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WO2023099886A1 - Signalling protein - Google Patents

Signalling protein Download PDF

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Publication number
WO2023099886A1
WO2023099886A1 PCT/GB2022/053030 GB2022053030W WO2023099886A1 WO 2023099886 A1 WO2023099886 A1 WO 2023099886A1 GB 2022053030 W GB2022053030 W GB 2022053030W WO 2023099886 A1 WO2023099886 A1 WO 2023099886A1
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WIPO (PCT)
Prior art keywords
domain
cell
dimerization
protein
chimeric protein
Prior art date
Application number
PCT/GB2022/053030
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French (fr)
Inventor
Marc MARTINEZ-LLORDELLA
Maria ALONSO-FERRERO
Evanthia NIKOLOPOULOU
Original Assignee
Quell Therapeutics Limited
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Publication date
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Publication of WO2023099886A1 publication Critical patent/WO2023099886A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex

Definitions

  • the present disclosure and invention relate to a chimeric protein useful in adoptive cell therapy (ACT).
  • the chimeric protein is a signalling protein, or a component thereof, which can provide a cell expressing the protein with a desired signal, e.g. a STAT-mediated signal.
  • the signal may confer a desired effect or property on the cell, e.g. increased function, activity, vitality, or survival, e.g. persistence in a transplanted host subject.
  • nucleic acid molecules encoding such a chimeric protein, recombinant constructs, vectors and cells containing the nucleic acid molecule, methods of producing such cells, and therapeutic uses thereof.
  • ACT Adoptive cell therapy
  • Tumour-infiltrating lymphocytes were initially shown to be effective in treating metastatic melanoma, and subsequently re-directed T-cells or NK cells expressing chimeric antigen receptors (CARs) or heterologous T-cell receptors (TCRs) to target different cellular target molecules have been developed and adopted for clinical use.
  • CARs chimeric antigen receptors
  • TCRs heterologous T-cell receptors
  • Tregs cytotoxic T-cells or NK cells, to target and kill unwanted or deleterious cells in the body, but more recently regulatory T cells (Tregs) have been developed for ACT.
  • Tregs have immunosuppressive function. They act to control cytopathic immune responses and are essential for the maintenance of immunological tolerance.
  • the suppressive properties of Tregs can be exploited therapeutically, for example to improve and/or prevent immune-mediated organ damage in inflammatory disorders, autoimmune diseases and in transplantation.
  • the transplanted, or administered, cells need to survive and persist in the recipient (the subject of the ACT therapy) in a functional state long enough to exert a useful therapeutic effect. Further, to be prepared in sufficient numbers for therapeutic use, the cells need to be generated (e.g.
  • IL-2 growth factor interleukin-2
  • Tregs generation, proliferation, survival
  • Tcons Activated conventional T cells
  • Tregs in contrast, cannot produce IL-2 and depend on paracrine access to IL-2 produced by Tcons present in the microenvironment.
  • the availability of IL-2 has a critical impact on the therapeutic effects of Tregs expanded in vitro and transferred into patients.
  • in vitro expansion protocols typically require high concentrations of IL-2, which renders Tregs highly dependent on this cytokine; 2) the concentration of IL-2 is often reduced in patients as a result of the administration of immunosuppressive drugs; and 3) within the inflamed tissue microenvironment access to IL-2 is often limited.
  • Liver transplantation constitutes a particularly challenging indication, given that the levels of IL-2 in the inflamed liver are known to be reduced, which is further aggravated by the routine use of calcineurin inhibitors, which substantially decrease the capacity of Tcons to produce IL-2.
  • IL-2 The administration of low doses of exogenous IL-2 restores the Treg dysfunction induced by calcineurin inhibitors and promotes the accumulation of Tregs in the liver.
  • a concern with the therapeutic use of low-dose IL-2 is the risk of simultaneously activating Tcons, which can enhance tissue damage.
  • Treg cells are engineered to express a CAR which has been modified such that it is capable of providing a productive IL-2 signal to the cell upon binding to its target antigen.
  • the intracellular signalling domain of the CAR includes sequences, or domains, derived from IL receptors, which allow it to transmit an “IL-2 signal” in the absence of endogenous IL-2, and without the need for IL-2 binding.
  • STAT5 Signal Transducer and Activator of Transcription 5
  • the CAR in WO 2020/044055 comprises an endodomain which comprises a STAT5 association motif and a JAK1- and/or a JAK2-binding motif.
  • other immune cells for ACT e.g. cytotoxic T-cells, or other Teffector cells, including CAR-T cells, may also require or benefit from additional signalling capacity being provided to the cell to increase survival or persistence of function.
  • additional signalling, or more particularly engineered signalling, whether to increase survival or persistence, or to improve the functional activity or therapeutic effect of cells for ACT is not limited to Treg cells.
  • WO 2020/04405 provides an important advance, there is a continuing need in the field of ACT for new and improved approaches, and in particular approaches which avoid or reduce the need to develop a modified CAR for each target, and which may have a more universal application.
  • Summary The present inventors have developed a new signalling protein which can be used to impart a desired signalling capacity to a cell, notably in which the signalling involves tyrosine kinase activity, and protein phosphorylation.
  • the signalling involves Janus kinase (JAK) phosphorylation and activity, and more particularly activation of the JAK-STAT signalling pathway involving JAK1.
  • the signalling protein may be inducible.
  • the signalling provided by the protein may be induced, when desired, by exposing a cell expressing the protein to an inducer molecule.
  • the protein may be responsive to an inducer molecule which may be provided to the cell to induce the desired signalling.
  • the new signalling protein is not limited to inducible systems, and a constitutive signalling capacity may be provided to the cell.
  • the signalling protein is based on dimerization – that is, the signalling protein is formed from monomers which are designed to dimerize, or more generally to multimerize, and in the dimerized/multimerized state to assume a signalling capacity, i.e. to become functional when dimerized to mediate signalling in a cell by which it is expressed.
  • the monomer of the signalling protein is a chimeric protein comprising a signalling domain and a dimerization domain, that is a domain capable of multimerization (i.e. at least dimerizing) with a cognate dimerization domain in another chimeric protein. More particularly, at least one dimerization domain may be contained in an interaction domain capable of interacting with (e.g. a binding domain capable of binding to) with an interaction domain in another chimeric protein. The interaction, or dimerization, between the domains may be direct or indirect. In other words, individual dimerization domains in different molecules of the chimeric protein may interact (e.g.
  • the signalling domain is capable of transmitting a signal, for example a STAT-5 or other STAT mediated signal, when the chimeric protein is dimerized (or more generally multimerized).
  • signalling may be induced by interacting with (e.g. binding to) the inducer molecule.
  • the chimeric protein is a dimerizable protein, or a protein capable of higher multimerization (oligomerization).
  • the signalling activity of the chimeric protein is induced, or enabled, by multimerization (e.g. dimerization).
  • Multimerization (e.g. dimerization) of the protein may be induced by an inducer, or may occur spontaneously when two molecules of the chimeric protein come into proximity.
  • a surprising feature of the chimeric signalling protein herein is that it is a cytoplasmic cell protein, or alternatively put, a soluble protein, that is, it is not associated in any way with the surface membrane (plasma membrane) of the cell in which it is present or expressed. It is a protein that is present or expressed inside a cell.
  • non-membrane associated protein is capable of mediating an effective signal
  • effective signalling including notably STAT- mediated signalling
  • STAT- mediated signalling is generally thought to require a membrane protein, or proximity to a membrane protein.
  • reports in the literature of JAK1 being predominantly located at the cell membrane have fuelled the design of previous STAT signalling constructs as transmembrane proteins (e.g. WO2020/044055).
  • Cytoplasmic signalling proteins as developed herein, demonstrating effective STAT signalling were therefore unexpectedly functional and are further associated with the benefit of not requiring successful transport to and through the membrane for function.
  • a first aspect provides a chimeric protein, said chimeric protein being a non-membrane associated protein comprising, linked together in any order: (i) an interaction domain comprising at least one dimerization domain; (ii) a signalling domain comprising a tyrosine kinase activating domain comprising a JAK1-binding motif and a tyrosine effector domain comprising one or more tyrosine residues that can be phosphorylated by JAK1; wherein the dimerization domain of said chimeric protein is capable of dimerizing with a cognate dimerization domain of at least a second, non-membrane associated, chimeric protein, which may be the same chimeric protein, or a different chimeric protein, and wherein dimerization of the chimeric protein with the at least a second chimeric protein provides a signal in a cell expressing the chimeric protein and the at least a second protein.
  • the chimeric protein defined above may accordingly be seen as a first chimeric protein.
  • the first and second, and optionally any further, chimeric proteins may thus be seen as monomers, or components, of a cytoplasmic multi-component signalling protein. Said dimerization may enable, facilitate or contribute to signalling by the signalling protein.
  • the second chimeric protein may also comprise a signalling domain. More particularly, dimerization of the dimerization domains of the two chimeric proteins (i.e. the first and the second chimeric proteins) may cause the signalling domains of the respective chimeric proteins to dimerize to enable, facilitate or contribute to signalling by the signalling protein.
  • the dimerization domain is able to dimerize with its cognate dimerization domain, e.g. they are designed or selected to be able to dimerize with one other. In other words, they are paired. Since the chimeric protein may comprise more than one dimerization domain, the possibility exists to create higher order oligomers (or alternatively termed, multimers). In effect, the chimeric protein may be viewed as a monomer, or subunit, of a higher order signalling protein, e.g. a dimeric signalling protein, or a trimer or 4-mer etc.
  • two molecules of the chimeric protein may dimerize together in order to constitute, or form, the signalling protein, without requiring a different, or second component (i.e. without requiring a separate component).
  • the chimeric protein is in itself capable of providing a signal in a cell expressing the protein, when it is dimerized.
  • the second chimeric protein may thus also be defined as being a non-membrane associated protein comprising, linked together in any order: (i) an interaction domain comprising at least one dimerization domain; (ii) a signalling domain comprising a tyrosine kinase activating domain comprising a JAK1-binding motif and a tyrosine effector domain comprising one or more tyrosine residues that can be phosphorylated by JAK1.
  • the two chimeric proteins need not necessarily be absolutely identical as long as they have domains (i) and (ii) as defined above.
  • the second chimeric protein may be a different chimeric protein.
  • the second chimeric protein may have a different signalling domain.
  • the chimeric protein herein may be seen as a first chimeric protein.
  • the signalling domain of the chimeric protein herein (that is the first chimeric protein) further comprises a JAK3-binding motif.
  • the signalling domain of the second chimeric protein comprises a JAK3- binding motif.
  • a chimeric protein said protein being a non-membrane associated protein comprising, linked together in any order: (i) an interaction domain comprising at least one dimerization domain; (ii) a signalling domain A comprising a tyrosine kinase activating domain comprising a JAK1-binding motif and a tyrosine effector domain comprising one or more tyrosine residues that can be phosphorylated by JAK1, or a signalling domain B comprising a tyrosine kinase activating domain comprising a JAK3-binding motif; wherein when at least two chimeric proteins are expressed in a cell, the dimerization domain of one chimeric protein is capable of dimerizing with the dimerization domain of another of the chimeric proteins, causing the signalling domains of the at least two chimeric proteins at least to dimerize (or in other words, to multimerize) to enable signalling by the
  • the chimeric protein forms a dimer.
  • the chimeric protein comprises, linked together in any order: (i) an interaction domain comprising at least one dimerization domain; (ii) a signalling domain A comprising a tyrosine kinase activating domain comprising a JAK1-binding motif and a tyrosine effector domain comprising one or more tyrosine residues that can be phosphorylated by JAK1, or a signalling domain B comprising a tyrosine kinase activating domain comprising a JAK3-binding motif; wherein the dimerization domain of one chimeric protein of a pair of chimeric proteins is capable of dimerizing with the dimerization domain of the other chimeric protein of the pair, causing the signalling domains of the pair of chimeric proteins to dimerize to enable signalling by the dimerized chimeric protein; and wherein the chimeric proteins of the pair of chimeric proteins may be the same
  • the chimeric protein may form higher multimers such as trimers, or 4-, 5- or 6-mers for example.
  • the signalling domain B does not comprise a tyrosine effector domain.
  • the signalling domain B is not capable of providing a signal to a cell expressing the chimeric protein in the absence of signalling domain A (e.g. in the absence of a chimeric protein comprising signalling domain A). It will be understood from the above that a chimeric protein comprising a signalling domain B is not capable of dimerizing with another chimeric protein containing signalling domain B to provide a functional signalling protein. At least one of two chimeric proteins capable of dimerizing comprises a signalling domain A.
  • a second aspect provides a pair of dimerizable chimeric proteins, which together when dimerized are capable of providing a signal in a cell expressing the proteins, each protein of the pair being a non-membrane associated protein, wherein the first chimeric protein of the pair comprises, linked together in any order: (i) an interaction domain comprising at least one dimerization domain; (ii) a signalling domain A comprising a tyrosine kinase activating domain comprising a JAK1-binding motif and a tyrosine effector domain comprising one or more tyrosine residues that can be phosphorylated by JAK1; and the second chimeric protein of the pair comprises, linked together in any order: (i) an interaction domain comprising at least one dimerization domain; and (ii) a signalling domain B comprising a tyrosine kinase activating domain comprising a JAK3-binding motif; and wherein the dimerization domain of the first chimeric protein is
  • the chimeric protein when dimerized or higher multimerized is capable of providing an inducible signal in a cell expressing the protein.
  • the dimerization domains i.e. cognate dimerization domains
  • the dimerization domains in two chimeric proteins are able to form a dimer in the presence of a dimerization inducer.
  • the chimeric protein may comprise, linked together in any order: (i) an interaction domain comprising at least one dimerization domain; (ii) a signalling domain comprising a tyrosine kinase activating domain comprising a JAK1-binding motif and a tyrosine effector domain comprising one or more tyrosine residues that can be phosphorylated by JAK1; wherein in the presence of a dimerization inducer, the dimerization domain of said chimeric protein is induced to dimerize with a cognate dimerization domain of a second, non-membrane associated, chimeric protein, wherein the second chimeric protein may be the same chimeric protein, or a different chimeric protein and wherein dimerization of the chimeric protein with the second chimeric protein provides a signal in a cell expressing the chimeric protein and the second chimeric protein
  • the chimeric protein comprises, linked together in any order: (i)
  • the chimeric protein may alternatively multimerize to form a higher order multimer.
  • the inducer may induce, or mediate, dimerization of the chimeric protein by binding to two separate sites, or dimerization domains, in the interaction domain, one in each of two chimeric proteins.
