a variant having at least 80% identity to SEQ ID NO: 24. Said variant may have at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to SEQ ID NO: 24.

[0121] In certain embodiments, the transmembrane domain may be derived from the EPOR transmembrane domain and may comprise or consist of the amino acid sequence shown as SEQ ID NO: 7, or a variant having at least 80% identity to SEQ ID NO: 7. The variant may have at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 7.

[0122] Alternatively, the transmembrane domain may be derived from the IL2RB transmembrane domain and may comprise or consist of the amino acid sequence shown as SEQ ID NO: 25, or a variant having at least 80% identity to SEQ ID NO: 25. Said variant may have at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to SEQ ID NO: 25.

[0123] Alternatively, the recombinant protein may comprise a domain derived from the CD8α transmembrane domain. Thus, the transmembrane domain may comprise or consist of the amino acid sequence shown as SEQ ID NO: 26, which represents amino acids 183 to 203 of human CD8α, or a variant having at least 80% identity to SEQ ID NO: 26. Suitably, said variant may have at least 85%, 90%, 95%, 97%, 98% or 99% identity to SEQ ID NO: 26.

[0124] Alternatively, the transmembrane domain may be derived from a transmembrane domain of a myeloid receptor protein, for example a TREM protein such as TREM1 or TREM2. Thus, the recombinant protein may comprise a transmembrane domain comprising or consisting of the amino acid sequence shown in SEQ ID NO:27 or SEQ ID NO:28, or a variant having at least 80% identity to SEQ ID NO:27 or SEQ ID NO:28. Suitably, said variant may have at least 85%, 90%, 95%, 97%, 98% or 99% identity to SEQ ID NO:27 or SEQ ID NO:28.

[0125] The recombinant proteins described herein can provide a signal to a cell expressing the recombinant protein (either alone in the case of homodimerization, or together with a second protein in the case of heterodimerization). The signal is typically provided to the cell by an endodomain. Thus, in one embodiment, the recombinant protein of the present invention further comprises an endodomain. In an alternative embodiment, the endodomain can be provided by a signaling protein that is a separate protein from the recombinant protein. In this aspect, the recombinant protein and the signaling protein together provide a signal to the cell. Thus, the recombinant proteins described herein can provide a signal to the cell directly (e.g., via its own endodomain) or indirectly (via the endodomain of the signaling protein).

[0126] Thus, a "signaling protein" as described herein refers to a protein that can associate with a recombinant protein as defined herein and transmit a signal to a cell. Thus, typically, a signaling protein comprises a transmembrane domain and an endodomain and can be expressed in a cell together with a recombinant protein (and / or a second protein). In this embodiment, it is particularly envisaged that the signaling protein may associate with the recombinant protein via the respective transmembrane domain, and such association may result in the transmission of a signal via the endodomain of the signaling protein. In one particular embodiment, the signaling domain may comprise a transmembrane domain derived from DAP10 or DAP12 as shown in SEQ ID NO: 27 or SEQ ID NO: 28, or a variant having at least 80% identity to SEQ ID NO: 27 or SEQ ID NO: 28. Suitably, said variant may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 27 or SEQ ID NO: 28. Those skilled in the art will appreciate that if the transmembrane domain of the signaling protein is derived from DAP10 or DAP10, the transmembrane domain of the recombinant protein may also be derived from a myeloid receptor, in particular a TREM receptor, as described above.

[0127] The "endodomain" described herein may be provided in a recombinant protein or in a separate signaling protein, as described above. However, in particular, the recombinant protein may comprise the endodomain. The endodomain comprises a tyrosine kinase activation domain, which comprises at least a JAK1 binding motif and / or a JAK2 binding motif, and a tyrosine effector domain, which can be phosphorylated by JAK1 kinase and / or JAK2 kinase. Phosphorylation of the tyrosine effector domain allows a signaling cascade to occur, for example, allowing other proteins in the signaling cascade to bind to the effector domain and / or be activated to transmit a signal in the cell. In other words, when the tyrosine effector domain is phosphorylated, it can recruit a signaling factor. As described herein, the recombinant protein can provide a signal to a cell containing the recombinant protein in the absence of a signal inducer molecule, and therefore, in another sense, produces a constitutive signal. A constitutive signal means that the cell is constantly receiving a signal from the recombinant protein. The signal may be more or less than that of the wild-type EPOR, but the recombinant protein is always capable of providing a signal. The terms "endodomain," "intracellular domain or region," and "cytoplasmic domain or region" are used interchangeably herein.

[0128] The tyrosine kinase activation domain may, in some embodiments, also contain a JAK3 binding motif, in particular a JAK1 binding motif and a JAK3 binding motif.

[0129] In one embodiment, for example, where the recombinant protein does not contain a JAK3 binding motif in an endodomain, the recombinant protein may be used in combination with a second protein that comprises an endodomain that includes a JAK3 binding motif.

[0130] The endodomain may signal through the JAK-STAT signalling pathway, in other words the signal may be mediated by activation of the JAK-STAT signalling pathway.

[0131] STAT proteins are transcription factors that are recruited to activated receptors, and therefore, in particular, the tyrosine effector domain may contain a STAT association motif that is a binding site for STAT. STAT may be STAT1, STAT2, STAT3, STAT4, STAT5, or STAT6, or any combination thereof. STAT association motifs may be obtained or derived from receptors, including cytokine receptors and receptor tyrosine kinases (RTKs). Tyrosine effector domains may contain one or more, such as two or more, such as three, four, five, or more, STAT association motifs, and these STAT association motifs may be the same or different.

[0132] As an example, STAT5 is a transcription factor involved in the IL-2 signaling pathway and plays a key role in Treg function, stability, and survival by promoting the expression of genes such as FOXP3, IL2RA, and BCLXL. To function and translocate to the nucleus, STAT5 needs to be phosphorylated. IL-2 ligation results in STAT5 phosphorylation by activating Jak1 / Jak2 and Jak3 kinases through specific signaling domains present in the IL-2Rβ and IL-2Rγ chains, respectively. JAK1 (or JAK2) can phosphorylate STAT5 without the need for JAK3, but the activity of STAT5 can be increased by transphosphorylation with both JAK1 / JAK2 and JAK3, stabilizing its activity.

[0133] As used herein, the term "STAT association motif" refers to an amino acid motif that contains tyrosine and can bind to a STAT polypeptide. Methods well known in the art for determining protein-protein interactions can be used to determine whether an association motif can bind to STAT. For example, co-immunoprecipitation can be followed by Western blotting.

[0134] The STAT association motif may be, for example, a STAT5 association motif that, upon phosphorylation, is capable of binding to a STAT5 polypeptide (as well as other STAT polypeptides).

[0135] In one embodiment, the STAT association motif is a STAT5 association motif.

[0136] Suitably, the endodomain may comprise two or more (e.g. at least two) STAT5 association motifs as defined herein. For example, the signalling domain may comprise two, three, four, five or more STAT5 association motifs as defined herein. In one embodiment, the signalling domain may comprise two or three STAT5 association motifs as defined herein.

[0137] Advantageously, the STAT5 association motif may be endogenously present in the cytoplasmic domain of a transmembrane protein that may be used to provide an endodomain herein. For example, the STAT5 association motif may be derived from an interleukin receptor (IL) receptor endodomain or a hormone receptor.

[0138] The endodomain may comprise an amino acid sequence selected from any of the chains of interleukin receptors of which STAT5 is a downstream component, for example, a cytoplasmic domain comprising amino acid numbers 266 to 551 of the IL-2 receptor β chain (National Center for Biotechnology Information (NCBI) Reference Sequence (REFSEQ): NP_000869.1, SEQ ID NO: 31), amino acid numbers 292 to 521 of the IL-9R chain (NCBI REFSEQ: NP_002177.2, SEQ ID NO: 33), amino acid numbers 257 to 825 of the IL-4R α chain (NCBI REFSEQ: NPJD00409.1, SEQ ID NO: 34), amino acid numbers 461 to 897 of the IL-3RB chain (NCBI REFSEQ: NP_000386.1, SEQ ID NO: 35), and / or amino acid numbers 314 to 502 of the IL-17R β chain (NCBI REFSEQ: NP_061195.2, SEQ ID NO: 36) may be used. It will be appreciated by those skilled in the art that any one or more of these sequences may be used. The entire region of the cytoplasmic domain of the interleukin receptor chain may be used.

[0139] The signaling domain may comprise one or more STAT5 association motifs, including an amino acid sequence set forth as SEQ ID NOs: 31-37, or a variant having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 31-37. For example, the variant may bind to STAT5 at a level that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the level of binding to STAT5 of an amino acid sequence set forth as one of SEQ ID NOs: 31-37. The variant or derivative may be capable of binding to STAT5 at a level close to or the same as that of one of SEQ ID NOs: 31-37, or may be capable of binding to STAT5 at a higher level (e.g., at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher) than the amino acid sequence set forth as one of SEQ ID NOs: 31-37.

[0140] For example, the STAT5-association motif may be derived from any one or more of IL2Rβ, IL-3Rβ (CSF2RB), IL-9R, IL-17Rβ, erythropoietin receptor (EPOR), thrombopoietin receptor, growth hormone receptor, and prolactin receptor. The endodomain may, for example, contain STAT-association motifs from both IL2RB and EPOR.

[0141] The STAT5 association motif may comprise the amino acid motif YXXF / L (SEQ ID NO: 38), where X is any amino acid.

[0142] Suitably, the STAT5 association motif may comprise the amino acid motif YCTF (SEQ ID NO: 39), YFFF (SEQ ID NO: 40), YLSL (SEQ ID NO: 41), or YLSLQ (SEQ ID NO: 42).

[0143] The endodomain may comprise one or more of the STAT5 association motifs described above, including the amino acid motifs YCTF (SEQ ID NO: 39), YFFF (SEQ ID NO: 40), YLSL (SEQ ID NO: 41), and / or YLSLQ (SEQ ID NO: 42).

[0144] The endodomain may comprise a first STAT5 association motif comprising the amino acid motif YLSLQ (SEQ ID NO: 42) and a second STAT5 association motif comprising the amino acid motif YCTF (SEQ ID NO: 39) or YFFF (SEQ ID NO: 40).

[0145] The endodomain may contain the following STAT5 association motifs: YLSLQ (SEQ ID NO: 42), YCTF (SEQ ID NO: 39), and YFFF (SEQ ID NO: 40).

[0146] Other STAT polypeptide association motifs are known in the art and can be used.For example, the tyrosine effector domain of the endodomain can comprise YXXQ (SEQ ID NO:57), where X is any amino acid, for example YRHQ (SEQ ID NO:58), to provide STAT3 signal to cells (especially Tcon cells).STAT3 association motifs are present in signal transduction proteins, for example IL-6R, IL10R and IL21R. In one embodiment, the endodomain as defined herein may comprise the cytoplasmic domain of the IL21Rα chain (e.g., comprising amino acids 256-538 of the IL-21Rα chain (NCBI RefSeq: NP_068570.1)) or a truncated fragment thereof comprising the box 1 motif (amino acids 266-274 of NCBI RefSeq: NP_068570.1) required for association with JAK1, and a STAT association motif comprising tyrosine residue 500 (amino acid number 519 of NCBI RefSeq: NP_000869.1) and the adjacent three residues C-terminal to tyrosine residue 500, i.e., YLRQ (SEQ ID NO: 59), required for STAT1 / 3 association. Alternatively, STAT1 or STAT4 signaling may be provided in a similar manner. For example, a STAT1 association motif may be found at amino acids 335-365 of IL2Rb (subdomain Aci2), as represented by the following sequence: QLLLQQDKVPEPASLSSNHSLTSCFTNQGYF (SEQ ID NO: 60)

[0147] "JAK1 binding motif" herein refers to a BOX motif that allows tyrosine kinase JAK1 association.Similarly, "JAK2 binding motif" herein refers to a BOX motif that allows tyrosine kinase JAK2 association.Suitable JAK1 binding motifs and JAK2 binding motifs are described, for example, by Ferrao & Lupardus (Frontiers in Endocrinology; 2017; 8(71); incorporated herein by reference).

[0148] As noted above, the JAK1 and / or JAK2 binding motifs may occur intrinsically within the cytoplasmic domain of a transmembrane protein.

[0149] For example, the JAK1 and / or JAK2 binding motif may be derived from interferon lambda receptor 1 (IFNLR1), interferon alpha receptor 1 (IFNAR), interferon gamma receptor 1 (IFNGR1), IL10RA, IL20RA, IL22RA, interferon gamma receptor 2 (IFNGR2), or IL10RB.

[0150] The JAK1 binding motif may comprise or consist of the amino acid motifs shown as SEQ ID NOs: 43-49 or variants thereof that are capable of binding to JAK1. KVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDK (SEQ ID NO:43) NPWFQRAKMPRALDFSGHTHPVATFQPSRPESVNDLFLCPQKELT (SEQ ID NO: 44) GYICLRNSLPKVLNFHNFLAWPFPNLPPLEAMDMVEVIYINR (SEQ ID NO: 45) PLKEKSIILPKSLISVVRSATLETKPESKYVSLITSYQPFSL (SEQ ID NO: 46) RRRKKLPSVLLFKKPSPFIFISQRPSPETQDTIHPLDEEAFLK (SEQ ID NO: 47) YIHVGKEKHPANLILIYGNEFDKRFFVPAEKIVINFITLNISDDS (SEQ ID NO: 48) RYVTKPPAPPNSLNVQRVLTFQPLRFIQEHVLIPVFDLSGP (SEQ ID NO: 49)

[0151] Variants of SEQ ID NOs: 43-49 may contain one, two, or three amino acid differences compared to any of SEQ ID NOs: 21-27 and may retain the ability to bind JAK1.

[0152] The above variants may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 43-49 and may retain the ability to bind to JAK1.

[0153] In a preferred embodiment, the JAK1-binding domain comprises or consists of SEQ ID NO:43, or a variant thereof, capable of binding to JAK1.

[0154] For example, the variant may bind to JAK1 at a level that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the binding level of the corresponding reference sequence. The variant or derivative may bind to JAK1 at a level close to or the same as the corresponding reference sequence, or may bind to JAK1 at a higher level than the corresponding reference sequence (e.g., at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher).

[0155] The JAK2 binding motif may comprise the amino acid motifs shown as SEQ ID NOs: 50-52 or variants thereof that are capable of binding to JAK2. NYVFFPSLKPSSSIDEYFSEQPLKNLLLSTSEEQIEKCFIIEN (SEQ ID NO:50) YWFHTPPSIPLQIEEYLKDPTQPILEALDKDSSPKDDVWDSVSIISFPE (SEQ ID NO:51) YAFSPRNSLPQHLKEFLGHPHHNTLLFFSFPLSDENDVFDKLSVIAEDSES (SEQ ID NO:52)

[0156] A variant of SEQ ID NO:50-52 may contain one, two, or three amino acid differences compared to any of SEQ ID NO:50-52 and may retain the ability to bind JAK2.

[0157] For example, the variant may bind to JAK2 at a level that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the binding level of the corresponding reference sequence. The variant or derivative may bind to JAK2 at a level close to or the same as the corresponding reference sequence, or may bind to JAK2 at a higher level than the corresponding reference sequence (e.g., at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher).

[0158] Methods well known in the art for determining protein-protein interactions may be used to determine whether a JAK1 or JAK2 binding motif can bind to JAK1 or JAK2, for example, co-immunoprecipitation followed by Western blotting.

[0159] Suitably, the endodomain may comprise the IL2Rβ endodomain shown as SEQ ID NO:31 or a variant having at least 80% sequence identity to SEQ ID NO:31.

[0160] The variant may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:31.

[0161] Suitably, said endodomain may comprise a truncated IL2Rβ endodomain as set forth in any one of SEQ ID NO: 53 or 54, or a variant having at least 80% sequence identity to any one of SEQ ID NO: 53 or 54. SEQ ID NO: 53 represents an IL2RB truncated variant with a Y510 mutation. SEQ ID NO: 54 represents an IL2RB truncated variant with a Y510 mutation and a Y392 mutation.

[0162] The variant may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:53 or 54.

[0163] In this embodiment, the invention provides a recombinant protein comprising (i) an exodomain derived at least in part from the extracellular region of EPOR, the portion comprising a modification to the extracellular region of EPOR that enables the recombinant protein to dimerize with a second protein in the absence of a signal inducer molecule and provide a signal to the T cell, and (ii) an endodomain comprising a truncated IL2Rβ endodomain of SEQ ID NO: 53 or SEQ ID NO: 54, or a variant having at least 80% sequence identity thereto.

[0164] In certain embodiments, the recombinant protein comprises an endodomain derived at least in part from the endodomain (cytoplasmic) region of EPOR (SEQ ID NO: 8). In certain embodiments, the endodomain may comprise the EPOR endodomain shown in SEQ ID NO: 8, or a variant having at least 40% sequence identity to SEQ ID NO: 8. For example, the variant may have at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identity to SEQ ID NO: 8.

[0165] Modifications of the endodomain can be selected depending on the desired level of STAT5 signaling.

[0166] The variant may have at least 80% sequence identity to SEQ ID NO:8. The variant may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:8. Any variant may be a functional variant and thus retain the ability to provide a signal to a cell in which the recombinant protein is expressed. In particular, in embodiments in which the endodomain of the recombinant protein does not contain any other functional (signaling) endodomain other than the variant EPOR endodomain, for example, in embodiments in which the endodomain of the recombinant protein consists of the variant EPOR endodomain, the variant is a functional variant and thus retains the ability to provide a signal to a cell in which the recombinant protein is expressed. The signal may in particular signal through the JAK-STAT signaling pathway, in particular the JAK2-STAT5 signaling pathway, and thus may comprise at least one JAK binding motif (e.g., a JAK2 binding motif) and at least one STAT association motif (e.g., a STAT5 association motif) as described herein. The variant EPOR endodomain may, for example, retain at least the tyrosine (Y) residue at position 368 of SEQ ID NO: 1 (Y95 of SEQ ID NO: 8) and / or at least the tyrosine (Y) residue at position 426 of SEQ ID NO: 1 (Y153 of SEQ ID NO: 8). For example, the variant EPOR endodomain may retain only one or both of the tyrosine (Y) residue at position 368 of SEQ ID NO: 1 (Y95 of SEQ ID NO: 8) and the tyrosine (Y) residue at position 426 of SEQ ID NO: 1 (Y153 of SEQ ID NO: 8).

[0167] Suitably, the variant may comprise at least one modification to reduce (e.g., eliminate) the binding of SHP1 to the EPOR endodomain sequence, and thus reduce the negative effect of SHP1 on JAK2. SHP1, also known as SHIP1, is a tyrosine phosphatase protein that comprises an SH2 domain and has the amino acid sequence shown in SEQ ID NO:61. SHP1 is known to associate with activated EPOR and negatively regulate the signal transduction induced by phosphorylation by JAK2. SHP1 can associate with Y181 and Y183 of the EPOR endodomain of SEQ ID NO:8. Thus, the present invention encompasses the use of EPOR endodomain sequences in which Y181 and / or Y183 of SEQ ID NO:8 have been modified to reduce the negative effects of SHP1 (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%, e.g., up to 100%). In another view, modification of Y181 and / or Y183 of SEQ ID NO:8 may increase signaling to cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In particular, Y181 and / or Y183 may be deleted from the EPOR endodomain sequences used in the endodomain herein or substituted with different amino acids. For example, the variant EPOR endodomain may comprise a deletion of a contiguous amino acid sequence encompassing Y181 and Y183 of SEQ ID NO: 8. The contiguous amino acid sequence encompassing Y181 and Y183 of SEQ ID NO: 8 may be at least 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids in length. For example, the contiguous amino acid sequence encompassing Y181 and Y183 of SEQ ID NO: 8 may be at least 55 amino acids in length, e.g., at least 60, 65, 70, or 75 amino acids in length.For example, the sequence of consecutive amino acids encompassing Y181 and Y183 of SEQ ID NO:8 may be up to 140, 135, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, or 80 amino acids in length. For example, the variant EPOR endodomain may comprise or consist of a deletion of amino acids 181-235 of SEQ ID NO:8 (amino acids 454-508 of SEQ ID NO:1). For example, the variant EPOR endodomain may comprise or consist of a deletion of amino acids 106-235 of SEQ ID NO:8 (amino acids 379-508 of SEQ ID NO:1). For example, the variant EPO endodomain may comprise or consist of a deletion of amino acids 161-235 of SEQ ID NO:8 (amino acids 434-508 of SEQ ID NO:1). Alternatively, Y181 and / or Y183 may be unmodified, particularly if increased signaling to the cell is not required.