  • the inducer may be capable of binding to two copies of the dimerization domain, one in each of two chimeric proteins. This brings the two proteins together and thereby allows the two signalling domains or a portion of a signalling domain, to dimerize, and thereby to be able to exert their function, namely transmission of a signal.
  • Higher multimers may similarly be created, for example where two dimerization domains in the interaction domain each dimerize with a separate chimeric protein.
  • an interaction domain may comprise more than two dimerization domains to achieve higher multimerization.
  • Dimerization, or higher multimerization, of the interaction domain by interacting with, or binding to, the inducer, allows the functional protein component of the chimeric protein, here the signalling domain, to be multimerized (e.g. dimerized) in order to exert its function.
  • the inducer may thus be seen as an inducer of multimerization, or more particularly an inducer of dimerization, which allows a protein which is active in dimeric form or higher multimeric form to be dimerized, or multimerized, thereby to induce the activity of the protein.
  • the inducer creates an interface between two or more binding sites, at least one in each copy of the protein, which in effect “multimerizes” the binding sites, e.g. “dimerizes” two binding sites.
  • Such an inducer may thus be a so-called chemical inducer of dimerization (CID).
  • a dimerization domain in the chimeric protein may be capable of homodimerization, that is two identical copies of a dimerization domain may dimerize. Such dimerization domains may be termed homodimerization domains.
  • a dimerization domain may be capable of heterodimerization, that is two different dimerization domains may dimerize.
  • Such dimerization domains may be termed heterodimerization domains.
  • the interaction domain may comprise a single dimerization domain, which may be a homodimerization domain or a heterodimerization domain.
  • the interaction domain may comprise two or more dimerization domains, which may be homodimerization and/or heterodimerization domains.
  • the chimeric protein may be configured such that a dimerization domain from each one of the pair of chimeric proteins binds to an inducer (e.g. CID) molecule. The pair of chimeric proteins in a dimer is thus bound together via binding of their respective dimerization domains to an inducer molecule, as depicted in Figure 1.
  • the interaction domain comprises: (i) a first dimerization domain D1; (ii) a second dimerization domain D2; wherein D1 and D2 may be the same or different; and wherein, when at least two chimeric proteins are expressed in a cell, and in the presence of a dimerization inducer, the D1 and D2 domains of one chimeric protein of the at least two chimeric proteins are induced to dimerize with the D1 and D2 domains of another chimeric protein, causing the signalling domains of the at least two chimeric proteins to dimerize to enable signalling by the chimeric protein; and wherein the chimeric proteins of the at least two chimeric proteins may be the same or different and the D1 and D2 domains of one chimeric protein may each bind with the D1 or D2 domain of another chimeric protein.
  • the configuration of the chimeric protein is such that D1 does not dimerize to any significant extent with D2 within the same chimeric protein in the presence of the inducer.
  • D1 of a first chimeric protein dimerizes with D2 of a second chimeric protein
  • D2 of a first chimeric protein dimerizes with D1 of a second chimeric protein.
  • the signal which may be induced by the chimeric protein, or the signalling protein may be a signal which improves or increases a functional property or activity of the cell.
  • the function or effect of a cell may be increased, which may be a function or effect in vitro or in vivo, that is during generation or expansion of a cell which is being prepared for ACT, or once the cell has been administered to a subject.
  • This may be, for example cell survival, persistence of the cell, persistence of function of the cell, vitality, functional effect (e.g. immunosuppressive or cytotoxic effect), phenotype of the cell, including memory phenotype, proliferation capacity and/or therapeutic efficacy of the cell.
  • the increase may be seen in a cell which comprises the protein relative to a cell which does not comprise the protein.
  • the signal is a pro-survival signal, which helps the cell to survive and to maintain its ability to function during and after culture, and to persist and maintain its functional ability following administration to a subject in the course of therapy. It may alternatively be referred to as a persistence signal.
  • the chimeric protein may be expressed in a cell to impart an inducible pro-survival signalling capacity to the cell. It has particular utility 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 a CAR, or any chimeric receptor. The protein thus has utility in the engineering of cells for ACT.
  • the signal is a STAT-mediated signal (e.g.
  • the tyrosine effector domain comprises at least one STAT association motif.
  • the tyrosine effector domain may alternatively be defined as comprising a STAT association motif.
  • the dimerization domains interact with an inducer of dimerization.
  • the dimerization domains bind to the inducer. In other words, they are, or they comprise, binding sites for the inducer.
  • the inducer may be a ligand, or more particularly a dimerization ligand. It will be seen therefore that in such an embodiment the dimerization domains allow, or mediate, dimerization by binding to the inducer (i.e. by inducer, or ligand, binding). Alternatively, the inducer may induce the dimerization domains to bind to one another by chemical cross-linking. In the situation where the dimerization domains of two interacting chimeric proteins are the same (including e.g. in the embodiment above in which the interaction domain comprises 2 dimerization domains, D1 and D2, and these are the same), the dimerization inducer may comprise two identical interaction sites (e.g.
  • the dimerization inducer may interact with (e.g. bind to) two different binding sites, one in (or provided by) the first dimerization domain (e.g. of a first chimeric protein, or D1) and the other in (or provided by) the second dimerization domain (e.g. of a second chimeric protein, or D2).
  • the dimerization inducer may comprise two different interaction sites (e.g.
  • D1 and D2 together provide a domain for interaction with the inducer, e.g. for binding to the inducer, i.e. an interaction domain. This may in an embodiment be referred to as the binding domain of the chimeric protein.
  • the interaction domain may be in one or in separate, e.g. two, parts.
  • D1 and D2 may be linked together.
  • D1 and D2 may be provided as a unit, or put another way, within a single interaction domain (e.g. a single, inducer-binding domain).
  • a single-unit interaction domain may be linked, or located, at the N- or the C-terminal of the signalling domain.
  • D1 and D2 may be provided as separate sites, or domains in the chimeric protein.
  • D1 and D2 may be provided as separate functional units in the chimeric protein. For example, they may each be linked, or located, at either end of the signalling domain.
  • the interaction domain e.g. an inducer-binding domain
  • a single unit (1-part) interaction domain comprises both D1 and D2. This allows “one-side” dimerization.
  • a 2-part binding domain comprises two separate parts, which are spaced apart in the chimeric protein, the first part comprising one of D1 or D2 and the second part comprising the other of D1 and D2. This allows “two-side dimerization”.
  • various configurations of the chimeric protein are possible, wherein different components of the chimeric protein are linked together in various orders, as described further below.
  • the various components of the chimeric protein may be linked to each other directly or indirectly, as described further below. Where applicable, this includes the individual components of the interaction domain (e.g. D1 and D2), and of the signalling domain.
  • one or more linkers may be present, said linkers being between any two components of the chimeric protein.
  • a third aspect provides a nucleic acid molecule (or alternatively termed, a polynucleotide) comprising a nucleotide sequence which encodes a chimeric protein as defined herein.
  • the nucleic acid molecule may be in the form of a construct, or more particularly, a recombinant construct, comprising the nucleic acid molecule and one or more other nucleotide sequences (a nucleotide sequence of interest).
  • the construct may comprise the nucleic acid molecule and a regulatory sequence, e.g. an expression control sequence, and/or a sequence encoding another functional protein (or more generally, a protein of interest), for example a receptor, e.g. a CAR or TCR etc.
  • the signalling protein comprises different chimeric proteins (i.e. where different chimeric proteins are dimerized)
  • nucleotide sequences encoding the different chimeric proteins may be provided in the same construct.
  • a separate nucleic acid molecule or construct may be provided for each separate, or different, chimeric protein.
  • the construct may comprise one or more co-expression sequences linking the nucleic acid molecule with one or more other coding nucleotide sequences.
  • a fourth aspect provides a vector comprising a nucleic acid molecule or construct as defined herein. Where separate chimeric proteins are encoded in separate molecules or constructs, they may each be contained in a separate vector. There may accordingly be a set of vectors, each comprising a sequence encoding a separate chimeric protein.
  • the vector may be a viral or non-viral vector.
  • the vector may comprise a nucleic acid molecule as defined herein and a further nucleotide sequence encoding a protein of interest, notably a receptor, e.g., a CAR or TCR.
  • a fifth aspect provides a cell which expresses a chimeric protein as defined herein, or which comprises a nucleic acid molecule, construct or vector as defined herein. The cell expresses the chimeric protein intracellularly, or more particularly in the cytoplasm, or cytosol, of the cell. Thus, the chimeric protein may be said to be expressed in a cell.
  • a cell population comprising a cell as defined herein.
  • the cell is an immune cell, or a precursor thereof.
  • the cell may be a stem cell, or more particularly a haemopoietic stem cell (HSC) or pluripotent stem cell (PSC), e.g. an induced pluripotent stem cell (iPSC).
  • the cell may be a lymphocyte, or more particularly a T-cell, NK cell, dendritic cell or myeloid-derived suppressor cell (MDSC).
  • the T-cell may be a Treg cell.
  • the cell may be a primary cell or from a cell line.
  • the cell may be a production host cell, that is a cell into which the nucleic acid molecule construct or vector is introduced in order to produce the chimeric protein, or a viral vector encoding the chimeric protein.
  • a sixth aspect provides a method of preparing a cell as defined herein (i.e. a cell according to the fifth aspect), said method comprising introducing into a cell (e.g. transducing or transfecting a cell with), a nucleic acid molecule, construct or vector as defined herein.
  • the method may include allowing the chimeric protein to be expressed in the cell. This may include, for example, culturing the cell.
  • Such a method may further comprise a preceding step of isolating, enriching, providing or generating a cell to be used in the method.
  • a cell may be isolated or enriched or generated after the step of introducing the nucleic acid molecule.
  • the nucleic acid molecule may be introduced into a precursor or progenitor cell, e.g. a stem cell, and the cell may then be induced or caused to differentiate, or change, into a desired cell type.
  • a precursor or progenitor cell e.g. a stem cell
  • an iPSC cell may be differentiated into an immune effector cell (e.g. a Treg or other T cell) or a Tcon cell may be converted into a Treg cell, etc.
  • This aspect may also include a method of preparing a chimeric protein as defined herein, said method comprising introducing into a cell, a nucleic acid molecule, construct or vector as defined herein, allowing the chimeric protein to be expressed by the cell, and optionally detecting and/or collecting the chimeric protein.
  • a seventh aspect provides a method of promoting the survival or persistence of a cell, said method comprising introducing into the cell, a nucleic acid molecule, construct or vector as defined herein.
  • This may include the step of allowing the chimeric protein to dimerize to a second chimeric protein, optionally wherein said second chimeric protein is a separate or different chimeric protein.
  • the step of allowing the chimeric protein to dimerize may include exposing the cell to (or contacting the cell with) an inducer.
  • the inducer induces dimerization, and signalling by the chimeric protein, or a signalling protein containing it, and provides a pro- survival signal to the cell.
  • This aspect may comprise administering a cell as defined herein to a subject, and administering a dimerization inducer to the subject.
  • the inducer may be administered before, during or after administration of the cell.
  • the method may be carried out in vivo.
  • the method of the seventh aspect may be carried out in vitro/ex vivo.
  • the chimeric protein may advantageously be expressed in a cell in the context of therapy.
  • an eighth aspect provides a method of preparing a cell for use in adoptive cell transfer therapy (ACT), said method comprising providing said cell with a chimeric protein as defined herein.
  • this method may comprise introducing into said cell a nucleic acid molecule, construct or vector as defined herein.
  • the method may also comprise introducing into the cell a separate nucleic acid molecule, construct or vector, for example which comprises a nucleotide sequence which encodes a separate (e.g. second) chimeric protein, or a therapeutic protein, notably a receptor, e.g. a CAR or TCR.
  • a ninth aspect provides a pharmaceutical composition comprising a cell, cell population or a vector as defined herein, together with at least one pharmaceutically acceptable carrier or excipient.
  • the cell or the vector comprises an additional nucleotide sequence encoding a further protein, notably a second chimeric protein, or a receptor, e.g.
  • the cell comprises a separate nucleic acid molecule, construct or vector which comprises a nucleotide sequence which encodes a further protein, notably a second chimeric protein, or a receptor, e.g. a CAR or TCR.
  • a tenth aspect provides a cell or cell population as defined herein, or a pharmaceutical composition as defined herein, or a vector as defined herein for use in therapy.
  • the cell, cell population or a pharmaceutical composition comprising the cell or cell population may be for ACT.
  • the vector or pharmaceutical composition comprising the vector may be for gene therapy.
  • the ACT or gene therapy may be for the treatment or prevention of any condition which is responsive to ACT or gene therapy, in particular immunotherapy by ACT or gene therapy.
  • An eleventh aspect provides a cell, cell population, vector or pharmaceutical composition as defined herein for use in the treatment of or prevention of cancer, or an infectious, neurodegenerative, inflammatory, autoimmune or allergic disease or any condition associated with an unwanted or deleterious immune response.
  • the cell may be used for inducing immunosuppression (i.e. for suppressing an unwanted or deleterious immune response), for example to improve and/or prevent immune- mediated organ damage in inflammatory disorders, autoimmune or allergic diseases or conditions, and in transplantation.
  • This aspect also provides a method of adoptive cell transfer therapy, said method comprising administering to a subject in need of said therapy a cell, cell population, or pharmaceutical composition as defined herein, particularly an effective amount of said cell, cell population or pharmaceutical composition.
  • a method of treating or preventing cancer, or an infectious, neurodegenerative, inflammatory, autoimmune or allergic disease or a condition associated with an unwanted or deleterious immune response said method comprising administering to a subject in need thereof a cell, cell population, vector or pharmaceutical composition as defined herein, particularly an effective amount of said cell, cell population, vector or pharmaceutical composition.
  • a cell, cell population or vector as defined herein in the manufacture of a medicament for use in treating or preventing cancer, or an infectious, neurodegenerative, inflammatory, autoimmune or allergic disease or a condition associated with an unwanted or deleterious immune response.
  • the use may be in induction of tolerance to a transplant; treating and/or preventing cellular and/or humoral transplant rejection; treating and/or preventing graft-versus-host disease (GvHD), an autoimmune or allergic disease; to promote tissue repair and/or tissue regeneration; or to ameliorate inflammation.
  • the cell may be a Treg cell.
  • a twelfth aspect provides a combination product comprising (a) a cell, cell population, vector or pharmaceutical composition as defined herein, and (b) an inducer (e.g. a CID), for use in therapy, particularly ACT or gene therapy.
  • the therapy may be any therapy as defined above, and further described herein.
  • the components (a) and (b) of the combination product may be for separate, sequential or simultaneous use.
  • Figure 1 depict formats of the chimeric protein which comprise a single dimerization domain, showing in (A) one-sided heterodimerization and (B) one-sided homodimerization.
  • the signalling domain comprises, N-terminal to C-terminal, JAK1- STAT5 motifs.