[0168] Alternatively, the endodomain may comprise or consist of a truncated EPOR endodomain as shown in SEQ ID NO: 62, or a variant having at least 80% sequence identity to SEQ ID NO: 62. The variant may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 62. Thus, typically, the truncated EPOR endodomain may be truncated at least in the portion of the endodomain comprising Y181 and Y183 of SEQ ID NO: 8. The variant may, for example, comprise the same truncation as SEQ ID NO: 62, and thus the only difference from SEQ ID NO: 62 is a deletion, substitution, or insertion within the sequence of SEQ ID NO: 62. As mentioned above, any variant may be a functional EPOR endodomain variant.

[0169] The endodomain of the recombinant protein may comprise or consist of a truncated EPOR endodomain shown as SEQ ID NO: 106, or a variant having at least 80% sequence identity to SEQ ID NO: 106. The variant may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 106. The variant may be truncated at least in the portion of the endodomain comprising Y181 and Y183 of SEQ ID NO: 8. The variant may, for example, comprise the same truncation as SEQ ID NO: 106, and thus the only difference from SEQ ID NO: 106 is a deletion, substitution, or insertion within the sequence of SEQ ID NO: 106. As mentioned above, any variant may be a functional EPOR endodomain variant.

[0170] The endodomain of the recombinant protein may comprise or consist of a truncated EPOR endodomain shown as SEQ ID NO: 107, or a variant having at least 80% sequence identity to SEQ ID NO: 107. The variant may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 107. The variant may be truncated at least in the portion of the endodomain comprising Y181 and Y183 of SEQ ID NO: 8. The variant may, for example, comprise the same truncation as SEQ ID NO: 107, and thus the only difference from SEQ ID NO: 107 is a deletion, substitution, or insertion within the sequence of SEQ ID NO: 107. As mentioned above, any variant may be a functional EPOR endodomain variant.

[0171] The variant EPOR endodomain may, for example, comprise an insertion of one or more amino acids. In certain embodiments, the insertion may be at the C-terminus of the variant EPOR endodomain, which may be at the C-terminus of the recombinant protein if the variant EPOR endodomain is located at the C-terminus of the recombinant protein. However, the insertion may be elsewhere within the variant EPOR endodomain. For example, the variant EPOR endodomain may comprise an insertion of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. For example, the variant EPOR endodomain may comprise an insertion of 1 to 30 amino acids, such as 2 to 20, for example 5 to 15 amino acids. For example, the variant EPOR endodomain may comprise an insertion of at least 5, 6, 7, 8, 9, or 10 amino acids, for example at its C-terminus. The at least 5 amino acids may have (may comprise or consist of) the sequence MDTVP (SEQ ID NO: 108) or a sequence that differs from SEQ ID NO: 108 by no more than 1 or 2 amino acids (e.g. deletion and / or substitution). The at least 6 amino acids may have the sequence SMDTVP (SEQ ID NO: 109) or a sequence that differs from SEQ ID NO: 109 by no more than 1 or 2 amino acids (e.g. deletion and / or substitution). The at least 7 amino acids may have (may comprise or consist of) the sequence ASMDTVP (SEQ ID NO: 110) or a sequence that differs from SEQ ID NO: 110 by no more than 1 or 2 amino acids (e.g. deletion and / or substitution). The at least 8 amino acids may have (may comprise or consist of) the sequence LASMDTVP (SEQ ID NO: 111) or a sequence that differs from SEQ ID NO: 111 by no more than 1 or 2 amino acids (e.g. deletion and / or substitution). The at least 9 amino acids may have (may comprise or consist of) the sequence ALASMDTVP (SEQ ID NO:112), or a sequence which differs from SEQ ID NO:112 by no more than 1 or 2 amino acids (e.g., deletions and / or substitutions).The at least 10 amino acids may have (may comprise or consist of) the sequence PALASMDTVP (SEQ ID NO: 113), or a sequence that differs from SEQ ID NO: 113 by no more than 1 or 2 amino acids (e.g., deletion and / or substitution). For example, the variant EPOR endodomain may comprise, e.g., at its C-terminus, an insertion of 5 amino acids (e.g., SEQ ID NO: 108), 6 amino acids (e.g., SEQ ID NO: 109), 7 amino acids (e.g., SEQ ID NO: 110), 8 amino acids (e.g., SEQ ID NO: 111), 9 amino acids (e.g., SEQ ID NO: 112), or 10 amino acids (e.g., SEQ ID NO: 113). In certain embodiments, the variant EPOR endodomain may comprise, e.g., at its C-terminus, an insertion of at least 10, e.g., 10 amino acids, e.g., the at least 10 amino acids have the sequence of SEQ ID NO: 113, or a sequence that differs from SEQ ID NO: 113 by no more than 1 or 2 amino acids. The inserted amino acid may be located at the C-terminus of a wild-type EPOR endodomain (i.e., at the C-terminus of SEQ ID NO: 8) or at the C-terminus of an EPOR endodomain that also has other modifications, for example at the C-terminus of a truncated EPOR endodomain described herein (for example at the C-terminus of SEQ ID NO: 62, SEQ ID NO: 106, or SEQ ID NO: 107). Alternatively, the insertion may be located within an EPOR endodomain that would otherwise be a wild-type EPOR endodomain (other than the insertion) or within an EPOR endodomain that also has other modifications, for example within a truncated EPOR endodomain (for example within SEQ ID NO: 62, SEQ ID NO: 106, or SEQ ID NO: 107).

[0172] One or more amino acids inserted at the C-terminus of the variant EPOR endodomain can, for example, increase or stabilize cell surface expression of the recombinant protein, and / or increase sensitivity to EPO, and / or increase activation of the JAK-STAT signaling pathway when expressed in a cell (e.g., Treg cell) compared to the same cell expressing the same recombinant protein without the insertion. The increase can be at least 10%, 20%, 30%, 40%, or 50%. Thus, one or more amino acids inserted at the C-terminus of the variant EPOR endodomain can be particularly useful in embodiments in which a portion of the exodomain derived from the extracellular domain of EPOR retains the ability to bind EPO, as described in exemplary embodiments herein. Increased sensitivity to EPO can be determined by measuring activation of JAK-STAT signaling, as described elsewhere herein. A recombinant protein with increased sensitivity to EPO provides a greater JAK-STAT signal at the same EPO concentration (particularly at low EPO concentrations, such as about 0.1 U / ml or less, e.g., about 0.01 U / ml or less, or about 0.001 U / ml or less).

[0173] The endodomain of the recombinant protein may, for example, comprise or consist of a variant EPOR endodomain as shown in SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 116, or a variant thereof having at least 80% sequence identity to SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 116. The variant may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 116, respectively. The variant may be truncated at least in the portion of the endodomain comprising Y181 and Y183 of SEQ ID NO: 8. As mentioned above, any variant may be a functional EPOR endodomain variant.

[0174] Transphosphorylation of both JAK1 / JAK2 and JAK3 increases the activity of STAT, e.g., STAT5, thereby stabilizing its activity. As described above, the endodomain, more specifically its tyrosine kinase activation domain, may further comprise a JAK3 binding motif. The "JAK3 binding motif" herein refers to a BOX motif that provides the tyrosine kinase JAK3. Suitable JAK3 binding motifs are described, for example, by Ferrao & Lupardus (Frontiers in Endocrinology; 2017; 8(71); incorporated herein by reference).

[0175] Methods well known in the art for determining protein-protein interactions may be used to determine whether the motif is capable of binding to JAK3, for example, co-immunoprecipitation followed by Western blot.

[0176] The JAK3 binding motif may occur intrinsically within the cytoplasmic domain of a transmembrane protein.

[0177] For example, the JAK3 binding motif may be derived from an IL-2Rγ polypeptide. A functional truncated or variant IL2Rγ polypeptide may be used within the endodomain, where the functional truncated or variant IL2Rγ polypeptide retains JAK3 binding activity (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the binding activity of IL2Rγ). In particular, a truncated IL2Rγ containing a JAK3 binding motif and a truncated IL2Rβ containing a STAT5 association motif, as well as a JAK1 binding motif, may be included in the endodomain defined herein. A functional truncate may provide the advantage of reducing the construct size for expression.

[0178] The JAK3 binding motif may comprise or consist of the amino acid motif sequence shown as SEQ ID NO:55 or SEQ ID NO:56, or a variant thereof capable of binding to JAK3 (e.g., a functional variant or fragment having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:55 or 56).

[0179] The variant may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:55 or SEQ ID NO:56.

[0180] In certain embodiments, the signaling domain comprises one or more JAK1 binding domains and at least one JAK3 binding domain / motif (eg, at least two or three JAK3 binding domains / motifs).

[0181] Those skilled in the art will understand that the polynucleotide sequence encoding the JAK3 binding domain may be located upstream or downstream (5' or 3') of the polynucleotide sequence encoding the tyrosine effector domain, e.g., STAT association motif, such as STAT5, and JAK1 and / or JAK2 binding motif. Typically, the JAK1 and / or JAK2 binding motif is upstream (5') of the tyrosine effector domain, e.g., STAT / STAT5, but this can vary. In particular, the polynucleotide encoding the JAK3 binding domain may be located downstream (3') of the polynucleotide encoding the STAT association motif and JAK1 / JAK2 binding motif. Thus, in another view, in the endodomains described herein, the JAK3 binding domain may be N-terminal or C-terminal, preferably C-terminal, to the tyrosine effector domain (e.g., STAT association motif) and JAK1 and / or JAK2 binding domain. In one embodiment, the JAK3 binding domain and the STAT association motif / JAK1 / 2 binding domain are located directly adjacent to each other (i.e., not distally separated by sequence). In certain embodiments, the JAK3-binding domain is translated in reverse, and thus the JAK3-binding motif may comprise the reverse sequence of SEQ ID NO: 55 or SEQ ID NO: 56 (e.g., the sequence set forth in SEQ ID NO: 63). Thus, a polynucleotide encoding the signaling domain may comprise the following base sequences: 5'-3' JAK1, 5'-3' STAT association motif, 3'-5' JAK3.

[0182] In certain embodiments, there may be a linker or hinge between the JAK3 binding motif and the STAT association motif / JAK1 or JAK2 binding motif, said linker or hinge may comprise or consist of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 amino acids, for example at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 glycine residues. In a most specific embodiment, the endodomain comprises a first amino acid sequence derived from IL2Rγ that includes a JAK3 binding domain (e.g., SEQ ID NO: 55 or SEQ ID NO: 56) and a second amino acid sequence derived from IL2Rβ that includes a STAT5 association motif and a JAK1 binding motif (e.g., SEQ ID NO: 43 or SEQ ID NO: 44), wherein the first and second amino acid sequences are connected or joined by a linker or hinge.

[0183] The endodomain may provide other signaling functions (e.g., signaling functions that can provide pro-survival or sustaining signals, which are signals that maintain the phenotype or induce activation or function of a cell, other than providing a STAT signal), and therefore may contain additional domains that can provide such signaling functions.

[0184] The endodomain may further comprise an intracellular signaling domain, such as, for example, the zeta chain endodomain of a T cell receptor or its homologues (e.g., eta chain, FcεR1 gamma and beta chains, MB1 (Igα) chain, B29 (Igβ) chain, etc.), CD3 polypeptide domains (Δ, δ, and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction such as CD2, CD5, and CD28. The intracellular signaling domain may comprise the human CD3 zeta chain endodomain, FcyRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), or a combination thereof.

[0185] Thus, the endodomain may comprise the intracellular signalling domain of the human CD3 zeta chain, which in one embodiment comprises or consists of the following sequence: UNIPROT:P20963, CD3Z_human, 31-143 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 64)

[0186] In one embodiment, the endodomain comprises an intracellular signaling domain comprising an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO:64.

[0187] The intracellular signaling domain of the chimeric protein may comprise the CD28 signaling domain described below. RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 65)

[0188] In one embodiment, the intracellular signaling domain comprises a signaling motif having at least 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO:65.

[0189] The intracellular signalling domain of the endodomain may comprise the CD27 signalling domain described below. QRRKYRSNKGESVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 66)

[0190] In one embodiment, the intracellular signaling domain comprises a signaling motif having at least 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO:66.

[0191] Additional intracellular signaling domains will be apparent to those of skill in the art and may be used in connection with alternative embodiments of the invention.

[0192] In this embodiment, the endodomain may contain additional domains or sequences that provide the cell in which the endodomain is expressed with transcription factor activity, e.g., a transcription factor important for the phenotype or function of the cell. For example, in the case of Treg, the signaling domain may further provide the cell with FOXP3, c-Rel, Runx, Ets-1, CREB, NFAT, and / or JunB (directly or indirectly). In particular, the endodomain may be capable of providing the cell with a FOXP3 activation or induction signal. In one embodiment, the endodomain may comprise FOXP3 (or any functional variant, truncated, or isoform thereof), where FOXP3 may be cleavable from the chimeric protein upon induction by CID (e.g., using the Notch system). In this case, any cleavable portion (e.g., FOXP3) is present at the C-terminus of the endodomain. As described above, the various domains, and individual portions of the domains (e.g., motifs within the endodomain) may be linked to each other by linkers.

[0193] As used herein, a linker is an amino acid sequence that connects one domain or part of a protein to another domain or part. A linker sequence can be any amino acid sequence that has the function of linking or connecting two domains or parts of them so that they can perform their functions. Thus, a linker can space the elements that are connected.

[0194] The nature of the linker can vary and is not limited with respect to its amino acid composition and / or sequence. However, the linker may be a flexible linker. Thus, the linker may comprise or consist of amino acids known to confer flexibility to the linker (as opposed to a rigid linker).

[0195] Flexible linker is a category of linker sequence that is well known and described in the art.Linker sequence is generally known as a sequence that can be used to link or join proteins or protein domains, for example, to create fusion or chimeric proteins or multifunctional proteins or polypeptides.They have different characteristics, for example, they can be flexible, rigid, or cleavable.Protein linkers are reviewed, for example, in Chen et al., 2013, Advanced Drug Delivery Reviews 65, 1357-1369, where the category of flexible linker is compared with the category of rigid linker and cleavable linker. Flexible linkers are also described in Klein et al., 2014, Protein Engineering Design and Selection, 27(10), 325-330; van Rosmalen et al., 2017, Biochemistry, 56,6565-6574; and Chichili et al., 2013, Protein Science, 22, 153-167.

[0196] A flexible linker is a linker that allows some degree of movement between the linked domains or components. Flexible linkers are generally composed of small, non-polar (such as Gly) or polar (such as Ser or Thr) amino acid residues. The small size of the amino acids allows them to be flexible and allow mobility of the connected moieties (domains or components). The incorporation of polar amino acids can maintain the stability of the linker in an aqueous environment by forming hydrogen bonds with water molecules. The most commonly used flexible linkers have a sequence that is mainly composed of Ser and Gly residues (the so-called "GS linker"). However, many other flexible linkers have been described (e.g., Chen et al, 2013, supra), which may contain additional amino acids (e.g., Thr and / or Ala and / or Lys and / or Glu) that may improve solubility. Any flexible linker known and reported in the art can be used.

[0197] GS linkers, and more specifically the use of GS ("Gly-Ser") domains in the linker, represent one preferred class of linkers, as such linkers may allow the length of the linker to be easily varied by altering the number of repeats of the GS domain. However, flexible linkers are not limited to those based on "GS" repeats, and other linkers containing Ser and Gly residues dispersed throughout the linker sequence have been reported, including by Chen et al. (supra).

[0198] In one embodiment, the linker sequence comprises at least one Gly-Ser domain consisting of only Ser and Gly residues. In such an embodiment, the linker may comprise 15 or less other amino acid residues, e.g., 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 6 or less, 7 or less, 5 or less, or 4 or less other amino acid residues.

[0199] The Gly-Ser domain may have the formula: (S)q-[(G)m-(S)m]n-(G)p Here, q is 0 or 1, m is an integer from 1 to 8, n is an integer of 1 or greater (eg, 1 to 8, more specifically, 1 to 6), and p is an integer of 0 or 1 to 3.

[0200] More specifically, the Gly-Ser domain may have the formula: (i) S-[(G)mS]n; (ii) [(G)mS]n; or (iii) [(G)mS]n-(G)p Here, m is an integer of 2 to 8 (eg, 3 to 4), n is an integer of 1 or greater (eg, 1 to 8, more specifically, 1 to 6), and p is 0 or an integer of 1 to 3. In a representative example, the Gly-Ser domain may have the formula: S-[GGGGS]n Here, n is an integer of 1 or more (preferably 1 to 8, or 1 to 6, 1 to 5, 1 to 4, or 1 to 3). In the above formula, the sequence GGGGS is SEQ ID NO:70.

[0201] However, not all linkers need to be flexible, and in some cases the linker sequence may not be a flexible linker sequence. If the linker connects an interaction domain, or D1 / Ht1 or D2 / Ht2 to a signaling domain, it is preferably a flexible linker.

[0202] The length of the linker is not critical, although it may be desirable to have a shorter or longer linker sequence, depending on which domains etc. are being linked.

[0203] In some cases, the linker may be from any one of 2, 3, 4, 5, or 6 amino acids in length to any one of 24, 23, 22, or 21. In other cases, the linker may be from any one of 2, 3, 4, 5, or 6 amino acids in length to any one of 21, 20, 19, 18, 17, 16, or 15 amino acids in length. In other cases, the linker may be intermediate between these ranges, e.g., 6-21, 6-20, 7-20, 8-20, 9-20, 10-20, 8-18, 9-18, 10-18, 9-17, 10-17, 9-16, 10-16, etc. Thus, the linker may be within a range consisting of any of the above integers.

[0204] In other cases, the linker may be longer, for example, from any one of 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 amino acids in length to any one of 100, 90, 80, 70, 60, 50, 45, 40, 30, 28, 25, or 24 amino acids in length. In other cases, it may be intermediate between this range and any of the ranges listed above. Thus, it may be within a range consisting of any of the integers listed above.

[0205] GS linkers, and more specifically the use of GS ("Gly-Ser") domains in the linker, represent an advantageous type of linker to use because such linkers may allow one to easily vary the length of the linker by varying the number of repeats of the GS domain. However, flexible linkers are not limited to those based on "GS" repeats, and other linkers containing Ser and Gly residues dispersed throughout the linker sequence have been reported, including by Chen et al. (supra).

[0206] The linker sequence may consist only of, i.e. consist of, one or more Gly-Ser domains as described or defined above. However, as described above, the linker sequence may comprise one or more Gly-Ser domains and additional amino acids. The additional amino acids may be present at one or both ends of the Gly-Ser domain or at one or both ends of a repeated section of the Gly-Ser domain. Thus, the additional amino acids, which may be other amino acids, may be located at one or both ends of the linker sequence, for example flanking the Gly-Ser domain(s). In other embodiments, the additional amino acids may be present between the Gly-Ser domains. For example, two Gly-Ser domains may flank a stretch of other amino acids in the linker sequence. Furthermore, as described above, in other linkers, the GS domain may not be repeated, but only G and / or S residues, or short domains such as GS, distributed along the length or sequence.

[0207] Representative examples of linker sequences are shown below. ETSGGGGSRL (SEQ ID NO:68) SGGGGSGGGGSGGGGS (SEQ ID NO:69) S(GGGGS)1-5 (wherein GGGGS is SEQ ID NO: 70) (GGGGS)1-5 (wherein GGGGS is SEQ ID NO: 70) SGGGGSGGGGS (SEQ ID NO:71) S(GGGS)1-5 (wherein GGGS is SEQ ID NO: 67) (GGGS)1-5, where GGGS is SEQ ID NO:67 SGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:72) SGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 73) S(GGGGGS)1-5 (wherein GGGGGS is SEQ ID NO: 74) (GGGGGS)1-5 (wherein GGGGGS is SEQ ID NO:74) S(GGGGGGS)1-5 (wherein GGGGGGS is SEQ ID NO: 75) (GGGGGGS)1-5 (wherein GGGGGGS is SEQ ID NO: 75) G6 (SEQ ID NO: 76) G8 (SEQ ID NO:77) KESGSVSSEQLAQFRSLD (SEQ ID NO: 78) EGKSSGSGSESKST (SEQ ID NO:79) GSAGSAAGSGEF (SEQ ID NO: 80) SGGGGSAGSAAGSGEF (SEQ ID NO: 81) SGGGLLLLLLLLGGGS (SEQ ID NO:82) SGGGAAAAAAAAGGGS (SEQ ID NO:83) SGGGAAAAAAAAAAAAAAAAGGGS (SEQ ID NO:84) SGALGGLALAGLLLAGLGLGAAGS (SEQ ID NO: 85) SLSLSPGGGGGPAR (SEQ ID NO:86) SLSLSPGGGGGPARSLSLSPGGGGG (SEQ ID NO: 87) GSSGSS (SEQ ID NO:88) GSSSSSS (SEQ ID NO: 89) GGSSSS (SEQ ID NO: 90) GSSSSS (SEQ ID NO: 91) SGGGGS (SEQ ID NO:92)

[0208] For linking motifs within a signaling domain, the following linkers can be mentioned: GGGGSGGGGSGGGGS (SEQ ID NO:93) GGGGG (SEQ ID NO:94) GGGGSGGGGS (SEQ ID NO:95) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 96) GGGGGGG (SEQ ID NO:97)

[0209] As used herein, the terms "polynucleotide" and "nucleic acid" are intended to be synonymous with each other.

[0210] Those skilled in the art will understand that many different polynucleotides and nucleic acids can code for the same polypeptide as a result of the degeneracy of the genetic code.Furthermore, it should be understood that those skilled in the art can use routine techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of the particular host organism in which the polypeptide is expressed.The sequences encoding the various domains and motifs, etc. described herein are known and available in the art, any of which can be used or modified for use herein.

[0211] The nucleic acids of the present invention may include DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides containing synthetic or modified nucleotides therein. Many different types of modifications are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains to the 3' and / or 5' ends of the molecule. For the purposes of the uses described herein, it should be understood that polynucleotides may be modified by any method available in the art. Such modifications may be made to improve the in vivo activity or life span of the polynucleotide of interest. The terms "variant", "homolog", or "derivative" in reference to a base sequence include any substitution, mutation, modification, replacement, deletion, or addition of one (or more) nucleic acid from or to the sequence.

[0212] Nucleic acid molecules / polynucleotides / sequences, such as DNA nucleic acid molecules / polynucleotides / sequences, may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned using standard techniques. Longer nucleic acid molecules / polynucleotides / sequences will generally be made using recombinant means, for example using polymerase chain reaction (PCR) cloning techniques. This will involve making a pair of primers (e.g., of about 15-30 nucleotides) that flank the target sequence one wishes to clone, contacting the primers with mRNA or cDNA obtained from an animal or human cell, carrying out a polymerase chain reaction under conditions that result in amplification of the desired region, isolating the amplified fragment (e.g., by purifying the reaction mixture on an agarose gel), and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

[0213] The nucleic acid construct may comprise the nucleic acid molecule together with one or more other base sequences, for example, regulatory sequences, such as expression control sequences, and / or other coding sequences. In particular, the other coding sequences may code for a protein of interest, which may be a therapeutic protein.

[0214] As described above, the recombinant protein may be co-expressed with another protein of interest, such as a second protein or receptor, in particular an antigen receptor, such as a CAR or TCR or a derivative thereof (such as a TCR-CAR construct, or a single chain TCR construct, etc.). The coding sequence of such an additional protein, such as a receptor, may be included within the construct.

[0215] The recombinant protein may be co-expressed with a safety switch polypeptide, which provides a suicide moiety to the cell in which it is expressed. This is useful as a safety mechanism to allow cells administered to a subject to be removed if the need arises, or indeed more generally, as desired or necessary, for example, after the cells have exerted or completed their therapeutic effect. Alternatively, as described above, the recombinant protein may include a suicide moiety.

[0216] A suicide moiety has the ability to induce cell death, or more generally, cell elimination or deletion. One example of a suicide moiety is a suicide protein encoded by a suicide gene, which can be expressed in or on a cell together with a desired transgene, in this case a recombinant protein (and optionally a CAR or other receptor co-expressed by the cell with the recombinant protein), and when expressed, causes the cell to be deleted and turns off the expression of the transgene (CAR). As used herein, a suicide moiety is a suicide polypeptide, which is a polypeptide that can cause a cell to be deleted under permissive conditions, i.e., under induced or turned-on conditions.

[0217] The suicide moiety may be a polypeptide or amino acid sequence that can be activated to exert cell elimination activity by an activating agent administered to the subject, or that is active in the presence of a substrate that can be administered to the subject and exerts cell elimination activity. In certain embodiments, the suicide moiety may be targeted by a separate cell elimination agent administered to the subject. The cell elimination agent can target the cells to be eliminated by binding to the suicide moiety. In particular, the suicide moiety may be recognized by an antibody, and upon binding of the antibody to the safety switch polypeptide when expressed on the cell surface, the cells are eliminated or eliminated.

[0218] The suicide moiety may be HSV-TK or iCasp9, as known in the art, however, in other instances, the suicide moiety may be or contain an epitope recognized by a cell-depleting antibody or other binding molecule capable of inducing cell depletion.

[0219] The term "delete" as used herein in the context of cell removal is synonymous with "remove" or "ablate" or "eliminate". The term is used to encompass cell death or cell proliferation inhibition such that the number of cells in a subject is reduced. 100% complete removal is desirable but not always achieved. Reducing the number of cells or inhibiting cell proliferation in a subject may be sufficient to have a beneficial effect.

[0220] In particular, the suicide moiety may be a CD20 epitope recognized by the antibody rituximab. Thus, in the safety switch polypeptide, the suicide moiety may comprise a minimal epitope based on the epitope from CD20 recognized by the antibody rituximab. More specifically, the polypeptide may comprise two CD20 epitopes R1 and R2 spaced apart by a linker L.

[0221] A safety switch based on the Rituximab epitope is described in WO2013 / 15339. Peptides that mimic the epitopes recognized by Rituximab (so-called mimotopes) have been developed and are used in WO2013 / 15339 as suicide moieties in a combined suicide-marker polypeptide construct that also contains a CD34 minimal epitope as a marker moiety. In particular, WO2013 / 15339 discloses a polypeptide called RQR8 having the sequence shown in SEQ ID NO: 99, which comprises two CD20 minimal epitopes separated from each other by a spacer sequence and an intervening CD34 marker sequence, and further linked to a stalk sequence that allows the polypeptide to protrude from the surface of the cell in which it is expressed. The safety switch polypeptide may be RQR8 or a variant having at least 80% sequence identity to RQR8, such as at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to RQR8. Other safety switch polypeptides that may be used as the basis for the safety switch domain include those described in our co-pending PCT Patent Application No. PCT / EP2021 / 064053 (WO2021 / 239812).

[0222] Other polypeptides that may be co-expressed with the chimeric or recombinant proteins described herein include transcription factors, growth factors, or other factors that may assist in enhancing the functionality of cell survival. For example, the transcription factor FOXP3 may be used to maintain the suppressive phenotype of Treg cells. "FOXP3" is an abbreviation for Forkhead Box P3 protein. FOXP3 is a member of the FOX protein family of transcription factors and functions as a master regulator of regulatory pathways in the development and function of regulatory T cells. "FOXP3" herein encompasses variants, isoforms, and functional fragments of FOXP3. "FOXP3 polypeptide" is a polypeptide that has FOXP3 activity, i.e., a polypeptide that can bind to FOXP3 target DNA and function as a transcription factor to regulate Treg development and function. Expressing FOXP3 in Tregs together with the recombinant proteins described herein may further aid in maintaining the Treg phenotype.

[0223] Co-expression of FOXP3 with a constitutively active recombinant receptor increases FOXP3 expression in cells and may help maintain the suppressive phenotype of Treg cells or cells with a regulatory phenotype.

[0224] "Increasing FOXP3 expression" means increasing the level of FOXP3 mRNA and / or protein in a cell (or a population of cells) compared to a corresponding cell (or a population of cells) that is not modified by introducing a nucleic acid molecule or vector.For example, the level of FOXP3 mRNA and / or protein in a cell (or a population of such cells) modified according to the present invention can be increased at least 1.5 times, at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 150 times the level in a corresponding cell (or a population of such cells) that is not modified according to the present invention.Preferably, the cell is a Treg and the population of the cells is a population of Tregs.

[0225] Suitably, the level of FOXP3 mRNA and / or protein in the modified cell (or population of such cells) may be increased by at least 1.5-fold, at least 2-fold, at least 5-fold over the level in a corresponding cell (or population of such cells) that has not been so modified. Preferably, said cell is a Treg and said population of cells is a population of Tregs.

[0226] The techniques for measuring the level of specific mRNA and protein are well known in the art.The mRNA level in a population of cells such as Treg can be measured by techniques such as Affymetrix's ebioscience prime flow RNA assay, Northern blotting, serial analysis of gene expression (SAGE), or quantitative polymerase chain reaction (qPCR).The protein level in a population of cells can be measured by techniques such as flow cytometry, high performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LC / MS), Western blotting, or enzyme-linked immunosorbent assay (ELISA).

[0227] "FOXP3 polypeptide" is a polypeptide having FOXP3 activity, i.e., a polypeptide that can bind to FOXP3 target DNA and function as a transcription factor to regulate the development and function of Treg. In particular, FOXP3 polypeptide may have the same or similar activity as wild-type FOXP3 (SEQ ID NO: 129), for example, at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, or 150% of the activity of wild-type FOXP3 polypeptide. Thus, FOXP3 polypeptides encoded by the base sequences in the nucleic acids or vectors described herein may have increased or decreased activity compared to wild-type FOXP3. Techniques for measuring transcription factor activity are well known in the art. For example, transcription factor DNA binding activity can be measured by ChIP. Transcriptional regulatory activity of a transcription factor can be measured by quantifying the expression level of the gene it regulates. Gene expression can be quantified by measuring the level of mRNA and / or protein produced by the gene using techniques such as Northern blotting, SAGE, qPCR, HPLC, LC / MS, Western blotting, or ELISA. Genes regulated by FOXP3 include cytokines such as IL-2, IL-4, and IFN-γ (Siegler et al. Annu. Rev. Immunol. 2006, 24: 209-26, incorporated herein by reference). As described in more detail below, FOXP3 or FOXP3 polypeptide includes functional fragments, variants, and isoforms thereof, for example, SEQ ID NO:32.

[0228] "Functional fragment of FOXP3" may refer to a part or region of a FOXP3 polypeptide or a polynucleotide encoding a FOXP3 polypeptide (i.e., a nucleotide sequence) that has the same or similar activity as a full-length FOXP3 polypeptide or polynucleotide. The functional fragment may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the activity of a full-length FOXP3 polypeptide or polynucleotide. A person skilled in the art will be able to generate functional fragments based on the known structural and functional characteristics of FOXP3. These are described, for example, in Song, X. et al., 2012. Cell reports, 1(6), pp. 665-675; Lopes, JE et al., 2006. The Journal of Immunology, 177(5), pp. 3133-3142; and Lozano, T. et al., 2013. Frontiers in oncology, 3, p. 294. In addition, N- and C-terminal truncated FOXP3 fragments having the sequence of SEQ ID NO: 37, as described below, are described in WO2019 / 241549 (herein incorporated by reference).

[0229] A "FOXP3 variant" may comprise an amino acid or nucleotide sequence that may have at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identity, preferably at least 95% or at least 97% or at least 99% identity, to a FOXP3 polypeptide or a polynucleotide encoding a FOXP3 polypeptide, e.g., SEQ ID NO: 129. A FOXP3 variant may have the same or similar activity as a wild-type FOXP3 polypeptide or polynucleotide, e.g., at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, or 150% of the activity of a wild-type FOXP3 polypeptide or polynucleotide. Those skilled in the art can generate FOXP3 variants based on the known structural and functional characteristics of FOXP3 and / or using conservative substitutions. FOXP3 variants may have similar or the same turnover time (or degradation rate) in Treg cells compared to wild-type FOXP3, for example, at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of the turnover time (or degradation rate) of wild-type FOXP3 in Treg. Some FOXP3 variants may have reduced turnover time (or degradation rate) compared to wild-type FOXP3, for example, FOXP3 variants with amino acid substitutions at amino acid 418 and / or 422 of SEQ ID NO:32, such as S418E and / or S422A, as described in WO2019 / 241549 (herein incorporated by reference).

[0230] Suitably, the FOXP3 polypeptide encoded by the nucleic acid molecule or vector described herein may comprise or consist of the polypeptide sequence of human FOXP3, such as UniProtKB Accession No. Q9BZS1 (SEQ ID NO: 32), or a functional fragment or variant thereof.

[0231] In some embodiments of the invention, the FOXP3 polypeptide comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 32, or a functional fragment thereof. Suitably, the FOXP3 polypeptide comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 32, or a functional fragment thereof. In some embodiments, the FOXP3 polypeptide comprises or consists of SEQ ID NO: 32, or a functional fragment thereof.

[0232] In some embodiments, the FOXP3 polypeptide may include a mutation at residues 418 and / or 422 of SEQ ID NO:32, as described above.

[0233] In some embodiments of the invention, FOXP3 polypeptides may be truncated at the N-terminus and / or C-terminus to produce functional fragments. In particular, N- and C-terminally truncated functional fragments of FOXP3 may comprise or consist of the amino acid sequence of SEQ ID NO:37, or a functional variant having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:37.

[0234] Suitably, the FOXP3 polypeptide may be a variant of SEQ ID NO: 32, such as a naturally occurring variant. Suitably, the FOXP3 polypeptide is an isoform of SEQ ID NO: 32. For example, the FOXP3 polypeptide may comprise a deletion of amino acids at positions 72 to 106 relative to SEQ ID NO: 32. Alternatively, the FOXP3 polypeptide may comprise a deletion of amino acids at positions 246 to 272 relative to SEQ ID NO: 32.

[0235] The nucleic acid molecule or construct may further comprise a nucleic acid sequence encoding a selectable marker. Suitable selectable markers are well known in the art and include, but are not limited to, fluorescent proteins such as GFP. Suitably, the selectable marker may be a fluorescent protein, such as GFP, YFP, RFP, tdTomato, dsRed, or variants thereof. In some embodiments, the fluorescent protein is GFP or a GFP variant.

[0236] Suitably, the selectable marker / reporter domain may be a luciferase-based reporter, a PET reporter (e.g., sodium iodide symporter (NIS)), or a membrane protein (e.g., CD34, low affinity nerve growth factor receptor (LNGFR)).

[0237] The use of a selectable marker is advantageous because it allows cells (e.g., Tregs) into which a nucleic acid molecule, construct, or vector has been successfully introduced (so that the encoded chimeric protein or other encoded protein or polypeptide is expressed) to be selected and isolated from the starting cell population using common methods such as, for example, flow cytometry.

[0238] In yet a further embodiment, the recombinant protein may be co-expressed with a mutant calcineurin protein that is resistant to at least one calcineurin inhibitor, in particular a mutant calcineurin protein that is resistant to at least one calcineurin inhibitor and sensitive to at least one calcineurin inhibitor. Such calcineurin mutants are further described below. In such an embodiment, the nucleic acid molecule or construct may further comprise a base sequence that codes for such a mutant calcineurin.

[0239] When two or more coding sequences are expressed from a single nucleic acid molecule or construct, the two or more coding sequences may be linked by a sequence that allows their co-expression. In particular, this co-expression sequence (also called co-expression site) may allow the encoded proteins or polypeptides to be expressed as separate entities. For example, the construct may include an internal promoter, an internal ribosome entry sequence (IRES) sequence, or a sequence that codes for a cleavage site.

[0240] In particular, the co-expression sequence may code for a self-cleaving sequence between the encoded polypeptides. In particular, the self-cleaving sequence may be a self-cleaving peptide. Such a sequence self-cleaves during protein production. Self-cleaving peptides that may be used are known in the art and are described, for example, in Donnelly et al., Journal of General Virology, 2001, 82, 1027-1041 (incorporated herein by reference). It is believed that the 2A peptide and 2A-like peptides cause ribosome skipping, resulting in a form of cleavage in which the ribosome skips the formation of a peptide bond between the end of the 2A peptide and the downstream amino acid sequence. The "cleavage" occurs between the glycine and proline residues at the C-terminus of the 2A peptide. That is, the upstream cistron will have some additional residues added to its end, and the downstream cistron will start with a proline.

[0241] Suitable self-cleavage domains include the P2A, T2A, E2A, and F2A sequences shown in SEQ ID NOs: 100-103, respectively. These sequences may be modified to include the amino acid GSG at the N-terminus of the 2A peptide. Thus, sequences corresponding to SEQ ID NOs: 100-103, but with GSG at the N-terminus, are also included as possible options. Such modified alternative 2A sequences are known and reported in the art. Alternative 2A-like sequences that may be used are shown in Donnelly et al. (supra), e.g., TaV sequences.

[0242] The self-cleaving sequences contained in the nucleic acid molecules may be the same or different.

[0243] The self-cleavage sequence may contain additional cleavage sites that can be cleaved by common enzymes present in cells. This may help achieve complete removal of the 2A sequence after translation. Such additional cleavage sites may include, for example, furin cleavage sites. Such cleavage sites are known in the art and may include, for example, RXXR (SEQ ID NO: 104), for example RRKR (SEQ ID NO: 105).

[0244] The nucleic acid molecules / polynucleotides used herein may be codon-optimized. Codon optimization has been previously described in WO1999 / 41397 and WO2001 / 79518. Different cells use different codons. This codon bias corresponds to the bias in the relative abundance of certain tRNAs in cell types. Expression can be increased by changing codons in the sequence to match the relative abundance of the corresponding tRNA. Similarly, expression can be decreased by deliberately selecting codons whose corresponding tRNAs are known to be scarce in a particular cell type. In this way, the degree of translational control can be further increased.

[0245] The recombinant protein-encoding sequences and other coding sequences may be provided in a construct in which they are operably linked to a promoter. In some cases, different sequences may be operably linked to the same promoter. A "promoter" is a region of DNA that initiates transcription of a gene. The promoter is located upstream (towards the 5' region of the sense strand) in the DNA near the transcription start site of the gene. Any suitable promoter may be used, and the selection can be easily made by one of skill in the art. The promoter may be from any source, may be a viral promoter, or may be a eukaryotic promoter, including a mammalian promoter or a human promoter (i.e., a physiological promoter). In one embodiment, the promoter is a viral promoter. Particular promoters include LTR promoter, EFS (or functional truncates thereof), SFFV, PGK, and CMV. In one embodiment, the promoter is an SFFV or viral LTR promoter. "Operatively linked to the same promoter" means that transcription of each polynucleotide sequence may be initiated from the same promoter, and each sequence is positioned and oriented so that transcription is initiated from the promoter. A polynucleotide that is operably linked to a promoter is under the transcriptional control of the promoter.