  • the interaction domain comprising a single dimerization domain, depicted as FKBP or FRB, which each bind to the inducer, depicted as rapamycin, is linked to the N-terminus of the signalling domain.
  • the dimerization domain in each chimeric protein is the same.
  • Figure 2 depicts various formats of the chimeric protein which comprises two dimerization domains, D1 and D2, in various formats.
  • the signalling domain comprises, N-terminal to C-terminal, JAK1-STAT5 motifs (double construct) or JAK1- STAT5-JAK3 motifs (triple construct; JAK3 is reverse orientation), wherein: in (A), the chimeric protein comprises a 1-part interaction (inducer-binding) domain linked to the N-terminus of the signalling domain (double construct/one-sided dimerization); in (B), the chimeric protein comprises a 2 part interaction (inducer-binding) domain wherein D1 is linked to the N-terminal of the signalling domain and D2 is linked to the C-terminal of the signalling domain (double construct/two-sided dimerization); in (C), the chimeric protein comprises a 2 part interaction (inducer-binding) domain wherein D1 is linked to the N-terminal of the signalling domain and D2 is linked to the C-terminal of the signalling domain (triple construct/two-sided dimerization).
  • FIG. 3 shows Rapamycin and rapalogs.
  • Figure 4 shows the structures of inducer molecules (A) cyclosporine (CsA); (B) FK506; (C) FKCsA; (D) Fusicoccin and (E) HaSX8.
  • FIG. 5 shows schematic diagrams of representative cytoplasmic constructs, pQTX-0340 and pQTX-0370. Both constructs contain the SFFV (Spleen focus forming virus) promoter followed by the rapamycin binding domains FRB (FKBP Rapamycin Binding) which is linked to the other rapamycin binding domain FKBP by a 5 amino acid linker. This is then linked by 12 amino acids to the truncated IL2RB signaling domain containing the Y510 amino acid. In pQTX-340 this is followed by the P2A self-cleaving peptide and the GFP reporter gene.
  • SFFV Single focus forming virus
  • FRB FKBP Rapamycin Binding
  • FIG. 6 shows a schematic diagram of the pSTAT5 assay using Treg cells.
  • Figure 7 shows that Human Tregs transduced with pQTX-340 demonstrate an increased pSTAT5 expression level upon Rapamycin treatment.
  • pQTX-340 transduced human Tregs (MOI 5) were rested for 24 hours prior to the assays, and cells were then treated with 0nM, 1nM, 10nM, 100nM, 1000nM or 2740nM of rapamycin for 48, 24 or 4 hours prior to staining.
  • FACS plots show GFP + cell populations and the pSTAT5 expression levels after 48 hour treatment of rapamycin with vehicle alone or with 1000nM.
  • the bar graph shows the mean fluorescence intensity (MFI) of pSTAT5 expression levels with the various indicated rapamycin concentrations treatments.
  • the line graph shows the percentage of pSTAT5 expression levels for pQTX-340 transduced Tregs within the GFP + or GFP- populations.
  • the bar graph shows the percentage of pSTAT5 expression levels of pQTX-340 transduced Tregs within the GFP + or GFP- populations or Mock Tregs. Data normalized to vehicle.
  • Figure 8 shows a schematic diagram of the survival assay.
  • Figure 9 shows that human Tregs transduced with pQTX-340 demonstrate an increased survival ability upon Rapamycin treatment.
  • pQTX-340 transduced human Tregs were co-cultured with CD3/28 beads or media alone for 10 days.1000nM rapamycin or vehicle alone treatments were added everyday to the co-cultures.
  • cells were stained with CD4, CD25, Annexin V and 7AAD to assess cell viability.
  • the FACS plots displayed show the lymphocytes gate, then within this population the single cells gate followed by cells which were negative for both Annexin V and 7AAD, thus the viable cells gate.
  • the viable cells’ population the CD4 + CD25 + cells where selected and then within this the GFP expression was assessed.
  • FIG. 12 shows that human Tregs transduced with pQTX-370 (pQTX-340 with a CAR) demonstrate an increased survival ability upon Rapamycin treatment.
  • a schematic diagram of pQTX-370 which is similar to pQTX-340 but with the addition of a CAR is shown.
  • pQTX-370 transduced human Tregs were co-cultured with CD3/28 beads for 6 days.1000nM rapamycin or vehicle alone treatments were added every day to the co-cultures. At day 6, cells were stained with CD4, CD25, Annexin V and 7AAD to assess cell viability.
  • the FACS plots displayed show the lymphocytes gate, then within this population the single cells gate followed by cells which were negative for both Annexin V and 7AAD, thus the viable cells gate.
  • the viable cells’ population the CD4 + CD25 + cells where selected and then within this the GFP expression was assessed.
  • the gates for GFP + and GFP- cells were set, and the ‘frequency of grandparent’ were reported.
  • Graph shows the percentage of live cells within the GFP + and GFP- populations.
  • Figure 13 shows cell counts under different treatment conditions (IL2, DMSO and different concentrations of Rapamycin) for cells transduced with pQTX-0340, in the presence of beads or media.
  • Tregs transduced with pQTX-0340 were seeded in a 96 well plate for a survival assay.
  • Cells were treated every day with varying concentrations of Rapamycin.
  • At Day 10 of the survival assay cells were stained and acquired by flow cytometry and cell counts were taken.
  • the live GFP positive population of cells were counted and plotted, below the bar chart are the conditions for each bar displayed.
  • the first chart displayed the data from the cells cultured with CD3/28 beads and the second chart shows the cells cultured with no beads and media alone.
  • Figure 14 shows schematic diagrams of additional cytoplasmic full-length constructs, pQTX-570, pQTX-571, pQTX-572, pQTX-573 and pQTX-574.
  • pQTX-570 FRB (FKBP Rapamycin Binding), 5aa linker, Cytoplasmic domain (beta), P2A self- cleaving peptide and the GFP reporter gene.
  • pQTX-571 FKBP, 5aa linker, Cytoplasmic domain (beta), P2A self-cleaving peptide and the GFP reporter gene.
  • pQTX-572 FKBP, 5aa linker, Cytoplasmic domain (gamma), P2A self-cleaving peptide and the mCherry reporter gene.
  • pQTX-573 FRB (FKBP Rapamycin Binding), 5aa linker, FKBP, 17aa linker, Cytoplasmic domain (beta), P2A self-cleaving peptide and the GFP reporter gene.
  • pQTX-574 FKBP, 5aa linker, FRB (FKBP Rapamycin Binding), 17aa linker, Cytoplasmic domain (gamma), P2A self-cleaving peptide and the mCherry reporter gene.
  • Figure 15 shows schematic diagrams of additional cytoplasmic full-length constructs pQTX-0583-0587, which additionally contain a furin sequence.
  • pQTX- 0583 FRB 100aa (SEQ ID NO.6), linker 5aa, IL2RB cytoplasmic domain 286aa (SEQ ID NO.9), furin 9aa, P2A 19aa and GFP 238 aa; pQTX-0584 FKBP 110aa (SEQ ID NO.8), linker 5aa, IL2RB cytoplasmic domain 286aa (SEQ ID NO.9), furin 9aa, P2A 19aa and GFP 238aa; pQTX-0585 FKBP 110aa (SEQ ID NO.8), linker 5aa, IL2RG cytoplasmic domain 86aa, furin 9aa, P2A 19aa, mCherry 236aa; pQTX- 0586 FRB 100aa (SEQ ID NO.6), linker 5aa, FKBP 109aa (SEQ ID NO.107), linker 17aa,
  • the subject of the products, methods and uses herein is a chimeric protein which can be used to promote the functionality or survival, or indeed any property, of a cell by which it is expressed.
  • the protein thus has utility in adoptive cell transfer, to assist in the preparation of cells for ACT, and/or to help keep the cells alive and functional following transfer to a subject. Therapeutic efficacy of the cell may be improved.
  • the chimeric protein is based on a dimerization system, in which a pair of cognate dimerizable domains are comprised within the protein, and when brought together in proximity, or in the presence of an inducer, a so-called dimerization inducer, the cognate dimerization domains of two protein molecules are brought together to form a dimer.
  • the “dimerization domain” may be any amino acid sequence that can dimerize, or even trimerize or multimerize. This brings together the signalling domains of two chimeric proteins, allowing them to dimerize and become functional, or in other words for signalling to be activated.
  • a feature of the present chimeric protein is that the interaction domain and the signalling domain are present in the same protein chain.
  • the chimeric protein monomer is a single chain protein.
  • dimerization of the chimeric protein provided herein activates a signalling pathway mediated by JAK kinase activity, including notably JAK1 activity, and especially the JAK1-STAT signalling pathway.
  • the chimeric protein may mimic the signalling which is induced by activation of a natural cytokine receptor, for example an interleukin (e.g. IL-2) receptor.
  • a natural cytokine receptor for example an interleukin (e.g. IL-2) receptor.
  • the signalling cascade activated by the chimeric protein of the present disclosure is similar to the signalling cascade activated by a natural cytokine receptor, while the magnitude of activation induced by the chimeric proteins of the present disclosure could be different from that of a natural cytokine receptor.
  • a “dimerization domain” as provided herein may be capable of supporting multimerization (e.g. in the event that multiple copies of the dimerization domain are provided in the same molecule); however, such domains may also still be considered “dimerization domains” as used herein, based on the ability of such domains to dimerize.
  • signalling can be effectively induced upon dimerization of chimeric proteins (i.e.
  • trimerization or other multimerization is not typically required, but it is not excluded).
  • two or more chimeric proteins dimerize to form a functional signalling protein. This includes that two chimeric proteins, which may be the same or different, form a dimer. In other embodiments, more than two chimeric proteins may associate together to form a higher order multimer, for example, where a chimeric protein contains two or more dimerization domains.
  • the signalling protein may accordingly be a simple dimer of two molecules of the same chimeric protein, or it may be a dimer between a chimeric protein as provided herein and another chimeric protein, which may be a different chimeric protein, and may for example confer another property on the protein, or on the cell, or may augment the functionality of the cell which expresses the signalling protein (e.g. by introducing a JAK3-binding motif, as mentioned above and described further below).
  • a functional signalling protein capable of signalling in a cell, or an activated signalling protein is thus a multi-component protein, in that it comprises at least two chimeric proteins.
  • Multi in this regard means two or more, e.g.3, 4 or more, for example, 2-6, 2-5, 2-4, 2-3 etc.
  • the chimeric proteins, and hence the signalling protein which is formed therefrom, are cytoplasmic proteins. When expressed, they are present in the cytosol or the cytoplasm of the cell, or in the soluble compartment of the cell.
  • the chimeric proteins are “non-membrane associated”. This means that they are not associated with the cell surface membrane of the cell in any way, and are not expressed on or in the cell surface membrane.
  • the chimeric proteins do not comprise a transmembrane domain or a portion thereof, or a membrane-targeting motif, e.g.
  • a myristoyl group or any moiety which is capable of associating with, e.g. binding to, or inserting into the cell membrane, or in any way associating with any component of a cell membrane.
  • the chimeric proteins therefore do not comprise an exodomain (i.e. a domain which is present on the outside of the cell).
  • the dimerization between dimerization domains in two chimeric proteins may be by any means, by which the respective domains are associated together. In the case of interaction domains which contain more than one dimerization domain, e.g. D1 and D2, this includes any means or method by which D1 and D2 in different proteins are associated together.
  • the two interacting domains each bind to a dimerization inducer.
  • the inducer thus creates an interface between two dimerization domains and brings them together as a dimer.
  • the inducer is a molecule which allows chemical cross-linking between two dimerization domains.
  • the inducer may permit the dimerization domains to each be covalently bonded to the inducer molecule.
  • the dimerization between two dimerization domains is induced, and the chimeric protein, or signalling protein of which it is a component, is an inducible chimeric protein, or inducible signalling protein.
  • the dimerization between two dimerization domains is constitutive, and the chimeric protein, or signalling protein of which it is a component is a constitutive signalling protein.
  • the dimerization may occur spontaneously when the two dimerization domains are brought together.
  • Chemically-induced dimerization systems are known in the art, using various inducer molecules, and different protein domains for dimerization, are described further below.
  • Direct systems of dimerization based on moieties which are able to bind to or interact with each other when in contact or proximity, for example based on affinity binding, or other forms of interaction or association (e.g. amphipathic interaction such as occurs with leucine zippers), are also known in the art, and described further below.
  • the interaction domain of the chimeric protein which comprises the dimerization domain(s), which as noted above may be in 1- or 2-part form, thus allows two chimeric protein molecules to interact with an inducer molecule.
  • a dimerization domain in one chimeric protein molecule binds to an inducer molecule which is also bound by a dimerization domain from another chimeric protein molecule, and in this way the dimerization domains in the two chimeric protein molecules become dimerized, and therefore the chimeric protein is also dimerized.
  • Figure 1 depicts hetero- and homo-dimerization between two chimeric proteins each comprising a single dimerization domain, which dimerize together in the presence of an inducer. Since there is only one dimerization domain in each chimeric protein, there is only dimerization on one side of the proteins (so-called “one-sided” dimerization).
  • each protein pair will bind to two molecules of the inducer.
  • D1 and D2 may heterodimerize, that is D1 and D2 may be different.
  • D1 and D2 may be referred to as heterodimerization domains Ht1 and Ht2.
  • Each inducer molecule is in this case bound by Ht1 from one protein and Ht2 from the other.
  • Various configurations of chimeric proteins comprising two dimerization domains are depicted in Figure 2.
  • the configuration of the chimeric protein is such that D1, and in particular Ht1, does not significantly dimerize with D2, in particular Ht2, within the same chimeric protein molecule.
  • D1/Ht1 from one chimeric protein dimerizes with D2/Ht2 from the other chimeric protein, causing dimerization of the two signaling domains.
  • the presence of the inducer causes a greater proportion of dimerization between two chimeric proteins, than dimerization within the same chimeric protein.
  • the amount of chimeric proteins which are dimerized within the same molecule in a cell or cell population, or in solution may be less than 50%, 40%, 30%, 20%, 10%, 5% or 1 % of the amount of chimeric proteins which are dimerized with a separate chimeric protein molecule, in the presence of the inducer.
  • 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 dimerization between proteins of interest that are fused to inducer-binding domains. Such systems have been described for use in cell biology for different applications, to bring proteins into proximity, for example to investigate signalling pathways and other biological mechanisms, in medicine to degrade or inactivate pathogenic proteins, and in gene and cell therapy.
  • a typical chemical inducer of dimerization (CID), or dimerization inducer to use the terminology herein, has the feature of being able to interact with, or bind to, two proteins or protein domains, one on either side of the molecule. It thus has two binding sites, or binding surfaces (or more generally, interaction sites).