[0246] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. As an example herein, some vectors used in recombinant nucleic acid technology can transfer an entity such as a segment of nucleic acid (e.g., a heterologous DNA segment, such as a heterologous cDNA segment) into a target cell. The vector can be a non-viral vector or a viral vector. Vectors used in recombinant nucleic acid technology include, but are not limited to, for example, plasmids, mRNA molecules (e.g., in vitro transcribed mRNA), chromosomes, artificial chromosomes, and viruses. The vector can also be, for example, naked nucleic acid (e.g., DNA). In the simplest form, the vector itself can be the base sequence of interest.

[0247] A vector as used herein may, for example, be a plasmid, mRNA or viral vector and may comprise a promoter (as described above) for the expression of a nucleic acid molecule / polynucleotide and, optionally, a regulator of that promoter.

[0248] In one embodiment, the vector is a viral vector, e.g., a retroviral vector, e.g., a lentiviral vector or a gamma retroviral vector.

[0249] The vector may further comprise an additional promoter, for example, in one embodiment, the promoter may be an LTR, such as a retroviral LTR or lentiviral LTR. Long terminal repeats (LTRs) are identical sequences of DNA that are repeated hundreds or thousands of times at both ends of proviral DNA formed by reverse transcription of retrotransposons or retroviral RNA. LTRs are used by viruses to insert genetic material into the host genome. The following signals for gene expression are present in the LTR: enhancers, promoters (which may have both transcription enhancers or regulatory elements), transcription initiation (e.g., capping), transcription terminators, and polyadenylation signals.

[0250] Suitably, the vector may comprise a 5'LTR and a 3'LTR.

[0251] The vector may contain one or more additional regulatory sequences that can act pre- or post-transcriptionally. "Regulatory sequence" refers to any sequence that promotes the expression of a polypeptide, for example, by increasing the expression of a transcript or enhancing mRNA stability. Suitable regulatory sequences include, for example, enhancer elements, post-transcriptional regulatory elements, and polyadenylation sites. Advantageously, the additional regulatory sequence may be present in the LTR.

[0252] Suitably, the vector may include, for example, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) operably linked to the promoter.

[0253] The vectors containing the nucleic acid molecules / polynucleotides may be introduced into cells using a variety of techniques known in the art, such as transformation or transduction. Several techniques are known in the art, including infection with recombinant viral vectors, such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, baculoviral vectors, herpes simplex viral vectors, direct injection of nucleic acids, and gene gun transformation.

[0254] Non-viral delivery systems include, but are not limited to, DNA transfer methods. Gene transfer includes the process of using non-viral vectors to deliver genes to target cells. Non-viral delivery systems can include liposomes or amphiphilic cell-penetrating peptides, preferably complexed with nucleic acid molecules or constructs.

[0255] Exemplary gene transfer methods include electroporation, DNA gene guns, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofection, cationic drug-mediated transfection, cationic facial amphiphiles (CFAs) (Nat. Biotechnol. (1996) 14: 556), and combinations thereof.

[0256] In some cases, the nucleic acid molecule may be designed to be used as a single construct encoding a recombinant protein and other polypeptides (e.g., a receptor or marker or other functional polypeptide or protein of interest), which will be contained in a single vector, although this does not exclude that the construct may be introduced into a cell in combination with other vectors encoding other polypeptides that may also be desired to be introduced into the cell, for example.

[0257] As described above, recombinant proteins may be co-expressed in or on cells in combination with CAR. The term "chimeric antigen receptor" or "CAR" herein refers to an engineered receptor that can confer antigen specificity to cells (e.g., Tregs). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. CARs typically include an extracellular domain that includes an antigen-specific targeting region, referred to herein as an antigen-binding domain, a transmembrane domain, an intracellular domain that optionally includes one or more costimulatory domains, and an intracellular signaling domain. The antigen-binding domain is typically linked to the transmembrane domain by a hinge domain. The design of CARs and the various domains that they may contain are well known in the art.

[0258] Binding of the CAR to a target antigen results in the delivery of an activation signal to the cell in which the CAR is expressed. Thus, the CAR directs the specificity of the engineered cell to the target antigen, and in particular to the cell expressing the target antigen.

[0259] The antigen-binding domain of the CAR may be derived from or obtained from any protein or polypeptide that binds (i.e. has affinity) to a desired target antigen, or more generally to a desired target molecule. It may be, for example, a ligand or receptor, or a physiological binding protein for the target molecule, or a part thereof, or a synthetic or derivative protein. The target molecule may generally be expressed on the surface of a cell, such as a target cell or a cell in the vicinity of the target cell (due to bystander effect), but may not be. Depending on the nature and specificity of the antigen-binding domain, the CAR may recognize a soluble molecule. This is the case, for example, when the antigen-binding domain is based on or derived from a cell receptor.

[0260] Antigen binding domains are most commonly derived from antibody variable chains (e.g. commonly in the form of an scFv), but may also be generated from other molecules such as T-cell receptor variable domains or, as discussed above, receptors for ligands or other binding molecules.

[0261] CAR is typically expressed as a polypeptide that also includes a signal sequence (also known as a leader sequence), in particular a signal sequence that targets CAR to the plasma membrane of a cell.Signal sequences are generally located next to or near the antigen-binding domain, generally upstream of the antigen-binding domain.Thus, the extracellular domain, or ectodomain, of CAR can include a signal sequence and an antigen-binding domain.

[0262] The antigen-binding domain provides the CAR with the ability to bind to a predetermined antigen of the binding partner. The antigen-binding domain preferably targets an antigen of clinical interest or an antigen at a disease site.

[0263] As described above, an antigen-binding domain may be a protein or peptide capable of specifically recognizing and binding to a biomolecule (e.g., a cell surface receptor or a component part thereof). Antigen-binding domains include naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partners for the binding partner biomolecule. Examples of antigen-specific targeting domains include antibodies or antibody fragments or derivatives, extracellular domains of receptors, ligands for cell surface molecules / receptors or their receptor binding domains, and tumor-binding proteins. As described below, the antigen-specific targeting domain may preferably be an antibody or antibody-derived, but may also include other antigen-specific targeting domains, such as antigen-specific targeting domains consisting of antigen peptide / MHC or HLA combinations capable of binding to the TCR of Tcon cells active at the site of transplantation, inflammation, or disease.

[0264] CAR can be directed to any desired target antigen or target molecule. This can be selected according to the intended therapy or condition to be treated. For example, it can be an antigen or molecule associated with a particular condition, or an antigen or molecule associated with the cell that is to be targeted to treat the condition. Typically, said antigen or molecule is a cell surface antigen or cell surface molecule.

[0265] The term "directed against" is synonymous with "specific for" or "anti". In other words, the CAR recognizes a target molecule. Thus, this term means that the CAR can specifically bind to a particular or given antigen, i.e., target. In particular, the antigen-binding domain of the CAR can specifically bind to a target molecule or target antigen (more specifically, when the CAR is expressed on the surface of a cell, particularly an immune effector cell). Specific binding can be distinguished from non-specific binding to a non-target molecule or non-target antigen. Thus, a cell expressing a CAR is directed or re-directed to specifically bind to a target cell expressing the target molecule or target antigen, particularly a target cell expressing the target antigen or target molecule on the cell surface.

[0266] Antigens targeted by the CAR include, but are not limited to, antigens expressed on cells associated with transplanted organs, autoimmune diseases, allergic diseases, and inflammatory diseases (e.g., neurodegenerative diseases). It will be understood by those skilled in the art that where the cells engineered to express a CAR are Treg cells or their precursors, the antigen will simply be present and / or expressed at the site of transplantation, inflammation, or disease due to the bystander effect of Treg cells.

[0267] Antigens expressed on cells associated with neurodegenerative diseases include antigens presented on glial cells, such as MOG.

[0268] Antigens associated with organ transplantation and / or cells associated with the transplanted organ include, but are not limited to, HLA antigens present in the transplanted organ but absent from the patient, and antigens whose expression is increased during transplant rejection, such as CCL19, MMP9, SLC1A3, MMP7, HMMR, TOP2A, GPNMB, PLA2G7, CXCL9, FABP5, GBP2, CD74, CXCL10, UBD, CD27, CD48, CXCL11, etc.

[0269] In one embodiment, the CAR is directed against an HLA antigen, in particular the HLA-A2 antigen.

[0270] Antibodies against such antigens are known in the art, and scFv can be conveniently obtained or generated based on known or available antibodies.In this regard, VH and VL sequences and CDR sequences are published to assist in the preparation of such antibody binding domains.For example, WO2020 / 044055 discloses, the disclosure of which is incorporated herein by reference.Any of the antigen binding domains disclosed in WO2020 / 044055, or CDR sequences, VH sequences, and / or VL sequences may be used.

[0271] As an example, the CAR may be a CAR that targets HLA-A2 (HLA-A2 is referred to herein as HLA-A * 02, HLA-A02, and HLA-A * 2). * 02 is a specific group of class I major histocompatibility complex (MHC) alleles at the HLA-A locus.

[0272] The antigen recognition domain may bind, preferably specifically bind, to one or more regions or epitopes in HLA-A2. An epitope, also known as an antigenic determinant, is the part of an antigen that is recognized by an antigen recognition domain (e.g., an antibody). That is, an epitope is the specific part of an antigen that an antibody binds to. Preferably, the antigen recognition domain binds, preferably specifically binds, to one region or epitope in HLA-A2.

[0273] Engineered cells, particularly T cells, may be generated by introducing a nucleic acid molecule, construct or vector defined herein by one of a number of means, such as transduction with a viral vector or gene transfer with DNA or RNA.

[0274] The cells are produced by introducing (eg, by transduction or transfection) a nucleic acid molecule, construct or vector defined herein into a cell.

[0275] Suitable cells are described further below, but the cells may be derived from a sample isolated from a subject, which may be a donor subject or a therapeutic subject (i.e., the cells may be autologous cells or donor cells, e.g., allogeneic cells, for introduction into another recipient).

[0276] The cells may be produced by a method comprising the steps of: (i) isolating or obtaining a cell-containing sample from a subject; and (ii) introducing (e.g., by transduction or transfection) a nucleic acid molecule, construct or vector defined herein into a cell-containing sample to obtain a population of engineered cells.

[0277] The cell into which the nucleic acid molecule, construct or vector is introduced may be referred to as a target cell. Before and / or after step (ii) of the above method, a target cell enriched sample may be isolated, enriched and / or generated from the cell-containing sample. For example, Treg (or other target cell) may be isolated, enriched and / or generated before and / or after step (ii) to isolate, enrich or generate a Treg enriched sample. Isolation and / or enrichment from the cell-containing sample may be performed after step (ii) to enrich cells and / or Treg (or other target cells) that contain the CAR, nucleic acid molecule / polynucleotide, construct and / or vector described herein.

[0278] Treg-enriched sample may be isolated or enriched by any method known to those skilled in the art, for example, by FACS and / or magnetic bead separation.Treg-enriched sample may be generated from a cell-containing sample by any method known to those skilled in the art, for example, by introducing DNA or RNA encoding FOXP3, and / or by ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells.Methods for isolating and / or enriching other target cells are known in the art.

[0279] The target cells may be Treg cells, or their precursor or progenitor cells.

[0280] "Engineered cells" refers to cells that have been modified to contain or express a polynucleotide that is not naturally encoded by the cell. Methods of engineering cells are well known in the art, including, but not limited to, genetic modification of cells, such as by transduction, e.g., retroviral or lentiviral transduction, gene transfer (e.g., DNA or RNA transient transformation), e.g., lipofection, polyethylene glycol, calcium phosphate, and electroporation. Any suitable method may be used to introduce a nucleic acid sequence into a cell. Non-viral techniques, such as amphiphilic cell-penetrating peptides, may be used to introduce nucleic acid. Cells may also be genetically modified, e.g., by using any known gene editing technique, e.g., CRISPR, Talen, or Zn finger, to insert a nucleotide, polynucleotide, or nucleic acid sequence described herein into the genome.

[0281] Thus, the nucleic acid molecules described herein are not naturally expressed by corresponding unmodified cells. Indeed, the nucleic acid molecules encoding recombinant proteins are artificial constructs and cannot exist or be expressed in nature. Preferably, the engineered cells are cells that have been modified, for example, by transduction or gene transfer. Preferably, the engineered cells are cells that have been modified or genomically modified, for example, by transduction or gene transfer. Preferably, the engineered cells are cells that have been modified or genomically modified, for example, by transduction or gene transfer. Preferably, the engineered cells are cells that have been modified or genomically modified, for example, by retroviral transduction. Preferably, the engineered cells are cells that have been modified or genomically modified, for example, by lentiviral transduction.

[0282] The term "introduced" as used herein refers to a method for inserting foreign nucleic acid, e.g., DNA or RNA, into a cell. The term "introduced" as used herein includes both transduction and gene transfer methods. Gene transfer is the process of introducing nucleic acid into a cell by non-viral methods. Transduction is the process of introducing foreign DNA or RNA into a cell via a viral vector. Engineered cells may be generated by introducing the nucleic acids described herein by one of a number of means, such as transduction with a viral vector or gene transfer with DNA or RNA.

[0283] The cells may be activated and / or expanded before or after introduction of the nucleic acid described herein, for example, by treatment with anti-CD3 monoclonal antibody, or with both anti-CD3 and anti-CD28 monoclonal antibodies. The cells may also be expanded in the presence of anti-CD3 and anti-CD28 monoclonal antibodies in combination with IL-2. IL-2 may suitably be replaced with IL-15. Other components that may be used in cell (e.g., Treg) expansion protocols include, but are not limited to, rapamycin, all-trans retinoic acid (ATRA), and TGFβ. "Activated" herein means that a cell is stimulated to cause proliferation of the cell. "Expanded" herein means that a cell or population of cells is induced to proliferate. The proliferation of a population of cells may be measured, for example, by counting the number of cells present in the population. The phenotype of the cells may be determined by methods well known in the art, such as flow cytometry. In one embodiment, the cells may be cultured and / or activated in the presence of EPO.

[0284] The cell may be an immune cell or a precursor thereof. The precursor cell may be a progenitor cell. Thus, representative immune cells include T cells, particularly cytotoxic T cells (CTL; ​​CD8+ T cells), helper T cells (HTL; CD4+ T cells), and regulatory T cells (Tregs). Other populations of T cells, such as naive T cells and memory T cells, are also useful herein. Other immune cells include NK cells, NKT cells, dendritic cells, MDSCs, neutrophils, and macrophages. Precursors of immune cells include pluripotent stem cells, such as induced pluripotent stem cells (iPSCs), or more committed progenitor cells, including multipotent stem cells or cells committed to a lineage. The precursor cells can be induced to differentiate into immune cells in vivo or in vitro. In one embodiment, the precursor cells may be somatic cells capable of transdifferentiation into the immune cell of interest.

[0285] Most notably, the immune cells may be NK cells, dendritic cells, MDSCs, or T cells, such as cytotoxic T lymphocytes (CTLs) or Treg cells.

[0286] In particular, the immune cells may be Treg cells. "Regulatory T cells (Treg) or T regulatory cells" are immune cells with immunosuppressive function that control cytopathic immune responses and are essential for maintaining immune tolerance. As used herein, the term Treg refers to T cells with immunosuppressive function.

[0287] As used herein, a T cell is a lymphocyte including any type of T cell, such as an alpha beta T cell (e.g., CD8 or CD4+), a gamma delta T cell, a memory T cell, a Treg cell, or the like.

[0288] Advantageously, the immune suppressive function may refer to the ability of Tregs to reduce or inhibit one or more of a number of physiological cellular events promoted by the immune system in response to stimuli such as pathogens, alloantigens, or autoantigens. Examples of such events include increased proliferation of normal T cells (Tcon) and secretion of inflammatory cytokines. Any such event may be used as an indicator of the strength of the immune response. A relatively weak immune response by Tconv in the presence of Tregs may indicate the ability of Tregs to suppress immune responses. For example, a relatively reduced secretion of cytokines indicates a weakened immune response and thus the ability of Tregs to suppress immune responses. Tregs can also suppress immune responses by regulating the expression of costimulatory molecules on antigen presenting cells (APCs) such as B cells, dendritic cells, and macrophages. The expression levels of CD80 and CD86 can be used to investigate the suppressive capacity of activated Tregs in vitro after co-culture.

[0289] Assays for measuring the strength of immune response and thus the suppressive ability of Treg are well known in the art. In particular, antigen-specific Tconv cells may be co-cultured with Treg, and peptides of the corresponding antigen may be added to the co-culture to stimulate the response from Tconv cells. The degree of proliferation of Tconv cells and / or the amount of cytokine IL-2 secreted by Tconv cells in response to the addition of the peptide may be used as an indicator of the suppressive ability of co-cultured Treg.

[0290] The proliferation of antigen-specific Tconv cells co-cultured with Tregs referred to herein may be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95%, or 99% less than the proliferation of the same Tconv cells cultured in the absence of Tregs. For example, the proliferation of antigen-specific Tconv cells co-cultured with the present Tregs may be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95%, or 99% less than the proliferation of the same Tconv cells cultured in the presence of unmanipulated Tregs. Cells, e.g., Tregs, comprising a nucleic acid, expression construct, or vector defined herein may have increased suppressive activity compared to non-manipulated Tregs (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% increased suppressive activity).

[0291] The antigen-specific Tconv cells co-cultured with Treg herein may express at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% less effector cytokines compared to the corresponding Tconv cells cultured in the absence of Treg (e.g., in the presence of unmanipulated Treg). The effector cytokines may be selected from IL-2, IL-17, TNFα, GM-CSF, IFN-γ, IL-4, IL-5, IL-9, IL-10, and IL-13. Suitably, the effector cytokines may be selected from IL-2, IL-17, TNFα, GM-CSF, and IFN-γ. Several different subpopulations of Tregs have been identified that may express different or different levels of specific markers. Tregs generally express the markers CD4, CD25, and FOXP3 (CD4 + CD25 + FOXP3 + ) T cells.

[0292] Tregs also express CTLA-4 (cytotoxic T-lymphocyte-associated molecule 4) or GITR (glucocorticoid-induced TNF receptor).

[0293] Treg cells are present in peripheral blood, lymph nodes, and tissues, and as used herein, Treg includes thymus-derived natural Treg (nTreg) cells, peripherally generated Treg, and induced Treg (iTreg) cells.

[0294] The cell surface markers CD4 and CD25 were expressed in the absence or in combination with low expression of the surface protein CD127 (CD4 + CD25 + CD127 - or CD4 + CD25 + CD127 low ), may be used to identify Tregs. The use of such markers for the identification of Tregs is well known in the art and is described, for example, in Liu et al (JEM; 2006; 203; 7(10); 1701-1711).

[0295] Tregs are CD4 + CD25 + FOXP3 + T cells, CD4 + CD25 + CD127 - T cells, or CD4 + CD25 + FOXP3 + CD127 - / low It may be a T cell.

[0296] Suitably, Tregs may be natural Tregs (nTregs). As used herein, the term "natural Tregs" refers to thymus-derived Tregs. Natural Tregs are CD4 + CD25 + FOXP3 + Helios + Neuropilin 1 +Compared with iTregs, nTregs highly express PD-1 (programmed cell death-1, pdcd1), neuropilin 1 (Nrp1), Helios (Ikzf2), and CD73. nTregs may be distinguished from iTregs based on the expression of Helios protein or neuropilin 1 (Nrp1), respectively.

[0297] Tregs may have a Treg-specific demethylation region (TSDR), which is a key methylation-sensitive element that controls the expression of Foxp3 (Polansky, JK et al., 2008. European journal of immunology, 38(6), pp. 1654-1663).

[0298] Further suitable Tregs include Tr1 cells (which do not express Foxp3 and produce high levels of IL-10), CD8 + FOXP3 + T cells, and γδFOXP3 + These include, but are not limited to, T cells.

[0299] Tregs include naive Tregs (CD45RA + FoxP3 low ), effector / memory Treg (CD45RA - FoxP3 high ), and cytokine-producing Tregs (CD45RA - FoxP3 low It is known that there are various subpopulations, such as "memory Tregs" that express CD45RO and +These cells have higher levels of CD45RO compared to naive Tregs (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more CD45RO) and preferably do not express CD45RA (mRNA and / or protein) or have lower levels of CD45RA compared to naive Tregs (e.g., at least 80%, 90%, or 95% less CD45RA compared to naive Tregs). "Cytokine-producing Treg" refers to Treg that does not express CD45RA (mRNA and / or protein) or has very low levels of CD45RA compared to naive Treg (e.g., at least 80%, 90%, or 95% less CD45RA compared to naive Treg) and has low levels of FOXP3 compared to memory Treg (e.g., less than 50%, 60%, 70%, 80%, or 90% FOXP3 compared to memory Treg). Cytokine-producing Treg may produce interferon gamma and may be less suppressive in vitro compared to naive Treg (e.g., less than 50%, 60%, 70%, 80%, or 90% suppressive compared to naive Treg). Expression level herein may refer to expression of mRNA or protein. In particular, for cell surface markers such as CD45RA, CD25, CD4, CD45RO, expression can refer to cell surface expression, i.e., the amount or relative amount of marker protein expressed on the cell surface.Expression level can be determined by any known method in the art.For example, mRNA expression level can be determined by Northern blotting / array analysis, and protein expression can be determined by Western blotting, preferably by FACS using antibody staining for cell surface expression.

[0300] In particular, Tregs may be naive Tregs. As used interchangeably herein, "naive regulatory T cells, naive T regulatory cells, or naive Tregs" refer to Treg cells that express CD45RA (particularly, express CD45RA on the cell surface). Thus, naive Tregs are CD45RA + Naive Treg generally refers to Treg that is not activated via endogenous TCR by peptide / MHC, whereas effector / memory Treg refers to Treg that is activated by stimulation via endogenous TCR. Typically, naive Treg may express at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more CD45RA than non-naive Treg cells (e.g., memory Treg cells). In another view, naive Treg cells may express at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, or 100-fold more CD45RA than non-naive Treg cells (e.g., memory Treg cells). The expression level of CD45RA can be easily determined by methods in the art, for example, flow cytometry using commercially available antibodies. Typically, non-naive Treg cells do not express CD45RA or express low levels of CD45RA.

[0301] In particular, naive Tregs may not express CD45RO, whereas CD45RO - Thus, naive Tregs may express at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less CD45RO than memory Tregs, or viewed alternatively, may express at least 2-, 3-, 4-, 5-, 10-, 50-, or 100-fold less CD45RO than memory Treg cells.

[0302] Naive Treg expresses CD25 as mentioned above, but depending on the origin of naive Treg, the expression level of CD25 may be lower than that of memory Treg.For example, for naive Treg isolated from peripheral blood, the expression level of CD25 may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower than that of memory Treg.Such naive Treg may be considered to have medium to low expression of CD25.However, those skilled in the art will understand that naive Treg isolated from umbilical cord blood may not show such difference.

[0303] Typically, naive Tregs as defined herein are CD4 + , CD25 + , FOXP3 + , CD127 low , CD45RA + It could be.

[0304] As used herein, low expression of CD127 refers to low expression of CD4 + Naïve Tregs express lower levels of CD127 compared to non-regulatory or Tcon cells. In particular, naïve Tregs express lower levels of CD4 + Compared to non-regulatory or Tcon cells, CD127 expression may be less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%. CD127 levels can be assessed by methods standard in the art, including flow cytometry of cells stained with anti-CD127 antibodies.

[0305] Typically, naive Tregs do not express or have low levels of CCR4, HLA-DR, CXCR3, and / or CCR6. In particular, naive Tregs may express lower levels of CCR4, HLA-DR, CXCR3, and CCR6 than memory Tregs, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower.

[0306] Naive Tregs express CCR7 + and CD31 + The vector may further express additional markers including:

[0307] The isolated naive Treg can be identified by methods known in the art, including the method of determining the presence or absence of any one or more panels of the above markers on the cell surface of isolated cells.For example, CD45RA, CD4, CD25, and CD127low can be used to determine whether a cell is naive Treg.The method of determining whether an isolated cell is naive Treg or has a desired phenotype can be performed as described below with respect to the additional steps that can be performed, and the method of determining the presence and / or expression level of cell markers is well known in the art, for example, flow cytometry using commercially available antibodies.

[0308] Suitably, cells such as Tregs are isolated from peripheral blood mononuclear cells (PBMCs) obtained from a subject. Suitably, the subject from which the PBMCs are obtained is a mammal, preferably a human. Suitably, the cells are matched (e.g. HLA-matched) to the subject to which the engineered cells are administered, or are the subject's own. Suitably, the subject to be treated is a mammal, in particular a human. The cells may be generated ex vivo, either from the patient's own peripheral blood (first party), or in the setting of a hematopoietic stem cell transplant from donor peripheral blood (second party), or from peripheral blood from an unrelated donor (third party). Suitably, the cells are the subject to which the engineered cells are administered.

[0309] Suitably, said Tregs are part of a population of cells. Suitably, the population of Tregs is at least 70% Tregs, such as at least 75%, 85%, 90%, 95%, 97%, 98%, or 99% Tregs. Such a population may be referred to as an "enriched Treg population".

[0310] In some embodiments, Tregs may be derived from ex vivo differentiation of inducible progenitor cells (e.g., iPSCs) or embryonic progenitor cells into Tregs. The nucleic acid molecules, constructs, or vectors described herein may be introduced into inducible progenitor cells or embryonic progenitor cells before or after differentiation into Tregs. Suitable methods for differentiation are known in the art and include those disclosed in Haque et al, J Vis Exp., 2016, 117, 54720 (incorporated herein by reference). In another embodiment, when the recombinant protein is an EPOR with a modification that allows dimerization and signal transduction in the absence of a signal inducer molecule, the endogenous EPOR nucleic acid may be modified by gene editing techniques (e.g., CRISPR) to provide the cell of the present invention.

[0311] The term "normal T cell" or Tcon or Tconv (used interchangeably herein) refers to a T lymphocyte cell that expresses an αβ T cell receptor (TCR) and a co-receptor that may be cluster of differentiation 4 (CD4) or cluster of differentiation 8 (CD8) and has no immunosuppressive function. Normal T cells are present in peripheral blood, lymph nodes, and tissues. Suitably, engineered Tregs may be generated from Tcon by introducing a nucleic acid comprising a sequence encoding FOXP3. Alternatively, engineered Tregs may be generated from Tcon by in vitro culture of CD4+CD25-FOXP3- cells in the presence of IL-2 and TGF-β.

[0312] Upon expression of the recombinant protein, the Tregs herein may have increased persistence compared to Treg cells that do not contain the recombinant protein. "Persistence" herein defines the length of time that Tregs can survive in a particular environment, e.g., in vivo (e.g., in a human patient or animal model). The Tregs disclosed herein may have at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% increased persistence compared to Tregs that do not express the recombinant protein of the present invention. Persistence can be measured, for example, by determining the amount or number of administered cells in a subject or patient over time, comparing cells expressing the recombinant protein of the present invention to a comparable cell type that does not express the recombinant protein, or to unmanipulated cells. The administered cells can be tracked using a marker protein, e.g., CD34 in the case of cells that also express the RQR8 safety switch.

[0313] In another embodiment, the target cell into which the nucleic acid molecule, construct or vector is introduced is not a cell intended for therapy. In one embodiment, the cell is a production host cell. The cell may be for the production of nucleic acids, for example for cloning, or for the production of vectors, or for the production of polypeptides.

[0314] Also provided herein is a cell population that comprises the cells defined or described herein.It will be understood that the cell population may comprise both the present cells that comprise the nucleic acid molecule, construct, or vector defined herein, and cells that do not comprise the nucleic acid molecule, construct, or vector, such as non-transduced or non-transduced cells.In certain embodiments, cell populations are provided in which at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells comprise the nucleic acid, expression construct, or vector, although all cells in the population may comprise the nucleic acid, expression construct, or vector.

[0315] Also provided is a pharmaceutical composition comprising the cell or cell population defined or described herein, the vector defined herein.The vector can be used in gene therapy.Thus, instead of administering cells, the vector can be administered to modify endogenous cells in the subject to express the introduced nucleic acid molecule.Vectors suitable for use in gene therapy are known in the art and include viral vectors.

[0316] A pharmaceutical composition is a composition that comprises or consists of a therapeutically effective amount of a pharma- ceutical active agent, i.e., the above-mentioned cells (e.g., Tregs), cell populations, or vectors. Preferably, the pharmaceutical composition comprises a pharma- ceutical acceptable carrier, diluent, or excipient (including combinations thereof). Carriers or diluents acceptable for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro, ed., 1985). The choice of pharmaceutical carrier, excipient, or diluent can be selected taking into account the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may comprise, as or in addition to the carrier, excipient, or diluent, a suitable binder, lubricant, suspending agent, coating agent, or solubilizing agent.

[0317] "Pharmaceutically acceptable" includes the meaning that the formulation is sterile and pyrogen-free. The carrier, diluent and / or excipient must be "acceptable" in the sense of being compatible with the cells or vectors and not deleterious to the recipient. Typically, the carrier, diluent and / or excipient will be a sterile, pyrogen-free physiological or infusion medium, although other acceptable carriers, diluents, excipients may be used.

[0318] Examples of pharma- ceutically acceptable carriers include, for example, water, saline, alcohol, silicone, wax, petrolatum, vegetable oils, polyethylene glycol, propylene glycol, liposomes, sugar, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, flavor oils, fatty acid monoglycerides, fatty acid diglycerides, petroleum fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0319] The cells, cell populations, or pharmaceutical compositions can be administered in a manner suitable for the treatment and / or prevention of the desired disease or condition. The dosage and frequency of administration are determined by factors such as the subject's condition and the type and severity of the subject's disease or condition, but the appropriate dosage may be determined by clinical trials. The pharmaceutical compositions can be formulated accordingly.

[0320] The cells, cell populations, or pharmaceutical compositions described herein may be administered parenterally, e.g., intravenously, or by infusion techniques. The cells, cell populations, or pharmaceutical compositions may be administered in the form of a sterile aqueous solution containing other substances, e.g., sufficient salts and glucose to make the solution isotonic with blood. The aqueous solution may be suitably buffered (preferably to a pH of 3-9). The pharmaceutical composition may be formulated accordingly. The preparation of suitable parenteral formulations under sterile conditions may readily be accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0321] The pharmaceutical composition may include cells in an infusion medium, such as a sterile isotonic solution. The pharmaceutical composition may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0322] The cells, cell populations, or pharmaceutical compositions may be administered in a single dose or multiple doses. In particular, the cells, cell populations, or pharmaceutical compositions may be administered in a single dose. The pharmaceutical compositions may be formulated accordingly.

[0323] The pharmaceutical composition may further comprise one or more active agents. The pharmaceutical composition may further comprise one or more other therapeutic agents, such as lymphodepleting agents (e.g., thymoglobulin, campath-1H, anti-CD2 antibodies, anti-CD3 antibodies, anti-CD20 antibodies, cyclophosphamide, fludarabine), mTOR inhibitors (e.g., sirolimus, everolimus), agents that inhibit costimulatory pathways (e.g., anti-CD40 / CD40L, CTAL4Ig), and / or agents that inhibit specific cytokines (IL-6, IL-17, TNFalpha, IL18).

[0324] Depending on the disease / condition or subject being treated, and the route of administration, the cells, cell populations, or pharmaceutical compositions may be administered at various dosages (e.g., measured as cells / kg, or cells / subject, etc.). In any event, the physician will determine the actual dosage most suitable for an individual subject, which will vary according to the age, weight, and response of the subject. Typically, however, for the cells herein, dosages are 5x10 cells / subject. 7 ~3x10 9 cells, or 10 8 ~2x10 9 The cells may be administered.

[0325] The cells may be appropriately modified for use in a pharmaceutical composition, for example, the cells may be cryopreserved and thawed at an appropriate time before being injected into a subject.

[0326] Further provided herein is a kit or combination product comprising the cells, cell populations, and / or pharmaceutical compositions of the present invention.Preferably, the kit is for use in the above-mentioned methods and uses described herein, such as the therapeutic methods described herein.Preferably, the kit includes instructions for the use of the kit components.The kit or composition may further include an inducer, such as rapamycin or an analog thereof.

[0327] The cells, cell populations, compositions, and vectors herein may be for use in therapy, i.e., treatment or prevention of a disease or condition. As described above, the cells in which the recombinant protein is expressed are typically cells that have been modified or engineered to express additional molecules (e.g., additional proteins), in particular receptors, such as CAR or TCR. Thus, the therapy may be for the prevention or treatment of a disease or condition that can be treated by or with cells expressing a receptor, such as CAR. The cells and compositions containing the cells are for adoptive cell therapy (ACT). A variety of conditions can be treated by administration of cells expressing a CAR according to the present disclosure, including in particular Treg cells. As described above, this may be a condition that responds to immunosuppression, in particular the immunosuppressive effect of Treg cells. Thus, the cells, cell populations, compositions, and vectors described herein may be used to induce or achieve immunosuppression in a subject. The Treg cells administered, or the Treg cells modified in vivo, may be targeted by expression of a receptor, such as a CAR. Conditions suitable for such treatment include infectious, neurodegenerative or inflammatory diseases, or more broadly, any condition associated with an unwanted or unnecessary or deleterious immune response.

[0328] The condition to be treated or prevented includes inflammation or inflammation-related or inflammation-associated condition.Inflammation can be chronic or acute.Furthermore, inflammation can be low-level inflammation or systemic inflammation.For example, inflammation can be inflammation that occurs under metabolic disorder, such as metabolic syndrome, or under conditions such as insulin resistance, type II diabetes, or obesity.

[0329] In particular, the cells, cell populations, vectors, and pharmaceutical compositions provide a means for inducing tolerance to transplantation, for treating and / or preventing cellular and / or humoral transplant rejection, for treating and / or preventing graft-versus-host disease (GvHD), autoimmune disease, or allergic disease, for promoting tissue repair and / or tissue regeneration, or for ameliorating inflammation. The cells, cell populations, vectors, and pharmaceutical compositions can be used in methods comprising administering to a subject the cells, cell populations, vectors, or pharmaceutical compositions described herein.

[0330] As used herein, "inducing tolerance to transplantation" refers to inducing tolerance to the transplanted organ in the recipient. In other words, inducing tolerance to transplantation means reducing the level of the recipient's immune response to the donor transplanted organ. Inducing tolerance to the transplanted organ may reduce the amount of immunosuppressant required by the transplant recipient or allow the withdrawal of immunosuppressant.

[0331] For example, engineered cells, such as Tregs, may be administered to a subject having a disease to alleviate, reduce, or ameliorate at least one symptom of the disease, such as jaundice, dark urine, itching, abdominal distension or tenderness, fatigue, nausea or vomiting, and / or loss of appetite. The at least one symptom may be alleviated, reduced, or ameliorated by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, or the at least one symptom may be completely alleviated.

[0332] The engineered cells, such as Tregs, may be administered to a subject having a disease to slow, reduce, or prevent the progression of the disease. The progression of the disease may be slowed, reduced, or prevented by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a subject not receiving the engineered cells, or the progression of the disease may be halted completely.

[0333] In one embodiment, the subject is a transplant recipient undergoing immunosuppressive therapy.

[0334] Preferably, the subject is a mammal. Preferably, the subject is a human.

[0335] The transplant may be selected from a liver transplant, a kidney transplant, a heart transplant, a lung transplant, a pancreas transplant, an intestine transplant, a stomach transplant, a bone marrow transplant, a vascularized composite tissue transplant, and a skin transplant.

[0336] Suitably, the cells may express a CAR that comprises an antigen binding domain capable of specifically binding to an HLA antigen present in the graft / transplant donor but absent in the graft / transplant recipient.

[0337] Suitably, the transplant is a liver transplant. In embodiments where the transplant is a liver transplant, the antigens include HLA antigens present in the transplanted organ but not in the patient, liver-specific antigens such as NTCP, or antigens whose expression is increased during rejection, such as CCL19, MMP9, SLC1A3, MMP7, HMMR, TOP2A, GPNMB, PLA2G7, CXCL9, FABP5, GBP2, CD74, CXCL10, UBD, CD27, CD48, CXCL11, etc.

[0338] As noted above, in one exemplary preferred embodiment, the antigen is HLA-A2.

[0339] The method for treating a disease or condition relates to the therapeutic use of the cells herein.In this regard, the cells may be administered to a subject having an existing disease or condition to relieve, reduce or ameliorate at least one symptom associated with the disease or condition, and / or to delay, reduce or prevent the progression of the disease.

[0340] Suitably, treating and / or preventing cellular and / or humoral transplant rejection may refer to administering an effective amount of cells (e.g., Tregs) such that the amount of immunosuppressant required by the transplant recipient is reduced, or may allow for discontinuation of the immunosuppressant.

[0341] Preventing disease or condition refers to the prophylactic use of cells herein.In this regard, the cells may be administered to a subject that has not yet suffered from or developed disease or condition and / or has not shown symptoms of disease or condition, in order to prevent disease or condition, or to reduce or prevent the development of at least one symptom associated with disease or condition.The subject may be predisposed to said disease or condition, or may be considered to be at risk of developing said disease or condition.

[0342] The autoimmune or allergic disease may be selected from inflammatory skin diseases such as psoriasis and dermatitis (e.g., atopic dermatitis); responses associated with inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis); dermatitis; allergic conditions such as food allergies, eczema, asthma; rheumatoid arthritis; systemic lupus erythematosus (SLE) (including lupus nephritis, cutaneous lupus); diabetes mellitus (e.g., diabetes mellitus type I and insulin-dependent diabetes mellitus); multiple sclerosis; neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS); chronic inflammatory demyelinating polyneuropathy (CIPD); and juvenile onset diabetes. As indicated above, recombinant protein is not limited to use in the context of immunosuppressive therapy, and may be expressed in cells to treat conditions such as cancer or infectious diseases. In such a situation, it may be desirable to kill or ablate cancer cells or infected cells, and in such a case, the chimeric protein may be expressed in cytotoxic cells, such as cytotoxic T cells or NK cells, or their precursors. In such a case, the receptor (e.g., CAR or TCR) co-expressed with the chimeric protein may be directed against a cancer antigen, or an antigen derived from a pathogen, etc.

[0343] The medical uses or methods herein may include the following steps. (i) isolating or providing a cell-containing sample; (ii) introducing a nucleic acid molecule, construct or vector as defined herein into a cell, and (iii) administering the cells obtained in (ii) to a subject. The cells may be Tregs as defined herein. An enriched Treg population may be isolated and / or generated from a cell-containing sample before and / or after step (ii) of the method. For example, the isolation and / or generation may be performed before and / or after step (ii) to isolate and / or generate an enriched Treg sample. Enrichment may be performed after step (ii) to enrich for cells and / or Tregs comprising the recombinant proteins, nucleic acid molecules, constructs, and / or vectors described herein.

[0344] Preferably, the cells are autologous cells. Preferably, the cells are allogeneic cells.

[0345] Suitably, the cells (e.g., engineered Tregs) may be administered in combination with one or more other therapeutic agents, such as lymphodepleting agents. The engineered cells, e.g., Tregs, may be administered simultaneously with the one or more other therapeutic agents or sequentially (i.e., before or after the one or more other therapeutic agents).