  • the inducer is capable of interacting with, or binding to, two different proteins or dimerization domains.
  • the inducer is capable of interacting with, or binding to, two copies, or molecules of the same dimerization domain (the same dimerization may be contained within two different chimeric proteins, for example where the signalling domains differ, as indicated above).
  • FK506 and rapamycin which are capable of binding to, and therefore inducing heterodimerization of, various different proteins or protein domains, including FK506-binding protein (FKBP), the FKBP-rapamycin domain of mTOR (FRB), calcineurin, and cyclophilin, which can be used in different combinations to achieve heterodimerization domain pairs and CID combinations, as detailed below.
  • FKBP FK506-binding protein
  • calcineurin FK506-binding protein
  • cyclophilin cyclophilin
  • homodimerization systems based on FK506 derivatives which are able to bind two FKBP molecules have been developed, i.e. based on symmetric or dimeric inducers, which comprise two binding sites for the same dimerization domain, as discussed further below.
  • the inducer is rapamycin or an analogue thereof, and the dimerization domains are protein domains which bind thereto. Rapamycin and rapamycin analogues induce heterodimerization by generating an interface between the FRB domain of mTOR and a FK506-bindng protein (FKBP). This association results in FKBP blocking access to the mTOR active site inhibiting its function.
  • rapamycin While mTOR is a very large protein, the precise small segment of mTOR required for interaction with Rapamycin is known and can be used.
  • the macrolides rapamycin and FK506 act by inducing the heterodimerization of cellular proteins. Each drug binds with a high affinity to the FKBP12 protein, creating a drug-protein complex that subsequently binds and inactivates mTOR/FRAP and calcineurin, respectively.
  • FKBP-rapamycin binding (FRB) domain of mTOR has been defined and applied as an isolated 89 amino acid protein moiety that can be fused to a protein of interest. Rapamycin can then induce the approximation of FRB fusions to FKBP12 or proteins fused with FKBP12.
  • one of the dimerization domains may be or comprise FRB, and the other dimerization domain (including the other of Ht2 or Ht1) may be or comprise FKBP.
  • 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.
  • FKBP includes FKBP12. Rapamycin has several properties of an ideal dimerizer: it has a high affinity (KD ⁇ 1 nM) for FRB when bound to FKBP, and is highly specific for the FRB domain of mTOR.
  • Rapamycin is an effective therapeutic immunosuppressant with a favourable pharmacokinetic and pharmacodynamics profile in mammals.
  • Pharmacological analogues of Rapamycin with different pharmacokinetic and dynamic properties such as Everolimus, Temsirolimus and Deforolimus (Benjamin et al, Nature Reviews, Drug Discovery, 2011) may also be used according to the clinical setting.
  • the surface of rapamycin which contacts FRB may be modified. Compensatory mutation of the FRB domain to form a surface that accommodates the "bumped" rapamycin restores dimerizing interactions only with the FRB mutant and not to the endogenous mTOR protein. Bayle et al.
  • rapamycin analogues or "rapalogs” and their corresponding modified FRB binding domains.
  • Bayle et al. (2006) describes the rapalogs: C-20-methyllyrlrapamycin (MaRap), C16(S)-Butylsulfonamidorapamycin (C16-BS-Rap) and C16-(S)-7- methylindolerapamycin (AP21976/C16-AiRap), as shown in Figure 3, in combination with the respective complementary binding domains for each.
  • Other rapamycins/rapalogs include sirolimus and tacrolimus (FK506).
  • a dimerization domain in one chimeric protein comprises FKBP and the cognate dimerization domain in another chimeric protein comprises FRB.
  • one of Ht1 and Ht2 comprises FKBP and the other comprises FRB.
  • the FKBP domain may comprise FKBP12.
  • Heterodimerization between the FRB domain of one chimeric protein molecule and the FKBP domain of another chimeric protein molecule causes dimerization of the signalling domains. Rapamycin is a standard pharmaceutical with well understood properties, excellent bioavailability and volume of distribution and which is widely available.
  • Rapamycin also does not aggravate the condition being treated, in fact, as it is an immunosuppressant it is likely to have a beneficial effect on unwanted toxicity. Furthermore, in cases where the cells expressing the protein are being used to achieve immunosuppression, notably with Treg cells expressing a CAR or TCR, the immunosuppressive properties of rapamycin and its analogues may be beneficial.
  • sequences for FRB and FKBP domains are known in the art.
  • FKBP/FRB may have or may comprise a sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 5, or a variant thereof.
  • SEQ ID NO: 1 is the native (wild-type) human FKBP12 domain
  • SEQ ID NO: 2 is the wild-type FRB segment of mTOR
  • SEQ ID NO: 3 is FRB with T to L substitution at 2098 which allows binding to AP21967
  • SEQ ID NO: 4 is a FRB segment of mTOR with T to H substitution at 2098 and W to F substitution at residue 2101 of the full mTOR which binds Rapamycin with reduced affinity to wild type
  • SEQ ID NO: 5 is a FRB segment of mTOR with K to P substitution at residue 2095 of the full mTOR which binds Rapamycin with reduced affinity.
  • Additional FRB and FKBP domain sequences include the following SEQ ID NOs: and variants thereof: SEQ ID NO: 6 represents a FRB sequence as shown as part of the protein of SEQ ID NO.1 of WO 2016/135470; SEQ ID NO: 7 represents a FKBP sequence which is contained in the protein of SEQ ID NO.4 of WO 2016/135470; SEQ ID NO: 8 is a so-called “codon-wobbled” FKBP sequence, in which the codons encoding the FKBP domain have been altered to prevent recombination; SEQ ID NO: 88 represents a FKBP sequence as shown as part of the protein of SEQ ID NO.1 of WO 2016/135470; SEQ ID NO: 89 represents a FKBP sequence as shown as part of the protein of SEQ ID NO.2 of WO 2016/135470.
  • SEQ ID NO: 107 represents a 109 amino acid FKBP sequence.
  • SEQ ID NOs: 112 and 113 represent 99 and 94 amino acid FRB sequences respectively.
  • Variant sequences may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 1 to 8, 88, 89, 107, 112 or 113 provided that the sequences provide an effective dimerization system. That is, provided that the sequences facilitate sufficient co-localization of the two chimeric proteins to allow dimerization of the two signalling domains.
  • FRB and/or FKBP variants may retain the ability to bind to rapamycin or an analogue thereof, e.g.
  • the "wild-type" FRB domain shown as SEQ ID NO: 2 comprises amino acids 2025-2114 of human mTOR.
  • the FRB sequence of the chimeric protein may comprise an amino acid substitution at one of more of the following positions: 2095, 2098 and 2101.
  • the variant FRB used in the chimeric protein of the invention may comprise one of the following amino acids at positions 2095, 2098 and 2101: 2095: K, P, T or A; 2098: T, L, H or F; 2101: W or F.
  • Bayle et al (as above) describe the following FRB variants, annotated according to the amino acids at positions 2095, 2098 and 2101 (see Table 1 of Bayle): KTW, PLF, KLW, PLW, TLW, ALW, PTF, ATF, TTF, KLF, PLF, TLF, ALF, KTF, KHF, KFF, KLF.
  • These variants are capable of binding rapamycin and rapalogs to varying extents, as shown in Table 1 and Figure 5A of Bayle et al.
  • the chimeric protein herein may comprise any one of these FRB variants.
  • FK506 is an inhibitor of the protein calcineurin.
  • calcineurin also binds to FK506 (when FK506 is bound by FKBP) and may also be used to provide, or is the basis for, a heterodimerization domain which binds to FK506 as inducer, along with FKBP as the cognate heterodimerization domain.
  • the inducer is FK506, and the cognate dimerization domains of two chimeric proteins, or Ht1 and Ht2 in a single chimeric protein, are protein domains which bind thereto.
  • one of the dimerization domains may be or comprise calcineurin or a FK506- binding fragment thereof
  • the dimerization domain may be or comprise FKBP (which may be as defined above, and includes for example FKBP12).
  • FKBP which may be as defined above, and includes for example FKBP12.
  • the term “calcineurin” includes variants and derivatives thereof. Such variants may include amino acid sequences having one or more amino acid modifications (e.g.
  • calcineurin retains the ability of the parent molecule to bind to FK506.
  • Calcineurin A is the 59 kDa catalytic subunit of the calcineurin protein and the dimerization domain may be or may comprise calcineurin A or a FK506-binding fragment thereof.
  • sequence of an isoform of human calcineurin A (UniprotKB –Q08209) is shown in SEQ ID NO.90.
  • the inducer may be the calcineurin inhibitor cyclosporine, also known as cyclosporine A (CsA), and the cognate dimerization domains of two chimeric proteins, or Ht1 and Ht2 in a single chimeric protein, may be protein domains which bind thereto.
  • cyclophilins are a family of proteins characterised by their ability to bind to CsA. Calcineurin activity is inhibited by CsA when bound to CyP (in other words, calcineurin binds to CsA when it is bound to CyP).
  • CyPs cyclophilins
  • CyPs are a family of proteins characterised by their ability to bind to CsA. Calcineurin activity is inhibited by CsA when bound to CyP (in other words, calcineurin binds to CsA when it is bound to CyP).
  • cyclophilins may be used to provide a heterodimerization domain capable of binding to CsA as inducer.
  • one of the dimerization domains may be or comprise calcineurin or a CsA-binding fragment thereof (more particularly a CsA-CyP binding fragment), and the other dimerization domain (including Ht2 or Ht1) may be or comprise cyclophilin or a CsA-binding fragment thereof.
  • the calcineurin may be a variant or derivative of a native, or wild-type calcineurin molecule. In this context a variant or derivative of calcineurin retains the ability of the parent molecule to bind to CsA.
  • the dimerization domain may be or may comprise calcineurin A or a CsA- (or CsA-CyP)- binding fragment thereof.
  • cyclophylin CyP
  • CyP CyP
  • Variants of CyP include amino acid sequences having one or more amino acid modifications (e.g. substitutions, additions and/or deletions) relative to the native sequence.
  • a variant or derivative of cyclophilin retains the ability of the parent molecule to bind to CsA.
  • a cyclophilin heterodimerization domain may be provided as a fusion protein with Fas (CyP-Fas).
  • Fas CyP-Fas
  • a representative sequence of cyclophilin, isoform 1 (UniprotKB P62937-1) is shown in SEQ ID NO.91.
  • a “heterodimeric inducer” molecule has been developed which combines a protein-binding interface from FK506 with a protein-binding interface from CsA, to form the molecule FKCsA (Belshaw, et al., 1996, PNAS, 93, 4604-4607).
  • the dimerization inducer is FKCsA
  • the cognate dimerization domains of two chimeric proteins, or Ht1 and Ht2 in a single chimeric protein are protein domains which bind thereto.
  • one of the dimerization domains may be or comprise cyclophilin or a CsA-binding fragment thereof
  • the other dimerization domain may be or comprise FKBP (Cyclophilin and FKBP and their respective fragments and variants may be as defined above, and include FKBP12 and CyP-Fas).
  • CID dimerization systems which may be used include those based on the self-labelling proteins SNAP-tag, a mutant of the DNA Repair protein O6- alkylguanine-DNA-alkyltransferase (available from New England Biolabs; and see Gautier et al., 2008, Chem. Biol.15, 128-136), and HALO-Tag, a haloalkane dehalogenase derivative designed to covalently bind to synthetic ligands (available from Promega; and see Los, et al., 2008. ACS Chem. Biol.3, 373-382).
  • the inducer is a HaXS molecule, e.g. HaXS8, and the cognate dimerization domains of two chimeric proteins, or Ht1 and Ht2 in a single chimeric protein, are protein domains which bind thereto.
  • one of the dimerization domains may be or comprise a SNAP-tag and the other dimerization domain (including Ht2 or Ht1) may be or comprise a HALO-tag.
  • a still further heterodimerization system which may be used is based on fusicoccin (FC) as CID, which binds simultaneously to a 14-3-3 protein and the C- terminal 52 amino acids (CT52) of a plant plasma membrane H+-ATPase (PMA) to stabilize the interaction between these two proteins.
  • the inducer is fusicoccin, and the cognate dimerization domains of two chimeric proteins, or Ht1 and Ht2 in a single chimeric protein, are protein domains which bind thereto.
  • one of the dimerization domains may be or comprise a 14-3-3 protein or a FC-binding fragment thereof
  • the other dimerization domain may be or comprise a C-terminal peptide of PMA, e.g. a CT52 peptide.
  • the 14-3-3 protein may for example be tobacco 14-3-3 (T14-3-3c) or a truncation thereof (T14-3-3c ⁇ C, residues 1-242).
  • the C-terminal peptide of PMA may be the CT52 fragment of tobacco H+ ATPase PMA2.
  • the terms “14-3-3 protein”, “C-terminal peptide of PMA” and “CT52” include variants and derivatives 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. In this context a variant or derivative retains the ability of the parent molecule to bind to fusicoccin.
  • CT52 of tobacco H+ ATPase PMA2 comprises three amino acid substitutions S939A, T955D and V956I.
  • the tobacco 14-3-3 and CT52 interaction partners been described in the literature (Ottman et al., 2007, Mol.
  • such a variant retains the desired or required property of the parent molecule from which it is derived, i.e. the reference sequence.
  • the variant sequence may have the stated % sequence identity provided that the variant sequence provides an effective dimerization system. That is, provided that the sequences facilitate sufficient co-localization of the two chimeric proteins to allow dimerization of the two signalling domains.
  • the variants may retain the ability to bind to their respective inducer, e.g. have at least 30, 40, 50, 60, 70, 80 or 90% of the binding affinity of the parent molecule to calcineurin, cyclophilin or fusicoccin.
  • Inducer-based homodimerization systems are also available, most notably based on FK1012, a homodimer of FK506, capable of binding to two molecules of FKBP.
  • Various derivatives of such dimeric inducer molecules have been developed, including various dimeric FK506-like analogues, including for example the molecules AP1903 (CAS 195514-63-7) and AP20187 (CAS 195514-80-8) which are commercially available from various sources.
  • Other multimeric inducer molecules capable of binding to FKBP have been developed.
  • the dimerization domain in a chimeric protein may be or may comprise FKBP, and homodimerization between two chimeric proteins each comprising a FKBP domain may be achieved using a dimeric inducer such as FK1012, AP1903 and AP20187.
  • AP1903 and AQP21087 may bind with high affinity to a FKBP12 variant sequence comprising the amino acid substitution F36V, as shown in SEQ ID NO: 35, which represents an exemplary homodimerization domain sequence for use herein.
  • FKBP12 F35V binds to AP1903 with higher affinity than wild-type FKBP12 binding to AP1903.