[0346] The cells, e.g., Tregs, may be activated and / or expanded before or after introduction of a nucleic acid molecule described herein, e.g., by treatment with an anti-CD3 monoclonal antibody, or by treatment with both an anti-CD3 monoclonal antibody and an anti-CD28 monoclonal antibody. Expansion protocols are as described above.

[0347] After each step of the above methods, particularly after expansion, the cells, eg Tregs, may be washed.

[0348] The population of engineered cells, e.g., Treg cells, may be further enriched by any method known to one of skill in the art, e.g., by FACS or magnetic bead separation.

[0349] The steps of the production method may be carried out in a closed, sterile cell culture system.

[0350] The present invention may also provide a method for increasing the stability and / or suppressive function of a cell, comprising introducing a nucleic acid molecule, expression construct, or vector provided herein into a cell. The increase in suppressive function can be measured, for example, by co-culturing activated antigen-specific Tconv cells with the cells of the present invention, as described above, and measuring, for example, the level of cytokines produced by Tconv cells. The increase in suppressive function can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% increase compared to unmanipulated Treg.

[0351] As defined herein, increased stability of cells, e.g., Tregs, refers to an increase in the persistence or survival of those cells compared to unmanipulated Tregs, or an increase in the percentage of cells that retain the Treg phenotype over a period of time (e.g., relative to cells that retain Treg markers such as FOXP3 or Helios). Increased stability can be at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% increase in stability, and can be measured by techniques known in the art, e.g., staining for Treg cell markers in the cell population and analysis by FACS.

[0352] A further aspect provided herein is a combination product comprising (a) a cell, a cell population, a vector, or a pharmaceutical composition as defined herein, and (b) EPO, for use in therapy, in particular ACT or gene therapy. The therapy may be any of the therapies defined above and further described herein.

[0353] Each component (a) and (b) of the above combination product may be for separate, sequential or simultaneous use.

[0354] Each component (a) and (b) of the combination product is typically provided as a separate composition, i.e., formulated separately. Thus, the combination product may be defined or referred to as a kit.

[0355] This disclosure is not limited by the exemplary methods and materials disclosed herein, and methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise specified, nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation.

[0356] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range is also specifically disclosed, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise. Each narrower range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is also encompassed by this disclosure. The upper and lower limits of these narrower ranges may independently be included or excluded in the range, and each range in which either, neither or both limits are included in the narrower range is also encompassed by this disclosure, unless a specifically excluded limit is within the stated range. Where a stated range includes one or both limits, ranges excluding either or both included limits are also included in this disclosure.

[0357] Please note that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0358] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," or "containing," and are inclusive or open-ended and do not exclude additional, unrecited elements, components, or method steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0359] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art to the appended claims. All publications mentioned herein are incorporated herein by reference. Sequence Listing SEQ ID NO:1 (wild type human EPOR) MDHLGASLWPQVGSLCLLLAGAAWAPPPNLPDPKFESKAALLAARGPEELLCFTERLEDLVCFWEEAASAGVGPGNYSFSYQLEDEPWKLCRLHQAPTARGAVRFWCSLPTADTSSFVPLELRVTAAS G APRYHRVIHINEVVLLDAPVGLVARLADESGHVVLRWLPPPETPMTSHIRYEVDVSAGNGAGSVQRVEILEGRTECVLSNLRGRTRYTFAVRARMAEPSFGGFWSAWSEPVSLLTPSDLDPLILTLSLILVVILVLLTVLALLSHRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWS PCTPFTEDPPASLEVLSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPEGASAASFEYTILDPSSQLLRPWTLCPELPPTPPHLKYLYLVVSDSGISTDYSSGDSQGAQGGLSDGPYSNPYENSLIPAAEPLPPSYVACS SEQ ID NO:2 (R130C modified human EPOR extracellular domain without signal peptide) APPPNLPDPKFESKAALLAARGPEELLCFTERLEDLVCFWEEAASAGVGPGNYSFSYQLE DEPWKLCRLHQAPTARGAVRFWCSLPTADTSSFVPLELRVTAASGAPRYHRVIHINEVVL LDAPVGLVACLADESGHVVLRWLPPPETPMTSHIRYEVDVSAGNGAGSVQRVEILEGRTE CVLSNLRGRTRYTFAVRARMAEPSFGGFWSAWSEPVSLLTPSDLDP SEQ ID NO:3 (wild type human EPOR extracellular domain without signal peptide) APPPNLPDPKFESKAALLAARGPEELLCFTERLEDLVCFWEEAASAGVGPGNYSFSYQLE DEPWKLCRLHQAPTARGAVRFWCSLPTADTSSFVPLELRVTAASGAPRYHRVIHINEVVL LDAPVGLVARLADESGHVVLRWLPPPETPMTSHIRYEVDVSAGNGAGSVQRVEILEGRTE CVLSNLRGRTRYTFAVRARMAEPSFGGFWSAWSEPVSLLTPSDLDP SEQ ID NO: 4 (R154C modified human EPOR extracellular domain including signal peptide) MDHLGASLWPQVGSLCLLLAGAAWAPPPNLPDPKFESKAALLAARGPEELLCFTERLEDLVCFWEEAASAGVGPGNYSFSYQLEDEPWKLCRLHQAPTARGAVRFWCSLPTADTSSFVPLELRVTAAS G APRYHRVIHINEVVLLDAPVGLVACLADESGHVVLRWLPPPETPMTSHIRYEVDVSAGNGAGSVQRVEILEGRTECVLSNLRGRTRYTFAVRARMAEPSFGGFWSAWSEPVSLLTPSDLDP SEQ ID NO:5 (wild type human EPOR extracellular domain without signal peptide) MDHLGASLWPQVGSLCLLLAGAAWAPPPNLPDPKFESKAALLAARGPEELLCFTERLEDLVCFWEEAASAGVGPGNYSFSYQLEDEPWKLCRLHQAPTARGAVRFWCSLPTADTSSFVPLELRVTAAS G APRYHRVIHINEVVLLDAPVGLVARLADESGHVVLRWLPPPETPMTSHIRYEVDVSAGNGAGSVQRVEILEGRTECVLSNLRGRTRYTFAVRARMAEPSFGGFWSAWSEPVSLLTPSDLDP SEQ ID NO:6 (wild type human EPOR signal peptide) MDHLGASLWPQVGSLCLLLAGAAW SEQ ID NO:7 (wild type human EPOR transmembrane domain) LILTLSLILVVILVLLTVLALLS SEQ ID NO:8 (wild type human EPOR cytoplasmic domain) HRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEV LSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPEGASAASFEYTILDPSSQLLRPWTLCPELPPTPPHLK YLYLVVSDSGISTDYSSGDSQGAQGGLSDGPYSNPYENSLIPAAEPLPPSYVACS SEQ ID NO:9 (wild type mouse EPOR) MDKLRVPLWPRVGPLCLLLAGAAWAPSPSLPDPKFESKAALLASRGSEELLCFTQRLEDLVCFWEEAASSGMDFNYSFSYQLEGESRKSCSLHQAPTVRGSVRFWCSLPTADTSSFVPLELQVTEASGSPRYHRIIHINEVVLLDAPAGLLARRAEEGSHVVLRWLPPPGAPMTTHIRYEVDVSAGNRAGGTQRVEVLEGRTECVLSNLRGGTRYTFAVRARMAEPSFSGFWSAWSEPASLLTASDLDPLILTLSLILVLISLLLTVLALLSHRRTLQQKIWPGIPSPESEFEGLFTTHKGNFQLWLLQRDGCLWWSPGSSFPEDPPAHLEVLSEPRWAVTQAGDPGADDEGPLLEPVGSEHAQDTYLVLDKWLLPRTPCSENLSGPGGSVDPVTMDEASETSSCPSDLASKPRPEGTSPSSFEYTILDPSSQLLCPRALPPELPPTPPHLKYLYLVVSDSGISTDYSSGGSQGVHGDSSDGPYSHPYENSLVPDSEPLHPGYVACS SEQ ID NO: 10 (R129C modified mouse EPOR extracellular domain including signal peptide MDKLRVPLWPRVGPLCLLLAGAAWAPSPSLPDPKFESKAALLASRGSEELLCFTQRLEDLVCFWEEAASSGMDFNYSFSYQLEGESRKSCSLHQAPTVRGSVRFWCSLPTADTSSFVPLELQVTEASGSPRYHRIIHINEVVLLDAPAGLLACRAEEGSHVVLRWLPPPGAPMTTHIRYEVDVSAGNRAGGTQRVEVLEGRTECVLSNLRGGTRYTFAVRARMAEPSFSGFWSAWSEPASLLTASDLDP SEQ ID NO:11 (wild type mouse EPOR extracellular domain including signal peptide) MDKLRVPLWPRVGPLCLLLAGAAWAPSPSLPDPKFESKAALLASRGSEELLCFTQRLEDLVCFWEEAASSGMDFNYSFSYQLEGESRKSCSLHQAPTVRGSVRFWCSLPTADTSSFVPLELQVTEASGSPRYHRIIHINEVVLLDAPAGLLARRAEEGSHVVLRWLPPPGAPMTTHIRYEVDVSAGNRAGGTQRVEVLEGRTECVLSNLRGGTRYTFAVRARMAEPSFSGFWSAWSEPASLLTASDLDP SEQ ID NO:12 (wild type mouse EPOR transmembrane domain) LILTLSLILVLISLLLTVLALLS SEQ ID NO:13 (wild type mouse EPOR cytoplasmic domain) HRRTLQQKIWPGIPSPESEFEGLFTTHKGNFQLWLLQRDGCLWWSPGSSFPEDPPAHLEVLSEPRWAVTQAGDPGADDEGPLLEPVGSEHAQDTYLVLDKWLLPRTPCSENLSGPGGSVDPVTMDEASETSSCPSDLASKPRPEGTSPSSFEYTILDPSSQLLCPRALPPELPPTPPHLKYLYLVVSDSGISTDYSSGGSQGVHGDSSDGPYSHPYENSLVPDSEPLHPGYVACS SEQ ID NO: 14 (EPO) MGVHECPAWL WLLLSLLSLP LGLPVLGAPP RLICDSRVLE RYLLEAKEAE NITTGCAEHC SLNENITVPD TKVNFYAWKR MEVGQQAVEV WQGLALLSEA VLRGQALLVN SSQPWEPLQL HVDKAVSGLR SLTTLLRALG AQKEAISPPD AASAAPLRTI TADTFRKLFR VYSNFLRGKL KLYTGEACRT GDR SEQ ID NO:15 - Native FKBP12 domain MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQE VIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE SEQ ID NO:16 - Wild-type FRB segment of mTOR MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFN QAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKLES FRB with T to L substitution in SEQ ID NO: 17-2098 MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFN QAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKLES FRB segment of mTOR with a T to H substitution in SEQ ID NO: 18-2098 and a W to F substitution at residue 2101 MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFN QAYGRDLMEAQEWCRKYMKSGNVKDLHQAFDLYYHVFRRISKLES SEQ ID NO:19 is the FRB segment of mTOR with a K to P substitution at residue 2095 MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFN QAYGRDLMEAQEWCRKYMKSGNVPDLTQAWDLYYHVFRRISKLES SEQ ID NO:20 - c-Jun leucine zipper RIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMN SEQ ID NO:21 Fos leucine zipper LTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEFILAAY SEQ ID NO:22 - BZip (RR) leucine zipper domain MDPDLEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGK SEQ ID NO:23 - AZip(EE) leucine zipper domain MDPDLEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK SEQ ID NO:24 - CD28 transmembrane domain FWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO:25 - IL2RB transmembrane domain IPWLGHLLVGLSGAFGFIILVYLLI SEQ ID NO:26 CD8a TM domain IYIWAPLAGTCGVLLLSLVIT SEQ ID NO: 27: TREM1 TM domain IVILLAGGFLSKSLVFSVLFA SEQ ID NO: 28: TREM2 TM domain ILLLLACIFLIKILAASALWA SEQ ID NO: 29: DAP10 TM domain LLAGLVAANAVASLLIVGAVF SEQ ID NO: 30: DAP12 TM domain GVLAGIVMGNLVLTVLIALAV SEQ ID NO: 31 (amino acids 266 to 551 of the receptor β chain (NCBI REFSEQ: NP_000869.1) NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV SEQ ID NO: 32 (Amino acid sequence of wild-type FOXP3 (UniProtKB accession Q9BZS1)) MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP SEQ ID NO:33 - IL9R (amino acids 292 to 521 of NP_002177.2) KLSPRVKRIFYQNVPSPAMFFQPLYSVHNGNFQTWMGAHGAGVLLSQDCAGTPQGALEPCVQEATALLTCGPARPWKSVALEEEQEGPGTRLPGNLSSEDVLPAGCTEWRVQTLAYLPQEDWAPTSLTRPAPPDSEGSRSSSSSSSSNNNNYCALGCYGGWHLSALPGNTQSSGPIPALACGLSCDHQGLETQQGVAWVLAGHCQRPGLHEDLQGMLLPSVLSKARSWTF SEQ ID NO:34 - IL4RA (amino acids 257 to 825 of NP_000409.1) KIKKEWWDQIPNPARSRLVAIIIQDAQGSQWEKRSRGQEPAKCPHWKNCLTKLLPCFLEHNMKRDEDPHKAAKEMPFQGSGKSAWCPVEISKTVLWPESISVVRCVELFEAPVECEEEEEVEEEKGSFCASPESSRDDFQEGREGIVARLTESLFLDLLGEENGGFCQQDMGESCLLPPSGSTSAHMPWDEFPSAGPKEAPPWGKEQPLHLEPSPPASPTQSPDNLTCTETPLVIAGNPAYRSFSNSLSQSPCPRELGPDPLLARHLEEVEPEMPCVPQLSEPTTVPQPEPETWEQILRRNVLQHGAAAAPVSAPTSGYQEFVHAVEQGGTQASAVVGLGPPGEAGYKAFSSLLASSAVSPEKCGFGASSGEEGYKPFQDLIPGCPGDPAPVPVPLFTFGLDREPPRSPQSSHLPSSSPEHLGLEPGEKVEDMPKPPLPQEQATDPLVDSLGSGIVYSALTCHLCGHLKQCHGQEDGGQTPVMASPCCGCCCGDRSSPPTTPLRAPDPSPGGVPLEASLCPASLAPSGISEKSKSSSSFHPAPGNAQSSSQTPKIVNFVSVGPTYMRVS SEQ ID NO:35 - IL3RB (amino acids 461 to 897 of NP_000386.1) RFCGIYGYRLRRKWEEKIPNPSKSHLFQNGSAELWPPGSMSAFTSGSPPHQGPWGSRFPELEGVFPVGFGDSEVSPLTIEDPKHVCDPPSGPDTTPAASDLPTEQPPSPQPGPPAASHTPEKQASSFDFNGPYLGPPHSRSLPDILGQPEPPQEGGSQKSPPPGSLEYLCLPAGGQVQLVPLAQAMGPGQAVEVERRPSQGAAGSPSLESGGGPAPPALGPRVGGQDQKDSPVAIPMSSGDTEDPGVASGYVSSADLVFTPNSGASSVSLVPSLGLPSDQTPSLCPGLASGPPGAPGPVKSGFEGYVELPPIEGRSPRSPRNNPVPPEAKSPVLNPGERPADVSPTSPQPEGLLVLQQVGDYCFLPGLGPGPLSLRSKPSSPGPGPEIKNLDQAFQVKKPPGQAVPQVPVIQLFKALKQQDYLSLPPWEVNKPGEVC SEQ ID NO:36 - IL17RB (amino acids 314 to 502 of NP_061195.2) RHERIKKTSFSTTTLLPPIKVLVVYPSEICFHHTICYFTEFLQNHCRSEVILEKWQKKKIAEMGPVQWLATQKKAADKVVFLLSNDVNSVCDGTCGKSEGSPSENSQDLFPLAFNLFCSDLRSQIHLHKYVVVYFREIDTKDDYNALSVCPKYHLMKDATAFCAELLHVKQQVSAGKRSQACHDGCCSL SEQ ID NO: 37 Amino acid sequence of an N- and C-terminally truncated FOXP3 fragment as described in WO2019 / 241549 <h2 style=";text-align:left;direction:ltr">GGAHASSSSL NPMPPSQLQL PTLPLVMVAP SGARLGPLPH LQALLQDRPH FMHQLSTVDA HARTPVLQVH PLESPAMISL TPPTTATGVF SLKARPGLPP GINVASLEWV SREPALLCTF PNPSAPRKDS TLSAVPQSSY PLLANGVCKW PGCEKVFEEP EDFLKHCQAD HLLDEKGRAQ EF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 38 STAT5 association motif <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> YXXF / L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 39 STAT5 association motif <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> YCTF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 40 STAT5 association motif <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> YFFF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 41 STAT5 association motif <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> YLSL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 42 STAT5 association motif <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> YLSLQ<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 43 JAK1 binding motif <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> KVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 44 JAK1 binding motif <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> NPWFQRAKMPRALDFSGHTHPVATFQPSRPESVNDLFLCPQKELT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 45 JAK1 binding motif <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GYICLRNSLPKVLNFHNFLAWPFPNLPPLEAMDMVEVIYINR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 46 JAK1 binding motif <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> PLKEKSIILPKSLISVVRSATLETKPESKYVSLITSYQPFSL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 47 JAK1 binding motif <h2 style=";text-align:left;direction:ltr"> RRRKKLPSVLLFKKPSPFIFISQRPSPETQDTIHPLDEEAFLK SEQ ID NO: 48 JAK1 binding motif YIHVGKEKHPANLILIYGNEFDKRFFVPAEKIVINFITLNISDDS SEQ ID NO: 49 JAK1 binding motif RYVTKPPAPPNSLNVQRVLTFQPLRFIQEHVLIPVFDLSGP SEQ ID NO:50 JAK2 binding motif NYVFFPSLKPSSSIDEYFSEQPLKNLLLSTSEEQIEKCFIIEN SEQ ID NO:51 JAK2 binding motif YWFHTPPSIPLQIEEYLKDPTQPILEALDKDSSPKDDVWDSVSIISFPE SEQ ID NO:52 JAK2 binding motif YAFSPRNSLPQHLKEFLGHPHHNTLLFFSFPLSDENDVFDKLSVIAEDSES SEQ ID NO:53 (IL2RB Truncate-Y510) NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLV SEQ ID NO:54 (IL2RB truncated - Y510 and Y392) NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV SEQ ID NO:55 JAK3 motif ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEI SEQ ID NO:56 JAK3 motif ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET SEQ ID NO:57: STAT3 signal YXXQ SEQ ID NO:58: STAT3 signal YRHQ SEQ ID NO:59 - STAT3 association motif YLRQ SEQ ID NO:60 - STAT1 association motif QLLLQQDKVPEPASLSSNHSLTSCFTNQGYF SEQ ID NO:6: SHP1 MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDF YDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQA ETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGG LETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYYATRVNAADIENRVLELNKKQESEDTA KAGFWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSD YINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNK CVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDH GVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDC DIDIQKTIQMVRAQRSGMVQTEAQYKFIYVAIAQFIETTKKKLEVLQSQKGQESEYGNIT YPPAMKNAHAKASRTSSKHKEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKRK SEQ ID NO: 62: Truncated EPOR endodomain HRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTTEDPPASLEV LSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPEGASAASFEYTILDPSSQLLRPWTLCPELPPTPPHLK SEQ ID NO:63-JAK3 reverse IESVLCLRESYDPQLSEALGKSVGSWASFNGHYETVLDELNKLTPIRPMTRE SEQ ID NO:64 CD3 zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:65 CD28 Intracellular Signaling RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO:66 CD27 intracellular signaling QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP SEQ ID NO:67 Linker sequence GGGS SEQ ID NO:68-98-Linker ETSGGGGSRL (SEQ ID NO:68) SGGGGSGGGGSGGGGS (SEQ ID NO: 69) S(GGGGS) 1-5 (wherein GGGGS is SEQ ID NO: 70) (GGGGS) 1-5 (wherein GGGGS is SEQ ID NO: 70) SGGGGSGGGGS (SEQ ID NO:71) S(GGGS)1-5 (wherein GGGS is SEQ ID NO: 67) (GGGS) 1-5 (wherein GGGS is SEQ ID NO: 67) SGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:72) SGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 73) S(GGGGGS) 1-5 (wherein GGGGGS is SEQ ID NO:74) (GGGGGS) 1-5 (wherein GGGGGS is SEQ ID NO:74) S(GGGGGGS) 1-5 (wherein GGGGGS is SEQ ID NO: 75) (GGGGGGS) 1-5 (wherein GGGGGS is SEQ ID NO: 75) G6 (SEQ ID NO: 76) G8 (SEQ ID NO:77) KESGSVSSEQLAQFRSLD (SEQ ID NO: 78) EGKSSGSGSESKST (SEQ ID NO:79) GSAGSAAGSGEF (SEQ ID NO: 80) SGGGGSAGSAAGSGEF (SEQ ID NO: 81) SGGGLLLLLLLLGGGS (SEQ ID NO:82) SGGGAAAAAAAAGGGS (SEQ ID NO:83) SGGGAAAAAAAAAAAAAAAAGGGS (SEQ ID NO:84) SGALGGLALAGLLLAGLGLGAAGS (SEQ ID NO: 85) SLSLSPGGGGGPAR (SEQ ID NO:86) SLSLSPGGGGGPARSLSLSPGGGGG (SEQ ID NO: 87) GSSGSS (SEQ ID NO:88) GSSSSSS (SEQ ID NO: 89) GGSSSS (SEQ ID NO: 90) GSSSSS (SEQ ID NO: 91) SGGGGS (SEQ ID NO:92) GGGGSGGGGSGGGGS (SEQ ID NO:93) GGGGG (SEQ ID NO:94) GGGGSGGGGS (SEQ ID NO:95) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 96) GGGGGGG (SEQ ID NO:97) GGGGGGGGG (SEQ ID NO:98) SEQ ID NO:99 RQR8 : ACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV SEQ ID NO:100 P2A peptide cleavage domain ATNFSLLKQAGDVEENPGP SEQ ID NO: 101 T2A peptide cleavage domain: EGRGSLLTCGDVEENPGP SEQ ID NO: 102 E2A peptide cleavage domain: QCTNYALLKLAGDVESNPGP SEQ ID NO: 103 F2A peptide cleavage domain: VKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 104 Furin cleavage site RXXR SEQ ID NO: 105 Furin cleavage site RRKR SEQ ID NO:106 - Truncated EPOR endodomain (amino acids 274 to 378 of SEQ ID NO:1) HRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTTEDPPASLEV LSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPR SEQ ID NO:107 - Truncated EPOR endodomain (amino acids 274 to 433 of SEQ ID NO:1) HRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTTEDPPASLEV LSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPEGASAASFEYTILDPSS SEQ ID NO:108 - C-terminal tail of endodomain from EPOR MDTVP SEQ ID NO:109 - C-terminal tail of endodomain from EPOR SMDTVP SEQ ID NO:110 - C-terminal tail of endodomain from EPOR ASMDTVP SEQ ID NO:111 - C-terminal tail of endodomain from EPOR LASMDTVP SEQ ID NO:112 - C-terminal tail of endodomain from EPOR ALASMDTVP SEQ ID NO:113 - C-terminal tail of endodomain from EPOR PALASMDTVP SEQ ID NO:114 - WT EPOR endodomain with a 10 amino acid tail at the C-terminus HRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEV LSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPEGASAASFEYTILDPSSQLLRPWTLCPELPPTPPHLK YLYLVVSDSGISTDYSSGDSQGAQGGLSDGPYSNPYENSLIPAAEPLPPSYVACSPALASMDTVP SEQ ID NO:115 - Truncated EPOR endodomain with a 10 amino acid tail at the C-terminus HRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEV LSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRPALASMDTVP SEQ ID NO:116 - Truncated EPOR endodomain with a 10 amino acid tail at the C-terminus HRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEV LSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPEGASAASFEYTILDPSSPALASMDTVP EXAMPLES