  • a dimerization domain may contain an amino acid sequence of an FKBP comprising a modification selected from the group consisting of: (i) a FKBP polypeptide containing one or more amino acid substitutions, (ii) two or three tandem repeats of an unmodified (naturally occurring) FKBP amino acid sequence, and (iii) two or three tandem repeats of a FKBP polypeptide containing one or more amino acid substitutions in some embodiments, the FKBP protein is a human FKBP protein (GenBank: CAG46965.1) and the modifications to FKBP described herein are made to the human FKBP protein in some embodiments, the one or more amino acid substitutions in FKBP include one or more of: F36V, L106P, E31G, R71G, and K105E, residues in reference to the human FKBP protein (GenBank: CAG46965.1).
  • each repeat may comprise a different mutation of that sequence.
  • the dimerization domain comprises three tandem repeats of an FKBP sequence, where one of the repeats comprises a natural FKBP sequence, a second repeat comprises FKBP containing F36V substitution, and a third repeat comprises FKBP containing F36V and L106P substitutions, in any order.
  • a dimerization domain may contain an amino acid sequence of an FKBP comprising a modification selected from the group consisting of: (i) a FKBP polypeptide containing F38V substitution, (ii) a FKBP polypeptide containing F36V and L106P substitutions, (iii) a FKBP polypeptide containing E31G, F38V, R71G, and K105E substitutions, and (iv) two or three tandem repeats of any of these FKBP polypeptides.
  • Representative dimerization domains may comprise a FKBP dimerization domain sequence selected from SEQ ID NOs: 36-40.
  • dimerization domains based on the amino-terminal subdomain of the B subunit of bacterial DNA gyrase (GyrB), which may be induced to homodimerize by the inducer courmermycin (Mohi et al., 1998 Mol. Biol. Cell 9, 3299-3308).
  • Homodimerizable dimerization domains may also be obtained from the extracellular binding regions of various receptors, such as cytokine receptors or TNFR-family receptors, or from binding proteins for various molecules. This may include for example steroid binding protein, estrogen binding protein, glucocorticoid binding protein, or tetracycline binding protein.
  • dimerization domains may be induced to dimerize in the presence of the ligand (e.g. cytokine etc.) or the binding target for the receptor/binding protein in question. Since the administration of a cytokine ligand to induce dimerization may have other effects, this may in some circumstances be less preferred. However, dimerization domains based on TNFR- family receptors such as TNFR2 or OX40, for example, may be used. Various such homodimerization systems which may be used are described in WO2019/169290 of Allogene Therapeutics Inc., the disclosure of which is incorporated herein by reference.
  • the sequence of a dimerization domain based on OX40 (1-214) is given in SEQ ID NO: 73.
  • Other homodimerization domains described in WO2019/169290 which may be used include those based on or derived from the extracellular domains of immune co-receptors or ligands such as CD80 and PD-1.
  • the dimerization inducer may be any molecule which induces dimerization between dimerization domains on separate chimeric molecules (including D1 and D2 on separate chimeric molecules having the same D1 and D2 domains). It may be a molecule to which both dimerization domains may bind in any way.
  • the inducer may be a ligand
  • the inducer may be a molecule which allows interaction between the dimerization domains by other means, such as chemical cross-linking.
  • the binding may include covalent bonding
  • the inducer may be a chemical cross- linker (i.e. a bifunctional molecule which comprises a first group reactive with a first dimerization domain (e.g. D1) and a second group reactive with a second dimerization domain (e.g. D2).
  • the inducer may be rapamycin or a rapamycin analogue ("rapalog") which has improved or differing pharmacodynamic or pharmacokinetic properties to rapamycin but have the same broad mechanism of action.
  • the inducer may be an altered rapamycin with engineered specificity for complementary FKBP12 or FRB - for example as shown in Figure 3.
  • Bayle et al 2006, as above describes various rapalogs functionalized at C16 and/or C20. Examples of such rapalogs in the first category include Sirolimus, Everolimus, Temsirolimus and Deforolimus.
  • rapalogs in the second category include C-20-methyllyrlrapamycin (MaRap); C16(S)-Butylsulfonamidorapamycin (C16-BS- Rap); C16-(S)-3-methylindolerapamycin (C16-iRap); and C16-(S)-7- methylindolerapamycin (AP21976/C16-AiRap).
  • Rapamycin is a potent immunosuppressive agent. Analogues of rapamycin are in every day clinical use. Modern rapalogs have excellent bioavailability and volumes of distribution.
  • rapamycin Although they are potent immunosuppressive agents, a short dose (to activate the signalling protein) would be expected to have minimal side- effects when treating conditions requiring an increased immune response, such as cancer (although long term dosing of rapamycin may not be desirable in such subjects/patients). As discussed above, the immunosuppressive effects would be beneficial when treating an inflammatory condition or disease, e.g. an autoimmune disease or transplant rejection, e.g. using a cell of the invention.
  • inducers include FK506, CsA, FKCsA and Fusicoccin, as discussed above and depicted in Figure 4A-D.
  • HaSX inducers may be represented by HaSX8, also depicted in Figure 4E.
  • inducers include dimeric FK506 molecules or derivatives or analogues thereof, including FK1012, AP1903 and AP21087. Still further inducers include ligands and binding partners for binding proteins as discussed above.
  • an inducer for dimerization is not required, and dimerization systems can be based on cognate dimerization domains which associate, e.g. bind or interact in any way, when in proximity. Again, such dimerization systems are known in the art, and any such system may be adopted, or adapted, for use herein. Particular mention may be made in this regard 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 leucine residues evenly spaced through an alpha-helix.
  • a dimerization domain herein is or comprises a leucine zipper sequence. These may be used for hetero- or homodimerization, according to the leucine zipper sequence which is used.
  • 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 Guatemalamann-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: 99 (this can include GG at N terminus) and a Fos leucine zipper sequence is shown in SEQ ID NO: 100.
  • Heterodimerization domains comprising Jun and Fos leucine zippers respectively may be used.
  • homodimerization domains comprising Jun leucine zippers may be used.
  • Other leucine zipper dimerization domains known in the art include those based on ZIP proteins, a class of transcription factors.
  • a ZIP domain is a region of alpha-helix containing leucines which line up to form the leucine zipper motif.
  • a ZIP domain can interact with leucines on other ZIP domains to reversibly hold the alpha- helices together (i.e. to dimerize them).
  • a dimerization domain herein can comprise a BZip or AZip leucine zipper domain.
  • a heterodimerization domain can be or comprise a BZip (RR) domain which heterodimerizes with an AZip (EE) domain.
  • Leucine zippers are an example of a coiled coil structural protein motif which may 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.
  • leucine zipper domains can include SYNZIP 1 to SYNZIP 48.
  • Other examples of leucine zipper domains include BATF, ATF4, ATF3, BACH1, JUND, NFE2L3, and HEPTAD.
  • the sequence of a BZip (RR) leucine zipper domain is shown in SEQ ID NO: 101.
  • the sequence of a AZip (EE) leucine zipper domain is shown in SEQ ID NO: 102.
  • a suitable pair of leucine zipper domains has a dissociation constant (Kd) of 1000 nM or less, for example 100 nM or less, 10 nM or less, or 1 nM or less.
  • Further exemplary pairs of dimerization domains can include PSD95- Dlgl-zo-1 (PDZ) domains, or a streptavidin domain and a streptavidin binding protein (SBP) domain.
  • Other dimerization domains may be obtained or derived from other proteins known to interact or bind to each other.
  • a heterodimerization domain pair can comprise CD80 and PDL-1.
  • a still further example of a homodimerization domain is the Fc region of immunoglobulin G.
  • the present chimeric protein comprises a signalling domain, also referred to as an endodomain, which comprises a tyrosine kinase activating domain comprising at least a JAK1-binding motif, and a tyrosine effector domain which can be phosphorylated by the JAK1 kinase.
  • the tyrosine kinase activating domain may in some embodiments also include a JAK2-binding motif and/or a JAK3-binding motif. In particular, it may include a JAK1- and a JAK3-binding motif.
  • the chimeric protein may be used in conjunction with a second chimeric protein which comprises a signalling domain comprising a JAK3-binding motif.
  • the signalling domain of the chimeric protein may signal through the JAK- STAT signalling pathway, or in other words, the signal may be mediated by activation of the JAK-STAT signalling pathway.
  • STAT proteins are transcription factors which are recruited to an activated receptor, and accordingly, in particular the tyrosine effector domain may comprise a STAT association motif, that is a binding site for a STAT.
  • the 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 (RTK).
  • the tyrosine effector domain may contain one or more, e.g. two or more, for example, 3, 4, 5 or more STAT association motifs, which may be the same or different.
  • STAT5 is a transcription factor involved in the IL-2 signalling pathway that plays a key role in Treg function, stability and survival by promoting the expression of genes such as FOXP3, IL2RA and BCLXL.
  • STAT5 In order to be functional and translocate into the nucleus, STAT5 needs to be phosphorylated. IL-2 ligation results in STAT5 phosphorylation by activating the JAK1/JAK2 and JAK3 kinases via specific signalling domains present in the IL-2R ⁇ and IL-2R ⁇ chain, respectively. Although JAK1 (or JAK2) can phosphorylate STAT5 without the need of JAK3, STAT5 activity is increased by the transphosphorylation of both JAK1/JAK2 and JAK3, which stabilizes their activity.
  • STAT association motif refers to an amino acid motif which comprises a tyrosine and, upon phosphorylation of the tyrosine, is capable of binding a STAT polypeptide.
  • the STAT association motif may for example be a STAT5 association motif which is capable, upon phosphorylation, of binding a STAT5 polypeptide (and similarly for the other STAT polypeptides).
  • the STAT association motif is a STAT5 association motif.
  • the signalling domain may comprise two (e.g. at least two) or more STAT5 association motifs as defined herein.
  • the signalling domain may comprise two, three, four, five or more STAT5 association motifs as defined herein.
  • the signalling domain may comprise two or three STAT5 association motifs as defined herein.
  • the STAT5 association motif may exist endogenously in a cytoplasmic domain of a transmembrane protein which may be used to provide the signalling domain of the chimeric protein herein.
  • the STAT5 association motif may be from an interleukin receptor (IL) receptor endodomain or a hormone receptor.
  • IL interleukin receptor
  • the signalling domain may comprise an amino acid sequence selected from any chain of the interleukin receptors where STAT5 is a downstream component, for example, the cytoplasmic domain comprising amino acid numbers 266 to 551 of IL-2 receptor ⁇ chain (NCBI REFSEQ: NP_000869.1, SEQ ID NO: 9), amino acid numbers 265 to 459 of IL- 7R ⁇ chain (NCBI REFSEQ: NP_002176.2, SEQ ID NO: 10), amino acid numbers 292 to 521 of IL-9R chain (NCBI REFSEQ: NP_002177.2, SEQ ID NO: 11), amino acid numbers 257 to 825 of IL-4R ⁇ chain (NCBI REFSEQ: NP_000409.1, SEQ ID NO: 12), amino acid numbers 461 to 897 of IL-3R ⁇ chain (NCBI REFSEQ: NP_000386.1, SEQ ID NO: 13) and/or amino acid numbers 314 to 502 of IL-17R ⁇ chain (NCBI REFSEQ
  • SEQ ID NO: 15 represents an IL7RA 2Y truncated sequence, which may also be used.
  • the signalling domain may comprise one or more STAT5 association motifs that comprise an amino acid sequence shown as SEQ ID NOs: 9-15 or a variant which is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NOs: 9-15.
  • the variant may be capable of binding STAT5 to 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 an amino acid sequence shown as one of SEQ ID NOs: 9-15.
  • the variant or derivative may be capable of binding STAT5 to a similar or the same level as one of SEQ ID NOs: 9-15 or may be capable of binding STAT5 to a greater level than an amino acid sequence shown as one of SEQ ID NOs: 9-15 (e.g. increased by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%).
  • the STAT5 association motif may be from any one or more of IL2R ⁇ , IL7R ⁇ , IL-3R ⁇ (CSF2RB), IL-9R, IL-17R ⁇ , erythropoietin receptor, thrombopoietin receptor, growth hormone receptor and prolactin receptor.
  • a chimeric protein may, for example, comprise STAT association motifs from both IL2R ⁇ and IL7R ⁇ .
  • the STAT5 association motif may comprise the amino acid motif YXXF/L (SEQ ID NO: 16); wherein X is any amino acid.
  • the STAT5 association motif may comprise the amino acid motif YCTF (SEQ ID NO: 17), YFFF (SEQ ID NO: 18), YLSL (SEQ ID NO: 19), or YLSLQ (SEQ ID NO: 20).
  • the signalling domain may comprise one or more STAT5 association motifs comprising the amino acid motif YCTF (SEQ ID NO: 17), YFFF (SEQ ID NO: 18), YLSL (SEQ ID NO: 19), and/or YLSLQ (SEQ ID NO: 20).
  • the signalling domain may comprise a first STAT5 association motif comprising the amino acid motif YLSLQ (SEQ ID NO: 20) and a second STAT5 association motif comprising the amino acid motif YCTF (SEQ ID NO: 17) or YFFF (SEQ ID NO: 18).
  • the signalling domain may comprise the following STAT5 association motifs: YLSLQ (SEQ ID NO: 20), YCTF (SEQ ID NO: 17) and YFFF (SEQ ID NO: 18). Association motifs for other STAT polypeptides are known in the art, and may be used.
  • the tyrosine effector domain of the signalling domain may comprise YXXQ (SEQ ID NO: 84), where X is any amino acid, for example YRHQ (SEQ ID NO: 85).
  • the STAT3 association motif is present in signalling proteins for example IL-6R, IL10R and IL21R.
  • the signalling domain of a chimeric protein may comprise the cytoplasmic domain of the IL21R alpha chain, e.g.
  • NCBI RefSeq amino acid numbers 256-538 of the IL-21R alpha chain (NCBI RefSeq: NP_068570.1), or a truncated fragment thereof comprising a box 1 motif (amino acid numbers 266 to 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 flanking 3 residues at the C-terminal side of tyrosine residue 500, i.e. YLRQ (SEQ ID NO: 114), required for STAT1/3 association.
  • YLRQ SEQ ID NO: 114
  • STAT1 or STAT4 signalling may be provided in a similar manner.
  • 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.105)
  • JAK1-binding motif refers to a BOX motif which allows for tyrosine kinase JAK1 association.
  • “JAK2-binding motif” as used herein refers to a BOX motif which allows for tyrosine kinase JAK2 association.
  • JAK1- and JAK2-binding motifs are described, for example, by Ferrao & Lupardus (Frontiers in Endocrinology; 2017; 8(71); which is incorporated herein by reference).
  • the JAK1- and/or JAK2-binding motif may occur endogenously in a cytoplasmic domain of a transmembrane protein.
  • the JAK1- and/or JAK2-binding motif may be 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.
  • the JAK1-binding motif may comprise or consist of an amino acid motif shown as SEQ ID NOs: 21-27 or a variant thereof which is capable of binding JAK1.
  • KVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERD K (SEQ ID NO: 21)
  • NPWFQRAKMPRALDFSGHTHPVATFQPSRPESVNDLFLCPQKELT SEQ ID NO: 22) GYICLRNSLPKVLNFHNFLAWPFPNLPPLEAMDMVEVIYINR (SEQ ID NO: 23)
  • PLKEKSIILPKSLISVVRSATLETKPESKYVSLITSYQPFSL RRRKKLPSVLLFKKPSPFIFISQRPSPETQDTIHPLDEEAFLK
  • SEQ ID NO: 25 YIHVGKEKHPANLILIYGNEFDKRFFVPAEKIVINFITLNISDDS
  • the variant may be at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to any one of SEQ ID NOs: 21-27 and retain the ability to bind JAK1.
  • the JAK1-binding domain comprises or consists of SEQ ID NO: 21 or a variant thereof which is capable of binding JAK1.
  • the variant may be capable of binding JAK1 to 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 a corresponding, reference sequence.
  • the variant or derivative may be capable of binding JAK1 to a similar or the same level as a corresponding, reference sequence or may be capable of binding JAK1 to a greater level than a corresponding, reference sequence (e.g. increased by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%).
  • a JAK2-binding motif may comprise or consist of an amino acid motif shown as SEQ ID NO: 28-30 or a variant therefore which is capable of binding JAK2.
  • SEQ ID NO: 28 NYVFFPSLKPSSSIDEYFSEQPLKNLLLSTSEEQIEKCFIIEN
  • SEQ ID NO: 28 YWFHTPPSIPLQIEEYLKDPTQPILEALDKDSSPKDDVWDSVSIISFPE
  • SEQ ID NO: 29 YAFSPRNSLPQHLKEFLGHPHHNTLLFFSFPLSDENDVFDKLSVIAEDSES
  • the variant of SEQ ID NOs: 28-30 may comprise one, two or three amino acid differences compared to any of SEQ ID NOs: 28-30 and retain the ability to bind JAK2.
  • the variant may be capable of binding JAK2 to 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 a corresponding, reference sequence.
  • the variant or derivative may be capable of binding JAK2 to a similar or the same level as a corresponding, reference sequence or may be capable of binding JAK2 to a greater level than a corresponding, reference sequence (e.g. increased by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%). Any method known in the art for determining protein:protein interactions may be used to determine whether a JAK1- or JAK2-binding motif is capable of binding to a JAK1 or JAK2.
  • the signalling domain may comprise an IL2R ⁇ endodomain shown as SEQ ID NO: 9; or a variant which has at least 80% sequence identity to SEQ ID NO: 9.
  • the variant may be at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 9.
  • the signalling domain may comprise a truncated IL2R ⁇ endodomain shown as any one of SEQ ID NO: 31 or 32 or a variant of any one of SEQ ID NO: 31 or 32 which has at least 80% sequence identity thereto.
  • SEQ ID NO: 31 represents a IL2RB truncated variant with a Y510 mutation.
  • SEQ ID NO: 32 represents a IL2RB truncated variant with Y510 and Y392 mutations.
  • the variant may be at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 31 or 32.
  • STAT e.g. STAT5
  • activity is increased by the transphosphorylation of both a JAK1/2 and JAK3, as this stabilizes their activity.
  • the signalling domain, or more particularly the tyrosine kinase activating domain thereof may further comprise a JAK3-binding motif.
  • “JAK3-binding motif” as used herein refers to a BOX motif which allows for tyrosine kinase JAK3.
  • JAK3-binding motifs are described, for example, by Ferrao & Lupardus (Frontiers in Endocrinology; 2017; 8(71); which is incorporated herein by reference). Any method known in the art for determining protein:protein interactions may be used to determine whether a motif is capable of binding to JAK3. For example, co- immunoprecipitation followed by western blot.
  • the JAK3-binding motif may occur endogenously in a cytoplasmic domain of a transmembrane protein.
  • the JAK3-binding motif may be from an IL-2R ⁇ polypeptide.
  • a functional truncated or variant IL2R ⁇ polypeptide may be used within the signalling domain of the chimeric protein, wherein the functional truncated or variant IL2R ⁇ polypeptide retains JAK3-binding activity (e.g. at least 20, 30, 40, 50, 60, 70, 80, 90 or 95% of binding activity of IL2R ⁇ ).
  • JAK3-binding activity e.g. at least 20, 30, 40, 50, 60, 70, 80, 90 or 95% of binding activity of IL2R ⁇ .
  • a truncated IL2R ⁇ comprising a JAK3-binding motif and a truncated IL2R ⁇ comprising a STAT5 association motif, and a JAK1-binding motif may be comprised in the signalling domain of a chimeric protein as defined herein.
  • Functional truncations may provide an advantage of reducing construct size for expression.
  • the JAK3-binding motif may comprise or consist of an amino acid motif sequence shown as SEQ ID NO: 33 or SEQ ID NO: 34 or a variant thereof which is capable of binding JAK3 (e.g. a functional variant or fragment having at least 80, 85, 90, 95 or 99% identity to SEQ ID NOs: 33 or 34).
  • the variant may be at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 33 or SEQ ID NO: 34.
  • the signalling domain comprises one or more JAK1-binding domains and at least one JAK3-binding domain/motif (e.g. at least 2 or 3 JAK3-binding domains/motifs).
  • the polynucleotide sequence encoding the JAK3-binding domain may be positioned upstream or downstream (5’ or 3’) of the polynucleotide sequence encoding the tyrosine effector domain, for example, the STAT, e.g. STAT5, association motif and JAK1-binding motif.
  • the JAK1- (and if present additionally the JAK2-) binding motif would be upstream (5’) of the tyrosine effector domain, e.g. STAT/STAT5, but this may be varied.
  • the polynucleotide encoding the JAK3-binding domain may be positioned downstream (3’) of the polynucleotide encoding the STAT association motif and the JAK1, and optionally JAK2, binding motifs.
  • the JAK3-binding domain may be N- or C-terminal to the tyrosine effector domain (e.g. STAT association motif) and the JAK1-binding domain, preferably C-terminal.
  • the JAK3-binding domain and the STAT association motif/JAK1-binding domains are positioned directly adjacent to one another (i.e. are not separated distally by sequence).
  • the JAK3-binding domain is translated in reverse orientation, thus the JAK3-binding motif may comprise a sequence in the reverse orientation to SEQ ID NOs: 33 or 34 (e.g. as shown in SEQ ID NO: 87).
  • the polynucleotide encoding the signalling domain may thus comprise nucleotide sequences in the following order: 5’-3’ JAK1, 5’-3’ STAT association motif, 3’-5’ JAK3.
  • the interaction domain may be linked to the N- or C-terminal of the signalling domain.
  • the interaction domain is a 2-part domain, comprising two separate parts each comprising a dimerization domain, the two parts may be separately linked to the N- and C-terminals of the signalling domain.
  • various configurations of the interaction domain, and individual dimerization domains (e.g. Ht1, Ht2), and signalling domains of the chimeric protein are possible. This may depend upon whether there is one or more than one dimerization domain, and when there are 2 or more dimerization domains, whether the interaction domain is a 1-part or 2-part domain.
  • the signalling domain may comprise, from N- to C-terminal: (i) JAK1 motif – STAT motif; (ii) JAK1 motif – STAT motif – JAK3 motif, particularly in reverse orientation; (iii) JAK1 motif – JAK2 motif – STAT motif; or (iv) JAK1 motif – JAK2 motif – STAT motif – JAK3 motif, particularly in reverse orientation; wherein the motifs are linked directly or indirectly to one another. Where the motifs are linked indirectly, they may be linked by a linker.
  • the STAT motif may in particular be a STAT5 motif.
  • a linker or a hinge may be present between the JAK3-binding motif and the STAT association motif/JAK1-, or JAK1- and JAK2-, binding motifs.
  • the 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, e.g. at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 glycine residues.
  • the signalling domain comprises a first amino acid sequence derived from IL2R ⁇ comprising a JAK3-binding domain (e.g.
  • the signalling domain may comprise SEQ ID NO: 33 and SEQ ID NO: 31 wherein SEQ ID NOs: 33 and 31 are connected by a linker or hinge.
  • the interaction domain or parts thereof and the signalling domain may be joined to each other via a linker.
  • the interaction domain comprises D1 and D2 (e.g.
  • Ht1 and Ht2 together in a single 1-part domain, these may also be joined together directly, or by a linker. Further, if the chimeric protein comprises other domains or sequences, for example an additional stabilising or functional domain at either end of the molecule, then such domains or sequences may be linked directly or indirectly via a linker. Linker sequences are discussed in more detail below.
  • a 1-part interaction domain may be linked to the N- or the C-terminal of the signalling domain.
  • a 1-part interaction domain may occur where there is a single dimerization domain (as shown in Figure 1), or where there are 2 or more dimerization domains.
  • Figure 2 shows various configurations for interaction domains which comprise 2 dimerization domains D1 and D2.
  • D1 and D2 may be homo- or heterodimerization domains, but typically they will be heterodimerization domains Ht1 and Ht2.
  • Figure 2A depicts a configuration wherein the binding domain is at the N-terminus of the signalling domain.
  • a 1-part interaction domain including a domain such as depicted here, allows a one-sided dimerization.
  • the signalling domain is depicted as comprising, N- to C-terminal JAK1-STAT5, but this may be modified to introduce a JAK3 domain linked at the C-terminus of STAT5, particularly in reverse orientation.
  • Ht1 and Ht2 may each be linked to an end of the signalling domain, i.e. one of Ht1 or Ht2 may be linked at the N- terminus of the signalling domain, and the other of Ht1 and Ht2 may be linked at the C-terminus of the signalling domain.
  • Figure 2B shows a configuration in which a JAK3 domain is added to signalling domain, particularly in reverse orientation.
  • a 2-part binding domain allows a 2-sided dimerization. It will be understood that the configurations depicted in Figure 2 are exemplary only, and may be modified or altered.
  • the STAT5 association motif may be replaced or supplemented by one or more other STAT association motifs, e.g. STAT3.
  • D1 and D2 are shown as Ht1 and Ht2 in Figure 2 but could be homodimerization domains.
  • the chimeric protein may have the following configurations: (i) the JAK1-, or JAK1- and JAK2-, binding motifs in the signalling domain may be linked N- or C-terminal to the STAT motif(s); (ii) where the signalling domain of the chimeric protein comprises a JAK3- binding motif, this is linked to the other end of the STAT motif than the JAK-, or the JAK1- and JAK2-, binding motifs.
  • the skilled person would be able to design an appropriate configuration for the chimeric protein, depending on the nature of the dimerization domains and the signalling domain, for example whether or not the signalling domain includes a JAK3- binding motif.
  • the signalling domain includes a JAK3-binding motif.
  • the purpose of the dimerization of the interaction domain is to bring together 2 chimeric proteins each comprising a signalling domain, whether this be 2 molecules of the same chimeric protein or 2 different chimeric proteins with different interaction and/or signalling domains, and to allow them to dimerize to form a functional signalling protein.
  • the dimerization between the signalling domains does not require a physical association between the two signalling domain monomers, in the sense of the 2 domains becoming physically linked or joined to one another – it suffices that they are brought into proximity together in such a manner that they can act together to mediate a signal in the cell.
  • the dimerization between the two signalling domains thus includes a functional association between them.
  • the skilled person is well aware of the requirements for dimerization of signalling domains, or more particularly the individual motifs thereof. Particular motifs may homo-dimerize with one another, but this is not necessarily the case.
  • the signalling domain may be designed such that individual motifs homo-dimerize.
  • the STAT5 association motif may homodimerize, as may the JAK1-binding motif.
  • the motifs do not need to homodimerize with one another, and whilst typically in a chimeric protein, STAT motifs will be designed to homo-dimerize with one another, STAT5 association motifs may for example heterodimerize with JAK1 or JAK2 (as depicted for example in Figure 2B).
  • JAK1 may heterodimerize with JAK3.
  • JAK1 dimerization with JAK3 is depicted in Figure 2C for example). To achieve functional dimerization of JAK1 and JAK3, from separate chimeric protein chains, JAK3 is typically translated in reverse orientation.
  • a chimeric protein comprising a signalling domain comprising a tyrosine kinase activating domain and a tyrosine effector domain as defined herein may dimerize with a second chimeric protein which is different, and in particular which comprises a different signalling domain.
  • the second chimeric protein may comprise a signalling domain comprising a JAK3-binding motif.
  • the first chimeric protein may not comprise a JAK3-binding motif.
  • a first chimeric protein as provided herein may comprise a signalling domain A comprising a tyrosine kinase activating domain comprising a JAK1-binding motif and a tyrosine effector domain comprising one or more tyrosine residues that can be phosphorylated by JAK1 (e.g. comprising one or more STAT association motifs).
  • the first chimeric protein may dimerize with a second chimeric protein comprising a signalling domain B comprising a tyrosine kinase activating domain comprising a JAK3-binding motif.
  • the first and second chimeric proteins may be provided as a pair, as indicated above.
  • the signalling domain B does not comprise a tyrosine effector domain.
  • the signalling domain B may not be capable on its own, when dimerized to another signalling domain B, of transmitting or mediating a signal to the cell.
  • the signalling domain here allows a chimeric protein comprising a signalling domain A to become dimerized to effect signalling activity, for example by dimerization of the JAK1-binding motif in signalling domain A with the JAK3-binding motif in signalling domain B.
  • the presence of the JAK3-binding motif, and recruitment and activation of JAK3 may augment the signalling by the dimerized signalling protein, as discussed above.
  • chimeric proteins herein may provide a so-called “split” signalling protein, wherein different functional parts of the signalling domain are provided on separate chains, or by separate chimeric proteins. Examples of such constructs, or chimeric proteins, are depicted in Figure 14.
  • Chimeric proteins A, B, and C have an interaction domain comprising a single dimerization domain; as depicted this is a heterodimerization domain, being either FKBP or FRB.
  • chimeric protein A comprises FRB and dimerizes either with construct B or construct C comprising FKBP.
  • Chimeric protein B comprises a signalling domain which is identical to that of protein A, based on the IL2RB chain, which comprises a JAK1- binding motif and a STAT5 association motif.
  • Chimeric protein C comprises a different signalling domain based on the IL2RG chain, which comprises a JAK3- binding motif.