[0360] Working Example Example 1 material and method Cloning: The construct of the present invention shown in Figure 2 was designed in-house. It can be cloned into a pMP71 backbone, and D5a high efficiency bacteria can be transformed with the plasmid and grown with the selection drug ampicillin. DNA can be extracted using a Miniprep Kit (Qiagen). Inserts can be introduced into the lentiviral backbone by PCR cloning.

[0361] Collection of PBMCs: Leukocyte cones are available from the UK NHS blood and transplant. PBMCs can be isolated using a density centrifugation protocol. Briefly, blood can be diluted 1:1 with 1xPBS and layered onto Ficoll-Paque (GE Healthcare). Samples can be centrifuged and the buffy cell layer removed and washed with PBS.

[0362] Treg and Tconv Isolation Protocol: Blood cones can be used to induce Treg and Teff populations. Blood cones can be subjected to CD4 enrichment by negative selection using RosetteSep™ Human CD4+ T Cell Enrichment Cocktail. CD4+ cells can then be isolated by density centrifugation. CD4+CD25+ T cells can then be isolated by positive selection using CD25 MicroBeads II (Miltenyi). The CD4+CD25- fraction of cells can be retained for use as a conventional T cell (Tconv) population. The CD4+CD25+ fraction can be stained with flow cytometry antibodies CD4 FITC (OKT4, Biolegend), CD25 PE-Cy7 (BC96, Biolegend), CD127 BV421 (A019D5, Biolegend), CD45RA BV510 (HI100, Biolegend), and LIVE / DEAD™ Fixable Near-IR-Dead Cell Stain (Thermofisher) followed by FACS sorting. Where indicated, CD4+CD25+CD127low (bulk Treg) or CD4+CD25+CD127low CD45RA+ (CD45RA+ Treg) can be used for sorting.

[0363] Tconv Culture Medium: Human Tconv can be grown in RPMI-1640 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum, penicillin, streptomycin, and L-glutamine (Gibco).

[0364] Treg culture medium and expansion: Human regulatory T cells can be cultured in Texmacs medium (Miltenyi) supplemented with IL-2 and activated with Human T-Activator CD3 / CD28 Dynabeads™ (Gibco). Cells can be re-fed with Treg medium supplemented with IL-2 every 2-3 days. A second stimulation with Dynabeads™ can be performed to promote further expansion of Treg cells.

[0365] Gene transfer and viral particle production HEK293T cells can be seeded in DMEM (Dulbecco's Modified Eagle Medium) + 10% fetal bovine serum (FBS) and cultured for 24 hours. The transfection reagent can be brought to room temperature and mixed with the DNA construct / plasmid of interest, packaging plasmid (pD8.91), and viral envelope (pVSV-G). The diluted DNA can be mixed with PEI and added to HEK293T. The supernatant can be collected 48 hours after transfection and filtered to concentrate the virus.

[0366] Transduction of T cells Tconv can be activated with anti-CD3 and anti-CD28 Dynabeads (Gibco) and resuspended in T cell culture medium. Non-tissue culture treated 24-well plates can be prepared by coating with Retronectin (Takahara-bio, Otsu, Japan) and the cell suspension can be added together with the lentiviral supernatant. Cells can be incubated, with medium changes every other day. Cells can be used for experiments 7 days after transduction.

[0367] Flow cytometry staining T cells can be removed from culture, washed with FACS buffer, and stained first with LIVE / DEAD™ Fixable Near-IR-Dead Cell Stain (Thermo Fisher) in PBS, followed by anti-CD4 AF700 (RPA-T4, BD) and anti-CD3 PE-Cy7 in FACS staining buffer. For intracellular staining of FOXP3, cells can be fixed, permeabilized, and stained with anti-FoxP3 PE (150D / E4, Thermo Fisher) antibody. Cells can be analyzed on a BD LSRII flow cytometer.

[0368] Flow cytometric phenotypic analysis of transduced cells T cells can be removed from culture and stained with LIVE / DEAD™ Fixable Near-IR-Dead Cell Stain as described above to identify live cells. Surface staining of cells can be performed with anti-CD4 AF700, anti-CD25 PE-Cy7 (BC96, Biolegend), anti-CD39 PerCPCy5.5 (A1, Biolegend), anti-CD62L PE-CF594 (DREG-56, BD), anti-TIM3 BV786 (7D3, BD), anti-TIGIT BV605 (A15153G, Biolegend), anti-CD45RO BUV395 (UCHL1, BD), anti-CD279 BUV737 (EH12.1, BD), and anti-CD223 BV711 (11C3C65, Biolegend). Cells can be permeabilized and stained with anti-FoxP3 PE (150D / E4, Thermo Fisher).

[0369] 4. Data Analysis Flow cytometry data can be analyzed using the flow cytometry analysis software FlowJo (FlowJo, LLC). All statistical analyses can be performed using Graphpad Prism v.5 (Graphpad, Software).

[0370] Example 1a: Expression in Jurkat The construct can be cloned into a lentiviral backbone encoding a puromycin resistance gene, as described above. Viral vectors can be generated and used to transduce Jurkat T cell lines. Two days after transduction, Jurkat cells can be selected with 4 μg / ml puromycin for one week. Cells are counted and 0.5×10 6 The cells can be stained with anti-EPOR antibody to determine the level of recombinant protein expressed in the cells, and expression can be assessed by flow cytometry as described above.

[0371] Example 2: STAT5 signaling of constructs The various constructs of recombinant proteins are cloned into lentiviral backbones that encode a puromycin resistance gene. Viral vectors are generated and used to transduce NFAT, NfkB, and STAT5 Jurkat reporter cell lines, where the NFAT, NfkB, or STAT5 response elements control the activity of the luc2 reporter gene. Two days after transduction, Jurkat cells are selected with 4 μg / ml puromycin for one week. Luciferase is evaluated in the various reporter cell lines using the ONE-Glo™ Luciferase Assay System (Promega).

[0372] Example 3: Generation of regulatory T cells expressing chimeric proteins Regulatory T cells are purified and FACS sorted as CD4+CD25+CD127- cells from healthy donors. Cells are activated using Human T-Activator CD3 / CD28 Dynabeads™ (ThermoFisher Scientific) in X-Vivo medium (Lonza) in the presence of interleukin-2 (1000IU / ml). After 48 hours of activation, cells are transduced with lentiviral particles encoding recombinant protein constructs. Cells are further expanded and proliferation rates are compared between different conditions. Cells are harvested and counted on day 14. 0.5×10 6 Cells were stained with anti-EPOR antibody. EPOR expression levels and transduction efficiency were assessed by flow cytometry using the percentage of anti-EPOR antibody. Treg phenotype was assessed by surface staining with anti-CD4, anti-CD25, anti-CD127, anti-CD8, anti-GITR, anti-CD39, anti-CD45RA, anti-CD45RO, and anti-ICOS, and intracellular staining with anti-FOXP3 and anti-HELIOS after fixation and permeabilization (Transcription Factor Staining Buffer Set, Thermo Fisher Scientific).

[0373] Example 4: STAT5 phosphorylation analysis as an indicator of IL2R signaling Chimeric protein-transduced Tregs were rested overnight in IL2-free medium, and STAT5 phosphorylation in Tregs was assessed by FACS analysis after 10 and 120 min of culture in medium alone or with rapamycin.

[0374] Example 5: Analysis of constructs 783, 784, 785, 786, 787, and 788 material and method Cloning: Each construct shown in Figure 3 was designed in-house. Cloning was performed in lentiviral vector backbones, and NEB Stable high efficiency bacteria were transformed with the plasmids and grown with the selective agent kanamycin. DNA was extracted using the Miniprep Kit and Maxiprep Kit (Qiagen). Inserts were introduced into other lentiviral or retroviral backbones by PCR cloning. Cloning was performed by site-directed mutagenesis or PCR-based methods.

[0375] Constructs: Construct pQTX 783 (EPOR WT) contains the wild-type EPO receptor operably linked to an HLA-A2-specific CAR and FoxP3 (separated by the T2A and P2A self-cleavage domains). Construct pQTX 784 (EPOR mut) comprises a mutated EPO receptor operably linked to an HLA-A2-specific CAR and FoxP3 (separated by T2A and P2A autocleavage domains). The mutated EPOR has a mutation in the exodomain (R to C mutation at position 154 of SEQ ID NO:1). The transmembrane and endodomains of the mutated EPOR are those of wild-type EPOR, respectively. Construct pQTX 785 (EPOR trunc V1) contains a mutant EPO receptor operably linked to an HLA-A2-specific CAR and FoxP3 (separated by the T2A and P2A autocleavage domains). The mutant EPOR has a mutation in the exodomain (R to C mutation at position 154 of SEQ ID NO:1) and the endodomain is truncated after position 378 of SEQ ID NO:1. The transmembrane domain is that of wild-type EPOR. Construct pQTX 786 (EPOR trunc V2) contains a mutant EPO receptor operably linked to an HLA-A2-specific CAR and FoxP3 (separated by the T2A and P2A autocleavage domains). The mutant EPOR has a mutation in the exodomain (R to C mutation at position 154 of SEQ ID NO:1) and the endodomain is truncated after position 433 of SEQ ID NO:1. The transmembrane domain is that of wild-type EPOR. Construct pQTX 787 (EPOR trunc V3 FS) contains a mutant EPO receptor operably linked to an HLA-A2-specific CAR and FoxP3 (separated by T2A and P2A autocleavage domains). The mutant EPOR has a mutation in the exodomain (R to C mutation at position 154 of SEQ ID NO:1), an endodomain truncated after position 433 of SEQ ID NO:1, and an additional 10 amino acid tail inserted at the C-terminus (PALASMDTVP). The transmembrane domain is that of wild-type EPOR. Construct pQTX 788 (WT EPOR trunc V3 FS) contains a mutant EPO receptor operably linked to an HLA-A2-specific CAR and FoxP3 (separated by the T2A and P2A autocleavage domains). The exodomain and transmembrane domain are those of wild-type EPOR, respectively. The endodomain is truncated after position 433 of SEQ ID NO:1, with an additional 10 amino acid tail inserted at the C-terminus (PALASMDTVP).

[0376] Collection of PBMCs: Leukopaks were obtained from BioIVT. PBMCs were isolated using a negative selection kit (StemCell Technologies). Briefly, unwanted fractions were targeted for removal by labeling with antibody conjugates and magnetic beads, and then separated using a magnet.

[0377] Treg and Tconv (Teff) Isolation Protocol: Leukopack was used to induce Treg and Tconv populations. Leukopack-derived PBMCs were subjected to CD25 positive selection followed by CD4 enrichment by negative selection. Human CD25 Positive Selection Cocktail and Human CD4+ T Cell Enrichment Cocktail (Stem Cell Technologies, Inc.), respectively, were used. Cells of interest were isolated using a magnet. The CD4+CD25- fraction of cells was retained for use as the Tconv population. CD127high Depletion Cocktail (Stem Cell Technologies, Inc.) was used to further separate the CD4+CD25+CD127- / low cell population and the cells of interest isolated using the magnet. Each cell fraction was stained with flow cytometry antibodies anti-CD4 VioBlue (M-T466, Miltenyi Biotec), anti-CD25 PE (3G10, Miltenyi Biotec), anti-CD127 APC (MB15-18C9, Miltenyi Biotec), anti-CD45RA FITC (T6D11, Miltenyi Biotec), and LD 7AAD (Biolegend) and then FACS sorted. Where indicated, CD4+CD25+CD127- / low (bulk Treg) or CD4+CD25+CD127- / low CD45RA+ (CD45RA+ Treg) sorting was used.

[0378] Treg culture medium and expansion: Human regulatory T cells were cultured in X-VIVO 15 medium (Lonza) supplemented with 5% human AB serum (heat-inactivated, Merck) and IL-2 (Proleukin, Clinigen Healthcare) and activated with anti-CD3 / CD28 beads. Cells were re-fed with Treg culture medium supplemented with IL-2 every 2–3 days. A second stimulation with anti-CD3 / CD28 beads was performed to promote further expansion of Treg cells.

[0379] Gene transfer and viral particle production: HEK293T cells were seeded in DMEM (Dulbecco's Modified Eagle Medium) + 10% fetal bovine serum (FBS) and cultured for 24 hours. The transfection reagent was brought to room temperature and mixed with the DNA construct / plasmid of interest, packaging plasmid (pMDLg pRRE), regulatory plasmid (PRSV-REV), and viral envelope (pMD2.G). The diluted DNA was mixed with FuGENE HD Transfection Reagent (Promega) and added to HEK293T. 48 hours after transfection, the supernatant was collected and filtered to concentrate the virus.

[0380] Transduction of T cells: Tregs were activated with anti-CD3 / CD28 beads and resuspended in T cell culture medium (X-Vivo 15 medium (Lonza), 5% human AB serum, and IL2). Non-tissue culture treated 24-well plates were prepared by coating with RetroNectin (Takahara-bio, Otsu, Japan) and the cell suspension was added together with the lentiviral supernatant. Cell cultures were spinoculated and medium was changed every other day. Cells were used for experiments 7 days after transduction or later.

[0381] Transduction efficiency: Tregs were first surface stained with HLA-A2 Dextramer- APC (Immudex) for 15 min at room temperature. Washed cells were then stained with LIVE / DEAD™ Fixable Near-IR-Dead Cell Stain (Thermo Fisher) in PBS for 20 min at room temperature. Washed cells were then stained with anti-CD4 BV510 (A161A1, Bio-Legend), anti-CD25 PE-Cy7 (BC96, Bio-Legend), and anti-EPOR PE (38409, Bio-Techne) in FACS staining buffer for 20 min at 4°C. After washing the cells, acquisition was performed on an Attune™ NxT flow cytometer. Cell surface expression was analyzed using FlowJo software. The following gating strategy was used: lymphocytes>single cells>viable cells>CD4+CD25+>HLA-A2 Dex+ EPOR+.

[0382] pSTAT5 Assay: On day 14 after Treg isolation, transduced Tregs were rested for 24–48 h by depletion of anti-CD3 / CD28 beads and removal of IL2 before assay setup. Tregs were treated with or without 1 IU / ml EPO (Stem Cell Technologies, UK) for 30 min at 37°C before staining began. Tregs were first surface stained with HLA-A2 Dextramer- APC (Immudex) for 15 min at room temperature. Washed cells were then stained with LIVE / DEAD™ Fixable Near-IR-Dead Cell Stain (Thermo Fisher) in PBS for 20 min at room temperature. Washed cells were then stained with anti-CD4 BV510 (A161A1, Biolegend) and anti-CD25 PE-Cy7 (BC96, Biolegend) in FACS staining buffer for 20 min at 4°C. Cells were then fixed and permeabilized using the PerFix Expose kit (Beckman Coulter) and stained intracellularly with pSTAT5 PE-CF594 (47 / Stat5 pY694, BD Bioscience). Cells were washed twice and then acquired on an Attune™ NxT flow cytometer. pSTAT5 expression was analyzed using FlowJo software. The following gating strategy was used: lymphocytes>single cells>viable cells>CD4+CD25+>HLA-A2 Dex+>pSTAT5+, except for mock Tregs, where the following gating strategy was used: lymphocytes>single cells>viable cells>CD4+CD25+>pSTAT5+.

[0383] Enrichment of transduced cells and measurement of FOXP3 expression: On day 14 after Treg isolation, transduced Tregs were rested for 24 hours by depletion of anti-CD3 / CD28 beads and removal of IL2 before assay setup. Different conditions were set up consisting of medium, CD3 / CD28 beads, 0IU / ml and 300IU / ml IL2, and readings were taken on days 0, 4, and 6. Tregs were first surface stained with HLA-A2 Dextramer- APC (Immudex) and then stained with LIVE / DEAD™ Fixable Near-IR-Dead Cell Stain (Thermo Fisher) in PBS. Washed cells were then stained with anti-CD4 BV510 (A161A1, Biolegend) and anti-CD25 PE-Cy7 (BC96, Biolegend) in FACS staining buffer for 30 minutes at 4°C. The washed cells were then permeabilized with FOXP3 / Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, UK) according to the manufacturer's instructions. The permeabilized cells were stained with anti-FoxP3 BV421 (206D, Biolegend) antibody. The cells were washed twice before acquisition on an Attune™ NxT flow cytometer. FoxP3 expression was analyzed using FlowJo software. The following gating strategy was used: lymphocytes>single cells>viable cells>CD4+CD25+>HLA-A2 Dex+>FoxP3+, except for mock Tregs, where the following gating strategy was used: lymphocytes>single cells>viable cells>CD4+CD25+>FoxP3+.

[0384] Cell viability / fold proliferation measurement: On day 14 after Treg isolation, transduced Tregs were rested for 24 hours by depletion of anti-CD3 / CD28 beads and removal of IL2 before assay setup. Different conditions were set up consisting of medium, A1+ K562 cells (which were A2-) or A2+ K562 cells in the absence of IL2 and reads were taken on days 0, 5, and 7. The K562:Treg ratio was 1:4. Tregs were surface stained with anti-EPOR PE (38409, Biotechni), anti-CD34 FITC (QBEND10, Life Technologies), anti-CD4 BV510 (A161A1, Biolegend), and anti-CD25 PE-Cy7 (BC96, Biolegend). The washed cells were then stained with Annexin V BV421 and 7-AAD in Annexin V Binding Buffer using the Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend). Equal volumes of cell suspension were acquired across all wells to allow for unbiased quantification of viable transduced cells in each well. Cells were acquired on an Attune™ NxT flow cytometer. Cell viability was analyzed using FlowJo software. The following gating strategy was used: lymphocytes>single cells>Annexin V-7-AAD-(viable cells)>CD4+CD25+>CD34 / QBEND+ or EPOR+.

[0385] Deep phenotyping: On day 14 after Treg isolation, transduced Tregs were rested for 24 hours by depletion of anti-CD3 / CD28 beads and removal of IL2 before staining. Tregs were first surface stained with HLA-A2 Dextramer APC (Immudex) for 15 minutes at room temperature. Washed cells were then stained with LIVE / DEAD™ Fixable eFluor455-Dead Cell Stain (Thermo Fisher) in PBS for 20 minutes at room temperature. Washed cells were then stained with anti-CCR4 BV480 (1G1, BD Biosciences), anti-CCR7 BUV395 (2-L1-A, BD Biosciences), and anti-CD183 / CXCR3 PE-Cy7 (G025H7, Biolegend) in Brilliant Stain Buffer Plus for 20 minutes at 37°C. The washed cells were then stained with anti-HLA-DR APC-Fire810 (G46-6, BD Biosciences), anti-PD-1 BV650 (EH12.2H7, Biolegend), anti-ICOS BV750 (DX29, BD Biosciences), anti-CD8a BUV805 (SK1, BD Biosciences), anti-TIM-3 / CD366 BV711 (7D3, BD Biosciences), anti-CD27 PE-AF700 (0323, Biolegend), anti-CCR6 BV605 (G034E3, Biolegend), anti-CD95 PE-Cy5 (DX2, BD Biosciences), anti-TIGIT APC-Cy7 (VSTM3, Biolegend), anti-CD70 BV786 (Ki-24, BD Biosciences), anti-CD4 Cells were stained with VioBlue (M-T466, Miltenyi Biotec), anti-CD34 PE (QBEND10, Life Technologies), and anti-CD25 BUV563 (2A3, BD Biosciences) for 30 min at 4°C. For intracellular staining of cells, cells were fixed and permeabilized using FOXP3 / Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, UK) according to the manufacturer's instructions.Permeabilized cells were stained with anti-Helios PE-Dazzle594 (22F6, Biolegend), anti-CTLA-4 BB700 (BNI3, BD Biosciences), and anti-FOXP3 AF647 (206D, BD Biosciences) in permeabilization buffer. Cells were washed twice and then acquired on a flow cytometer. Marker expression was analyzed using FlowJo software.

[0386] Inhibition assay: On day 14 after Treg isolation, transduced Tregs were rested for 24 hours by depletion of anti-CD3 / CD28 beads and removal of IL2 before assay setup. Tregs were stained using the CellTrace™ Violet (CTV) Cell Proliferation Kit (Thermo Fisher) and T effectors were stained using the CellTrace™ Yellow (CTY) Cell Proliferation Kit (Thermo Fisher) according to the manufacturer's protocol. CTV-stained Tregs were cultured with CTY-stained T effectors at different ratios as indicated. 120 Gy-irradiated HLA-A2+ or HLA-A2- B cells were cocultured at a B cell:T effector ratio of 1:3.3. Alternatively, Human T-Activator CD3 / CD28 Dynabeads™ (Gibco) were added at a bead:T effector ratio of 1:1. Cell cultures were incubated at 37°C, 5% CO2 for 4 days before staining. Cells were washed with PBS and then stained with Fixable Near IF-Dead Cell Stain (Thermo Fisher) in PBS for 20 min at room temperature. Washed cells were then stained with anti-EPOR PE (38409, Biotechni), anti-CD34 FITC (QBEND10, Life Technologies), and HLA-A3 (REA950, Miltenyi Biotec) for 20 min at 4°C. After washing the cells, acquisition was performed on an Attune™ NxT flow cytometer. Data were analyzed using FlowJo software. The following gating strategy was used: lymphocytes>single cells>viable cells>HLA-A3->CTY+.

[0387] Cytotoxicity Assay: The CellTrace™ Violet (CTV) Cell Proliferation Kit (Thermo Fisher) was used to label target cells, HLA-A2+ K562 or HLA-A2- K562, according to the manufacturer's protocol. The indicated Treg or natural killer (NK) effector cells were co-cultured with the target cells at various ratios as indicated. Anti-CD107a PE (H4A3, BD Biosciences) was added to the cultures and incubated for a total of 4 hours at 37°C and 5% CO2. After 1 hour of incubation, GolgiSTOP working solution (BD Biosciences) was added to the cultures. Washed cells were acquired on an Attune™ NxT flow cytometer. Data were analyzed using FlowJo software.

[0388] Intracellular cytokine staining: Treg or T cells were activated with Leukocyte Activation Cocktail with BD GolgiPlug™ for 5 hours at 37°C. Cells were then stained with LIVE / DEAD™ Aqua in PBS for 20 minutes at room temperature. Washed cells were then stained with anti-CD4 PerCPCy5.5 (SK3, BioLegend), anti-EPOR PE (38409, Biotechni), and anti-CD34 FITC (QBEND10, Life Technologies) for 30 minutes at 4°C. Washed cells were then fixed and permeabilized with FOXP3 / Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, UK) according to the manufacturer's instructions. The permeabilized cells were then stained with anti-IL-17A BV605 (BL168, Biolegend), anti-TNFα APC-Cy7 (Mab11, Biolegend), anti-IFN-γ PE-Cy7 (4S.B3, Biolegend), and anti-IL2 PE-PE / Dazzle 594 (MQ1-17H12, Biolegend). After washing, the cells were acquired using a flow cytometer.

[0389] Data Analysis: Flow cytometry data can be analyzed using the flow cytometry analysis software FlowJo (FlowJo, LLC). All statistical analyses can be performed using Graphpad Prism v.9.4.1 (Graphpad Prism Software).

[0390] result FIG. 4A - Constructs are well expressed in transduced human Tregs Also shown are FACS plots showing the transduction efficiency of fresh Tregs transduced with various constructs, namely 658 (no tech), 783, 784, 785, 786, and mock. Transduction efficiency was measured using HLA-A2 dextramer and EPOR expression. Similar transduction efficiency was obtained for each construct, which is also shown in the table below. [Table 2]

[0391] FIG. 4B - Constructs are well expressed in transduced human Tregs Also shown are FACS plots showing the transduction efficiency of frozen Tregs transduced with various constructs, namely 783, 784, 785, 786, 787, 788, and mock. Transduction efficiency was measured using HLA-A2 dextramer and EPOR expression. Similar transduction efficiency was obtained for each construct, which is also shown in the table below. [Table 3]

[0392] Figure 5 - Tregs transduced with EPOR-derived proteins specifically upregulate pSTAT5 levels Transduced Tregs were treated with or without EPO and then stained for pSTAT5 expression levels. The transduced fraction of cells was pre-gated, defined as HLA-A2 CAR+, whereas the non-transduced fraction is HLA-A2 CAR-. The transduced fraction of cells expressing one of constructs 783, 784, 785, and 786 had higher MFI levels of pSTAT5 compared to the non-transduced fraction of cells, indicating that EPOR-derived proteins promote JAK-STAT signaling. The transduced fraction of cells expressing one of constructs 783, 784, 785, and 786 also responded specifically to exogenous EPO, as detected by higher MFI levels of pSTAT5, whereas the non-transduced fraction did not respond to exogenous EPO, and this non-transduced fraction remained at a similar basal level of MFI of pSTAT5. Cells transduced with construct 658, which does not have EPOR-derived proteins, had similar MFI levels of pSTAT5 in both the transduced and non-transduced fractions. It is worth noting that the medium used (X-vivo + 5% human serum) likely contained EPO, as EPO is present in serum. This explains the increase in pSTAT5 in cells transduced with construct 783 (WT EPOR) and the further increase when EPO was added. However, when no additional EPO was used, the transduced fractions of 784, 785, and 786 had higher pSTAT5 than 783.

[0393] Figure 6A - Tregs transduced with EPOR-derived proteins are enriched over time and have a survival advantage compared to control Tregs Transduced Tregs were cultured with or without IL2. Cells transduced with constitutive EPOR technology, 784, 785, and 786, are enriched in the population over time. In contrast, 658 (no EPOR derived protein) and 783 (WT EPOR) constructs are not preferentially enriched over time. Cells transduced with constitutive EPOR (784, 785, and 786) show a survival advantage over 783 (WT EPOR) and 658 (no tech) in the absence of IL2. 786 (EPOR trunc V2) appears to have the greatest advantage as these transduced cells are increased in proportion over time compared to non-transduced cells. In the presence of IL2, the non-transduced fraction of cells is able to proliferate as a result of signaling through the native IL2R, and thus the constitutive EPOR technology transduced population is not enriched.

[0394] Figure 6B - Tregs transduced with EPOR-derived proteins maintain FoxP3 expression over time Transduced Tregs were cultured with or without IL2. The percentage of cells expressing FoxP3 was similar across all transduced fractions of 783, 784, 785, and 786 compared to 658. In the non-transduced fraction of cells, the percentage of FoxP3 expression was somewhat lower than in the transduced fraction of cells. Cells transduced with EPOR-derived proteins expressed and maintained FoxP3 expression levels over time, indicating that Tregs maintain their phenotype.

[0395] FIG. 6C-786 transduced Tregs recognize cognate A2 antigen and respond similarly to 658 control Tregs Survival assay showing 658- and 786-transduced Tregs co-cultured with A2+ K562 cells or A2- K562 (A1+ K562) cells. The data show that both 658- and 786-transduced Tregs specifically recognize and respond to the A2 antigen by changes in cell proliferation fold. Thus, transduction with constitutive EPOR technology (786) does not adversely affect the function of the cells.

[0396] Figure 7 - Tregs transduced with EPOR-derived proteins maintained expression of key Treg markers Transduced Tregs were deep phenotyped for various Treg-associated markers. Graphs show the percentage of expression of the indicated markers within mock Tregs, transduced (HLA-A2 Dex+), or non-transduced (HLA-A2 Dex-) fractions of cells. EPOR-transduced Tregs (both transduced and non-transduced fractions) maintained similar high expression levels of key Treg markers such as CD25, CD27, CD95, CTLA-4, CXCR3, and FoxP3 compared to mock-transduced Tregs. This result suggests that transduction with EPOR-derived proteins had no significant effect on Treg phenotype.

[0397] Figure 8 - Tregs transduced with EPOR-derived proteins maintained their suppressive capacity Functional assays were performed to examine the suppressive ability of transduced Tregs. The graph shows the suppressive rate of transduced Tregs (or mock Tregs) against the proliferation of T effector cells by different stimuli: aCD3 / CD28 beads, HLA-A2- B cells, and HLA-A2+ B cells. The results show that Tregs using EPOR technology have similar suppressive ability to mock Tregs.

[0398] Figure 9 - Tregs transduced with EPOR-derived proteins do not kill target cells The cytotoxicity of transduced Tregs against target cells was tested. HLA-A2- K562 cells or HLA-A2+ K562 cells were co-cultured with transduced Tregs at various ratios. HLA-A2+ natural killer (NK) cells were used as a positive control. The results show that mock- and EPOR-transduced Tregs do not kill either target or non-target cells, thus maintaining the regulatory phenotype.

[0399] Figure 10 - Tregs transduced with EPOR-derived proteins have similar intracellular cytokine profiles as control Tregs The presence of intracellular cytokines in 658- and 786-transduced Tregs was examined. After stimulating the cells with PMA / ionomycin, intracellular cytokines were detected. T effector cells were used as a positive control. 786-transduced Tregs had similar low levels of intracellular cytokines IL2, TNFα, IL-17, and IFNγ as 658-transduced Tregs in both transduced and non-transduced fractions. This suggests that the 786 construct does not induce an inflammatory response in the cells and that the cells maintain their regulatory phenotype.

Claims

1. 1. A T cell comprising a recombinant protein, wherein the recombinant protein comprises an exodomain derived at least in part from the extracellular region of EPOR, the exodomain comprising a dimerization domain that enables the recombinant protein to dimerize with a second protein and provide a signal to the T cell in the absence of a signal inducer molecule.

2. said at least a portion of said exodomain comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:3 or SEQ ID NO:5; and / or The T cell of claim 1, wherein the at least a portion of the exodomain derived from the extracellular region of EPOR comprises the dimerization domain. (i) the dimerization domain is a leucine zipper or a cysteine ​​residue; (ii) the dimerization is disulfide bond dimerization, and / or (iii) The T cell of claim 1, wherein the dimerization is homodimerization or heterodimerization.

4. The T cell of claim 1 , wherein the signal inducer molecule is EPO.

5. the exodomain being: (i) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2, or a variant having at least 80% sequence identity to SEQ ID NO: 2 and having a cysteine ​​residue at position 130, or comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4, or a variant having at least 80% sequence identity to SEQ ID NO: 4 and having a cysteine ​​residue at position 154; and / or (ii) comprises one or more domains or sequences heterologous to EPOR; and / or (iii) comprising one or more tag peptides, suicide moieties, and / or inducible dimerization domains; The T cell of claim 1. (i) the second protein is different from the recombinant protein, and / or (ii) the T cell further comprises the second protein; and / or (iii) the T cells further comprise a chimeric antigen receptor, a xenogeneic TCR, a safety switch polypeptide, a xenogeneic FOXP3 polypeptide, and / or a mutant calcineurin protein; and / or The T cell of claim 1, wherein (iv) the extracellular region of the EPOR is capable of binding to EPO, and / or (v) the recombinant protein comprises an endodomain or is capable of associating with a signaling protein comprising an endodomain, and in particular, the endodomain comprises a tyrosine kinase activation domain comprising a JAK1-binding motif and / or a JAK2-binding motif, and a tyrosine effector domain comprising one or more tyrosine residues that can be phosphorylated by JAK1 and / or JAK2.

7. In (v) of claim 6, (a) the tyrosine effector domain comprises at least one STAT association motif, preferably a STAT5 association motif; (b) the endodomain further comprises a JAK3 binding motif; (c) the endodomain comprises or consists of the EPOR endodomain set forth in SEQ ID NO: 8, or an EPOR endodomain variant having at least 40% sequence identity to SEQ ID NO: 8; as needed, (i) the EPOR endodomain variant comprises at least one modification to reduce binding of SHP1 to the EPOR endodomain sequence, in particular, the modification is to Y181 and / or Y183 of SEQ ID NO: 8; and / or (ii) the EPOR endodomain variant comprises or consists of a truncated EPOR endodomain in which at least a portion of the EPOR endodomain including Y181 and / or Y183 of SEQ ID NO: 8 has been truncated, for example, the truncated EPOR endodomain comprises or consists of the sequence of SEQ ID NO: 62, SEQ ID NO: 106, or SEQ ID NO: 107, or a variant having at least 80% sequence identity to SEQ ID NO: 62, SEQ ID NO: 106, or SEQ ID NO: 107; and / or (iii) the variant EPOR endodomain variant comprises an insertion of at least the sequence of SEQ ID NO: 108 or a sequence that differs by no more than two amino acids from SEQ ID NO: 8, e.g., the variant comprises an insertion of at least the sequence of SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, or SEQ ID NO: 113, or a sequence that differs by no more than one or two amino acids from SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, or SEQ ID NO: 113; Furthermore, if necessary, The T cell of claim 6, wherein the EPOR endodomain variant comprises or consists of the sequence of SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 116, or a variant having at least 80% sequence identity to the sequence of SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO:

116.

8. (i) a nucleotide sequence encoding the recombinant protein defined in claim 1; (ii) a construct comprising a nucleic acid molecule as defined in (i) and one or more additional base sequences; or (iii) a vector comprising a nucleic acid molecule as defined in (i) or a construct as defined in (ii); T cells comprising:

9. 9. The T cell of claim 8, wherein the additional base sequence (i) is a regulatory sequence and / or (ii) encodes a protein of interest, and / or the protein of interest is (iii) a therapeutic protein, (iv) an antigen receptor, (v) a CAR or TCR, (vi) a safety switch polypeptide, or (vii) a FOXP3 polypeptide.

10. A cell population comprising T cells as defined in claim 1. (i) a nucleic acid molecule comprising a nucleic acid sequence encoding a recombinant protein as defined in claim 1; (ii) a construct comprising a nucleic acid molecule as defined in (i) and one or more additional base sequences; or (iii) a vector comprising the nucleic acid molecule defined in (i) or the construct defined in (ii), The method for producing T cells according to claim 1, comprising the step of introducing the T cells into cells. (i) a nucleic acid molecule comprising a nucleic acid sequence encoding a recombinant protein as defined in claim 1; (ii) a construct comprising a nucleic acid molecule as defined in (i) and one or more additional base sequences; or (iii) a vector comprising the nucleic acid molecule defined in (i) or the construct defined in (ii), 1. A method for promoting cell survival or persistence, comprising introducing into a cell: Optionally, the method further comprises culturing the cells in the presence of EPO.

13. A pharmaceutical composition comprising a T cell defined in claim 1 or 8 or a cell population containing said T cell. (a) for treating cancer, an infectious disease, a neurodegenerative disease, or an inflammatory disease, or for inducing immunosuppression, or (b) for use in inducing tolerance to transplantation, treating and / or preventing graft-versus-host disease (GvHD), an autoimmune disease, or an allergic disease, promoting tissue repair and / or tissue regeneration, or ameliorating inflammation in a subject; 10. A pharmaceutical composition comprising a T cell as defined in claim 1 or 8 or a cell population comprising said T cell, Preferably, the cells are Treg cells. (a) for treating cancer, an infectious disease, a neurodegenerative disease, or an inflammatory disease, or for inducing immunosuppression; (b) for use in inducing tolerance to transplantation, treating and / or preventing graft-versus-host disease (GvHD), an autoimmune disease, or an allergic disease, promoting tissue repair and / or tissue regeneration, or ameliorating inflammation in a subject; 10. Use of a T cell or a cell population comprising said T cell as defined in claim 1 or 8 for the manufacture of a medicament, comprising: Preferably, the cells are Treg cells.

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