  • Chimeric proteins D and E comprising a 1-part interaction domain comprising 2 dimerization domains, Ht1 and Ht2, here represented by FRB and FKBP.
  • Protein D has a signalling domain comprising an IL2RB sequence and hence may dimerize with itself (i.e. it works alone) or with protein E, which comprises an IL2RG sequence. Protein E is only functional when dimerized with protein D.
  • the signalling domain may provide other signalling functions (e.g. those capable of providing a pro-survival or persistence signal, a signal which maintains cell phenotype or induces activation or function in addition to providing a STAT signal), and thus may comprise further domains which are capable of providing such signalling functions.
  • the signalling domain however may not comprise any suicide or safety switch moiety or domain, and thus particularly does not comprise any killing or suicide function.
  • the signalling domain may additionally comprise an intracellular signalling domain such as ⁇ chain endodomain of the T-cell receptor or any of its homologs (e.g., ⁇ chain, Fc ⁇ R1 ⁇ and ⁇ chains, MB1 (Ig ⁇ ) chain, B29 (Ig ⁇ ) chain, etc.), CD3 polypeptide domains ( ⁇ , ⁇ and ⁇ ), 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.
  • an intracellular signalling domain such as ⁇ chain endodomain of the T-cell receptor or any of its homologs (e.g., ⁇ chain, Fc ⁇ R1 ⁇ and ⁇ chains, MB1 (Ig ⁇ ) chain, B29 (Ig ⁇ ) chain, etc.), CD3 polypeptide domains ( ⁇ , ⁇ and
  • the intracellular signaling domain may comprise human CD3 zeta chain endodomain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors or combinations thereof.
  • ITAM immunoreceptor tyrosine-based activation motif
  • the additional intracellular signaling domain may comprise the intracellular signaling domain of a human CD3 zeta chain, which in one embodiment comprises or consists of the following sequence: UNIPROT: P20963, CD3Z_HUMAN, position 31-143 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR (SEQ ID NO: 81)
  • the intracellular signaling domain comprises at least 85, 90, 95, 97, 98 or 99% identity to SEQ ID NO: 81.
  • the intracellular signaling domain of the chimeric protein may comprise the following CD28 signaling domain: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 82) In one embodiment, the intracellular signaling domain comprises a signaling motif which has at least 85, 90, 95, 97, 98 or 99% identity to SEQ ID NO: 82.
  • the intracellular signaling domain of the chimeric protein may comprise the following CD27 signaling domain: QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 83).
  • the intracellular signaling domain comprises a signaling motif which has at least 85, 90, 95, 97, 98 or 99% identity to SEQ ID NO: 83. Additional intracellular signalling domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.
  • the signalling domain may comprise additional domains or sequences which provide transcription factor activity to the cell in which it is expressed, e.g. a transcription factor which has importance for phenotype or function of the cell.
  • the signalling domain may additionally be capable of providing the cell with FOXP3, c-Rel, Runx, Ets-1, CREB, NFAT and/or JunB (directly or indirectly).
  • the signalling domain may be capable of providing a FOXP3 activating or inducing signal to the cell.
  • the signalling domain may comprise FOXP3 (or any functional variant, truncation or isoform thereof), wherein the FOXP3 may be cleavable from the chimeric protein upon induction with a CID (for example, using a Notch system).
  • the signalling domain may be present at the C terminus of the chimeric protein, and any cleavable portion (e.g. FOXP3) would be present at the C-terminus of the signalling domain.
  • the chimeric protein may if desired comprise a further domain, for example at either or both ends of the protein.
  • Such an additional domain may be any desired domain, but since chimeric proteins and signalling proteins herein are cytoplasmic, non-membrane associated, proteins, it will not be a transmembrane domain, or any domain capable of associating with the cell membrane in any way (as discussed above). It may be a functional domain, or it may be non-functional. That is, the domain may be included simply to provide a structural element to the protein, without necessarily imparting any functional property or activity to it. As noted above the various domains, and individual parts of the domains (e.g. the motifs in the signalling domain) may be linked to one another by linkers. Thus, the chimeric protein may contain one or more linkers. Typically, it will contain at least one linker.
  • the protein may contain a linker linking the interaction domain, or a part thereof, to the signalling domain.
  • the chimeric protein may contain a linker linking a 1-part binding domain to the signalling domain, and a linker linking D1 to D2.
  • the chimeric protein may comprise a 2-part binding domain wherein each of D1 and D2 (e.g. Ht1 and Ht2) are linked by a linker to the signalling domain. Any additional domains may also be linked by linkers.
  • a linker as referred to herein is an amino acid sequence which links one domain or part of the protein to another.
  • the linker sequence may be any amino acid sequence which functions to link, or connect, two domains or parts thereof together, such that they may perform their function.
  • a linker may space apart the elements which are linked, for example to allow them to bind to their target.
  • the linker may allow D1 and D2 from separate chimeric protein molecules to bind to an inducer molecule.
  • different linkers may be required, for example to connect the interaction domain, or D1 and D2 individually to the signalling domain, or to one another, and/or to other domains.
  • the nature of the linker in terms of its amino acid composition and/or sequence of amino acids may be varied and is not limited. However, the linker may be a flexible linker.
  • Flexible linkers are a category of linker sequences well known and described in the art. Linker sequences are generally known as sequences which may be used to link, or join together, proteins or protein domains, to create for example fusion proteins or chimeric proteins, or multifunctional proteins or polypeptides. They can have different characteristics, and for example may be flexible, rigid or cleavable. Protein linkers are reviewed for example in Chen et al., 2013, Advanced Drug Delivery Reviews 65, 1357-1369, which compares the category of flexible linkers with those of rigid and cleavable linkers.
  • a flexible linker is a linker which allows a degree of movement between the domains, or components, which are linked. They are generally composed of small non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acid residues. The small size of the amino acids provides flexibility and allows for mobility of the connected parts (domains or components). The incorporation of polar amino acids can maintain the stability of the linker in aqueous environments by forming hydrogen bonds with water molecules.
  • GS linkers The most commonly used flexible linkers have sequences primarily composed of Ser and Gly residues (so-called “GS linkers”). However, many other flexible linkers have also been described (see Chen et al, 2013, supra, for example), which may contain additional amino acids such as Thr and/or Ala, and/or Lys and/or Glu which may improve solubility. Any flexible linker known and reported in the art may be used.
  • GS linkers, or more particularly GS (“Gly-Ser”) domains in linkers may allow the length of the linker readily to be varied by varying the number of GS domain repeats, and so such linkers represent one suitable class of linkers.
  • linker sequence comprises at least one Gly-Ser domain composed solely of Ser and Gly residues.
  • the linker may contain no more than 15 other amino acid residues, e.g. no more than 14, 13, 12, 11, 10, 9, 8, 6, 7, 5, or 4 other amino acid residues.
  • the Gly-Ser domain may have the formula: (S)q-[(G)m-(S)m]n-(G)p wherein q is 0 or 1; m is an integer from 1-8; n is an integer of at least 1 (e.g. from 1 to 8, or more particularly 1 to 6); and p is 0 or an integer from 1 to 3.
  • the Gly-Ser domain may have the formula: (i) S-[(G)m-S]n; (ii) [(G)m-S]n; or (iii) [(G)m-S]n-(G)p wherein m is an integer from 2-8 (for example 3-4); n is an integer of at least 1 (for example from 1 to 8, or more particularly 1 to 6); and p is 0 or an integer from 1 to 3.
  • the Gly-Ser domain may have the formula: S-[G-G-G-G-S]n wherein n is an integer of at least one (preferably 1 to 8, or 1-6, 1-5, 1-4, or 1- 3).
  • the sequence GGGGS is SEQ ID NO.46.
  • S(GGGGS)2 is SEQ ID NO.42 and S(GGGGS)4 is SEQ ID NO. 44.
  • the linker sequence is not a flexible linker sequence.
  • the linker connects an interaction domain, or D1/Ht1 or D2/Ht2, to a signalling domain, it is preferably a flexible linker.
  • the length of the linker may not be critical, it may in some cases be desirable to have a shorter linker sequence, or a longer linker sequence, depending on what domains etc. are being linked.
  • the linker sequence may have a length of no more than 30, e.g. no more than 25, 24, 23, 22, 21 or 20 amino acids.
  • the linker connecting D1/Ht1 and D2/Ht2 may be less than 15, or 10 amino acids or alternatively viewed may be between 5-15 or 5-10 amino acids in length (e.g. for chimeric proteins having a one-part binding domain, particularly for chimeric proteins having a one-part binding domain C terminal to the signalling domain).
  • a longer linker sequence may be desired. This may particularly be the case where the interaction domain is included internally, between two other domains.
  • a longer linker may be composed of, or may comprise, multiple repeats of a GS domain.
  • the linker may be from any one of 2, 3, 4, 5 or 6 to any one of 24, 23, 22 or 21 amino acids in length. In other cases, it may be from any one of 2, 3, 4, 5 or 6 to any one of 21, 20, 19, 18, 17, 16, or 15 amino acids in length.
  • the linker may be of longer length, for example, from any one of 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 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 indicated above. It may accordingly be within a range made up from any of the integers listed above.
  • linkers may be designed or selected to allow the various domains and parts of the chimeric protein to function or to exert their effects, and so that the dimerizations may take place as set out above, particularly in the case of a chimeric protein having an interaction domain with 2 or more dimerization domains, so that D1/Ht1 from one protein molecule may dimerize to D2/Ht2 from another protein molecule, and that the signalling domain monomers of the respective proteins may dimerize with one another. It is well within the routine skill of the person skilled in the art to design or select appropriate linkers. For example, a linker should provide sufficient flexibility so the signalling domains can dimerize, but not so much flexibility so that the energic barrier to such dimerization is not overcome.
  • GS linkers or more particularly GS (“Gly-Ser”) domains in linkers
  • Gly-Ser GS
  • flexible linkers are not limited to those based on “GS” repeats, and other linkers comprising Ser and Gly residues dispersed throughout the linker sequence have been reported, including in Chen et al., supra.
  • a linker sequence may be composed solely of, or may consist of, one or more Gly-Ser domains as described or defined above. However, as noted above, the linker sequence may comprise one or more Gly-Ser domains, and additional amino acids.
  • the additional amino acids may be at one or both ends of a Gly-Ser domain, or at one or both ends of a stretch of repeating Gly-Ser domains.
  • the additional amino acids which may be other amino acids, may lie at one or both ends of the linker sequence, e.g. they may flank the Gly-Ser domain(s).
  • the additional amino acids may lie between Gly-Ser domains.
  • two Gly- Ser domains may flank a stretch of other amino acids in the linker sequence.
  • GS domains need not be repeated, and G and/or S residues, or a short domain such as GS, may simply be distributed along the length or the sequence.
  • GGGGS (SEQ ID NO.46) GGGGSGGGGSGGGGS (SEQ ID NO: 67) GGGGG (SEQ ID NO: 68) GGGGSGGGGS (SEQ ID NO: 69) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO 70) GGGGGG (SEQ ID NO: 50) GGGGGGGG (SEQ ID NO: 51).
  • Illustrative constructs are represented by SEQ ID NO: 86, or more particularly by SEQ ID NO: 103 or SEQ ID NO: 104 as used in the Examples herein and shown in Figure 5.
  • constructs comprise an interaction domain comprising two dimerization domains FRB (94 amino acids) and FKBP (107 amino acids) separated from each other by a 5 amino acid linker.
  • the interaction domain is joined by a 12 amino acid linker to a signalling domain comprising an IL2RB truncated variant with a Y510 mutation (SEQ ID NO.31).
  • the depicted construct pQTX-0340 (SEQ ID NO: 103) is linked at the C-terminal to a P2A self-cleavage sequence and GFP for the purposes of expressing the construct and detecting it in the experiments described.
  • Construct pQTX-0370 is based on construct pQTX-0340 but additionally comprises an anti-HLA-A2 CAR linked by a T2A sequence to the N- terminal end of the IL2RB sequence (i.e. between the signalling domain and the P2A- GFP sequence).
  • Representative constructs for use herein may be based on the constructs of SEQ ID NOs:103 or 104, but omitting or substituting the GFP, for example for another protein of interest, as described in more detail below.
  • Other illustrative constructs which may be used as the basis for chimeric proteins as provided herein are shown in Figures 14 and 15 (see SEQ ID NOs:106, 108, 109, 110 and 111).
  • a third aspect herein provides a nucleic acid molecule comprising a nucleotide sequence which encodes the chimeric protein.
  • polynucleotide and “nucleic acid” are intended to be synonymous with each other. It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described here to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed.
  • Nucleotide sequences encoding the various domains and motifs etc. described herein are known and available in the art, and any of these may be used or modified for use herein.
  • Nucleic acids according to the second aspect may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.
  • polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.
  • variant refers to any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence.
  • Nucleic acid molecules/polynucleotides/nucleotide 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 by standard techniques. Longer nucleic acid molecules/polynucleotides/nucleotide sequences will generally be produced using recombinant means, for example using polymerase chain reaction (PCR) cloning techniques. This will involve making a pair of primers (e.g.
  • PCR polymerase chain reaction
  • a nucleic acid construct may comprise the nucleic acid molecule together with one or more other nucleotide sequences, for example, regulatory sequences, e.g.
  • the other coding sequence may encode a protein of interest.
  • This may be a therapeutic protein.
  • a chimeric protein may be co-expressed with another protein of interest, for example a receptor, particularly an antigen receptor, for example, a CAR or a TCR or a derivative thereof (e.g. a TCR-CAR construct, or single chain TCR construct etc.).
  • the coding sequence for such a further protein, e.g. receptor may be comprised within a construct as referred to above.
  • the chimeric protein may also be co-expressed with a safety switch polypeptide.
  • the additional domain of the chimeric protein may comprise a safety switch polypeptide.
  • a safety switch polypeptide provides a cell in or on which it is expressed with a suicide moiety. This is useful as a safety mechanism which allows a cell which has been administered to a subject to be deleted should the need arise, or indeed more generally, according to desire or need, for example once a cell has performed or completed its therapeutic effect.
  • a suicide moiety possesses an inducible capacity to lead to cellular death, or more generally to elimination or deletion of a cell.
  • a suicide moiety is a suicide protein, encoded by a suicide gene, which may be expressed in or on a cell alongside a desired transgene, in this case the chimeric protein (and optionally a CAR or other receptor which is co-expressed by the cell along with the present chimeric protein), which when expressed allows the cell to be deleted to turn off expression of the transgene (CAR).
  • a suicide moiety herein is a suicide polypeptide that is a polypeptide that under permissive conditions, namely conditions that are induced or turned on, is able to cause the cell to be deleted.
  • the suicide moiety may be a polypeptide, or amino acid sequence, which may be activated to perform a cell-deleting activity by an activating agent which is administered to the subject, or which is active to perform a cell-deleting activity in the presence of a substrate which may be administered to a subject.
  • the suicide moiety may represent a target for a separate cell-deleting agent which is administered to the subject.
  • the cell- deleting agent By binding to the suicide moiety, the cell- deleting agent may be targeted to the cell to be deleted.
  • the suicide moiety may be recognised by an antibody, and binding of the antibody to the safety switch polypeptide, when expressed on the surface of a cell, causes the cell to be eliminated, or deleted.
  • the suicide moiety may be HSV-TK or iCasp9 as is known in the art. However, in other examples the suicide moiety may be, or may comprise an epitope which is recognized by a cell-deleting antibody or other binding molecule capable of eliciting deletion of the cell.
  • the term “delete” as used herein in the context of cell deletion is synonymous with “remove” or “ablate” or “eliminate”.
  • the term is used to encompass cell killing, or inhibition of cell proliferation, such that the number of cells in the subject may be reduced.100% complete removal may be desirable but may not necessarily be achieved. Reducing the number of cells, or inhibiting their proliferation, in the subject may be sufficient to have a beneficial effect.
  • the suicide moiety may be a CD20 epitope which is recognised by the antibody Rituximab.
  • the suicide moiety may comprise a minimal epitope based on the epitope from CD20 that is recognized by the antibody Rituximab.
  • the polypeptide may comprise two CD20 epitopes R1 and R2 that are spaced apart by a linker L.
  • Safety switches based on Rituximab epitopes are described in WO2013/15339.
  • WO2013/15339 discloses a polypeptide termed RQR8, having the sequence set forth in SEQ ID NO:41, which comprises two CD20 minimal epitopes, separated from one another by spacer sequences and an intervening CD34 marker sequence, and further linked to a stalk sequence which allows the polypeptide to project from the surface of a cell on which it is expressed.
  • the safety switch polypeptide may be RQR8 or a variant thereof having at least 80% sequence identity thereto, e.g. at least 85, 88, 90, 95, 96, 97, 98, or 99% sequence identity thereto.
  • Other safety switch polypeptides which may be used as the basis of safety switch domains include those described in our co-pending PCT patent application No. WO 2021/239812.
  • Other polypeptides which may be co-expressed with the chimeric protein include transcription factors, growth factors or other factors which may assist in enhancing functionality of survival of the cell.
  • the transcription factor FOXP3 may be used to maintain the suppressive phenotype of Treg cells.
  • “FOXP3” is the abbreviated name of the forkhead box P3 protein.
  • FOXP3 is a member of the FOX protein family of transcription factors and functions as a master regulator of the regulatory pathway in the development and function of regulatory T cells.
  • FOXP3 as used herein encompasses variants, isoforms, and functional fragments of FOXP3.
  • a “FOXP3 polypeptide” is a polypeptide having FOXP3 activity i.e. a polypeptide able to bind FOXP3, target DNA and function as a transcription factor regulating development and function of Tregs.
  • the present nucleic acid molecule or construct may further comprise a nucleic acid sequence encoding a selectable marker.
  • selectable markers are well known in the art and include, but are not limited to, fluorescent proteins – such as GFP.
  • the selectable marker may be a fluorescent protein, for example GFP, YFP, RFP, tdTomato, dsRed, or variants thereof.
  • the fluorescent protein is GFP or a GFP variant.
  • 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)).
  • NIS Sodium Iodide Symporter
  • LNGFR low-affinity nerve growth factor receptor
  • Tregs in which a nucleic acid molecule, construct or vector has been successfully introduced (such that the encoded chimeric protein and any other encoded proteins or polypeptides are expressed) to be selected and isolated from a starting cell population using common methods, e.g. flow cytometry.
  • the chimeric protein may be co-expressed with a mutant calcineurin protein which is resistant to at least one calcineurin inhibitor, and in particular a mutant calcineurin protein which is resistant to at least one calcineurin inhibitor and sensitive to at least one calcineurin inhibitor.
  • calcineurin mutants are discussed further below.
  • the nucleic acid molecule or construct may further comprise a nucleotide sequence encoding such a mutant calcineurin.
  • two or more coding sequences are expressed from a single nucleic acid molecule or construct, they may be linked by a sequence allowing co-expression of the two or more coding sequences.
  • the co-expression sequence or alternatively termed, the co-expression site, may enable expression of an encoded protein or polypeptide as a discrete entity.
  • the construct may comprise an internal promoter, an internal ribosome entry sequence (IRES) sequence or a sequence encoding a cleavage site.
  • the co-expression sequence may encode a self-cleavage sequence in between encoded polypeptides.
  • the self-cleaving sequence may be a self-cleaving peptide.
  • Such sequences auto-cleave during protein production.
  • Self-cleaving peptides which may be used are 2A peptides or 2A-like peptides which are known and described in the art, for example in Donnelly et al., Journal of General Virology, 2001, 82, 1027-1041, herein incorporated by reference. 2A and 2A-like peptides are believed to cause ribosome skipping, and result in a form of cleavage in which a ribosome skips the formation of peptide bond between the end of a 2A peptide and the downstream amino acid sequence.
  • cleavage occurs between the Glycine and Proline residues at the C-terminus of the 2A peptide meaning the upstream cistron will have a few additional residues added to the end, while the downstream cistron will start with the Proline.
  • Suitable self-cleaving domains include P2A, T2A, E2A, and F2A sequences as shown in SEQ ID NOs: 73-77 respectively.
  • the sequences may be modified to include the amino acids GSG at the N-terminus of the 2A peptides.
  • sequences corresponding to SEQ ID NOs: 73-77 but with GSG at the N-termini thereof.
  • Such modified alternative 2A sequences are known and reported in the art.
  • the self-cleaving sequences included in the nucleic acid molecule may be the same or different.
  • the self-cleaving sequence may include an additional cleavage site, which may be cleaved by common enzymes present in the cell. This may assist in achieving complete removal of the 2A sequences after translation.
  • Such an additional cleavage site may for example comprise a Furin cleavage site.
  • Such cleavage sites are known in the art, and may include for example RXXR (SEQ ID NO: 78), for example RRKR (SEQ ID NO: 79).
  • a furin sequence is comprised within the constructs shown in Figure 15 – as shown in this case, the furin sequence may be linked via a linker sequence, for example at its C-terminal end. It has been shown that the inclusion of such a linker may enhance cleavage.
  • the nucleic acid molecule/polynucleotides used herein may be codon- optimised. Codon optimisation has previously been described in WO 1999/41397 and WO 2001/79518. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression.
  • nucleotide sequence encoding the chimeric protein, and any other coding nucleotide sequences may be provided in a construct in which they are operably linked to a promoter. In some cases, different nucleotide sequences may be operably linked to the same promoter.
  • a “promoter” is a region of DNA that leads to initiation of transcription of a gene.
  • Promoters are located near the transcription start sites of genes, upstream on the DNA (towards the 5’ region of the sense strand). Any suitable promoter may be used, the selection of which may be readily made by the skilled person.
  • the promoter may be from any source, and may be a viral promoter, or a eukaryotic promoter, including mammalian or human promoters (i.e. a physiological promoter).
  • the promoter is a viral promoter.
  • Particular promoters include LTR promoters, EFS (or functional truncations thereof), SFFV, PGK, and CMV.
  • the promoter is SFFV or a viral LTR promoter.
  • “Operably linked to the same promoter” means that transcription of the polynucleotide sequences may be initiated from the same promoter and that the nucleotide sequences are positioned and oriented for transcription to be initiated from the promoter.
  • Polynucleotides operably linked to a promoter are under transcriptional regulation of the promoter.
  • a vector is a tool that allows or facilitates the transfer of an entity from one environment to another.
  • some vectors used in recombinant nucleic acid techniques allow entities, such as a segment of nucleic acid (e.g. a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell.
  • Vectors may be non-viral or viral.
  • vectors used in recombinant nucleic acid techniques include, but are not limited to, plasmids, mRNA molecules (e.g. in vitro transcribed mRNAs), chromosomes, artificial chromosomes and viruses.
  • the vector may also be, for example, a naked nucleic acid (e.g. DNA).
  • the vector may itself be a nucleotide sequence of interest.
  • the vectors used herein may be, for example, plasmid, mRNA or virus vectors and may include a promoter (as described above) for the expression of a nucleic acid molecule/polynucleotide and optionally a regulator of the promoter.
  • the vector is a viral vector, for example a retroviral, e.g. a lentiviral vector or a gamma retroviral vector.
  • the vectors may further comprise additional promoters, for example, in one embodiment, the promoter may be a LTR, for example, a retroviral LTR or a lentiviral LTR.
  • LTRs Long terminal repeats
  • LTRs are identical sequences of DNA that repeat hundreds or thousands of times found at either end of retrotransposons or proviral DNA formed by reverse transcription of retroviral RNA. They are used by viruses to insert their genetic material into the host genomes.
  • LTRs signals of gene expression are found in LTRs: enhancer, promoter (can have both transcriptional enhancers or regulatory elements), transcription initiation (such as capping), transcription terminator and polyadenylation signal.
  • the vector may include a 5’LTR and a 3’LTR.
  • the vector may comprise one or more additional regulatory sequences which may act pre- or post-transcriptionally.
  • Regulatory sequences are any sequences which facilitate expression of the polypeptides, e.g. act to increase expression of a transcript or to enhance mRNA stability. Suitable regulatory sequences include for example enhancer elements, post-transcriptional regulatory elements and polyadenylation sites.
  • the additional regulatory sequences may be present in the LTR(s).
  • the vector may comprise a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), e.g. operably linked to the promoter.
  • WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
  • Vectors comprising the present nucleic acid molecules/polynucleotides may be introduced into cells using a variety of techniques known in the art, such as transformation and transduction. Several techniques are known in the art, for example infection with recombinant viral vectors, such as retroviral, lentiviral, adenoviral, adeno-associated viral, baculoviral and herpes simplex viral vectors; direct injection of nucleic acids and biolistic transformation. Non-viral delivery systems include but are not limited to DNA transfection methods.
  • transfection includes a process using a non-viral vector to deliver a gene to a target cell.
  • Non-viral delivery systems can include liposomal or amphipathic cell penetrating peptides, preferably complexed with a nucleic acid molecule or construct.
  • Typical transfection methods include electroporation, DNA biolistics, lipid- mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated transfection, cationic facial amphiphiles (CFAs) (Nat. Biotechnol. (1996) 14: 556) and combinations thereof.
  • CFAs cationic facial amphiphiles
  • the present nucleic acid molecules may be designed to be used as single constructs which encode the chimeric protein and any other polypeptide (e.g. receptor or marker or other functional polypeptide or protein of interest) and this would be contained in a single vector, it is not precluded that they are introduced into a cell in conjunction with other vectors, for example encoding other polypeptides it may be desired also to introduce into the cell.
  • any other polypeptide e.g. receptor or marker or other functional polypeptide or protein of interest
  • derivative or variant in relation to the present proteins or polypeptides includes any substitution of, variation of, modification of, replacement of, deletion of and/or addition of one (or more) amino acid residues from or to the sequence providing that the resultant protein or polypeptide retains the desired function.
  • the desired function may be the ability of that domain to signal (e.g. activate or inactivate a downstream molecule, where the derivative or variant is a dimerization domain, the desired function is interaction with the inducer molecule, e.g. binding to rapamycin or an analogue thereof etc., or interaction directly with another cognate dimerization domain.
  • variants or derivatives referred to herein are functional variants or derivatives.
  • 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% function compared to the corresponding, reference sequence.
  • the variant or derivative may have a similar or the same level of function as compared to the corresponding, reference sequence or may have an increased level of function (e.g. increased by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%).
  • amino acid substitutions may be made, for example from 1, 2 or 3 to 10 or 20 substitutions provided that the modified sequence retains the required activity or ability.
  • Amino acid substitutions may include the use of non-naturally occurring analogues.
  • 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% activity or ability compared to the corresponding, reference sequence.
  • the variant or derivative may have a similar or the same level of activity or ability as compared to the corresponding, reference sequence or may have an increased level of activity or ability (e.g. increased by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%).
  • Proteins or peptides may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues as long as the endogenous function is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.
  • Conservative substitutions may be made, for example according to Table 1 below. Table 1 The derivative may be a homologue.
  • homologue as used herein means an entity having a certain homology with the wild type amino acid sequence and the wild type nucleotide sequence.
  • the term “homology” can be equated with “identity”.
  • a homologous or variant sequence may include an amino acid sequence which may be at least 70%, 75%, 85% or 90% identical, preferably at least 95%, 96%, 97%, 98% or 99% identical to the subject sequence. Typically, the variants will comprise the same active sites etc. as the subject amino acid sequence.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context herein it is preferred to express homology in terms of sequence identity.
  • 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 percentage homology or identity between two or more sequences. Percentage homology or sequence identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension. Calculation of maximum percentage homology/sequence identity therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties.
  • a suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al. (1984) Nucleic Acids Res.12: 387). Examples of other software that can perform 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.
  • 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 BLAST 2 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). 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 pair comparison.
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix – the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see the user manual for further details).
  • the public default values for the GCG package or in the case of other software, the default matrix, such as BLOSUM62.
  • the percentage identity is determined across the entirety of the reference and/or the query sequence. Once the software has produced an optimal alignment, it is possible to calculate percentage homology, preferably percentage sequence identity.
  • “Fragment” typically refers to a selected region of the polypeptide or polynucleotide that is of interest functionally, e.g. is functional or encodes a functional fragment. “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion (or part) of a full-length polypeptide or polynucleotide. Such variants, derivatives and fragments may be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5' and 3' flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made.
  • flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut.
  • the DNA is then expressed in accordance with the invention to make the encoded protein.
  • These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.
  • the chimeric protein may be co-expressed in or on a cell in conjunction with a CAR.
  • CAR chimeric antigen receptor
  • CARs are also known as artificial T-cell receptors, chimeric T-cell receptors or chimeric immunoreceptors.
  • a CAR typically comprises an extracellular domain comprising an antigen-specific targeting region, termed herein an antigen-binding domain, a transmembrane domain, and an endodomain comprising optionally one or more co-stimulatory domains, and an intracellular signaling domain.
  • the antigen-binding domain is typically joined to the transmembrane domain by a hinge domain.
  • the design of CARs, and the various domains that they may contain, is well known in the art. When the CAR binds its target antigen, this results in the transmission of an activating signal to the cell in which it is expressed.
  • the CAR directs the specificity of the engineered cells towards the target antigen, particularly towards cells expressing the targeted antigen.
  • the antigen-binding domain of a CAR may be derived or obtained from any protein or polypeptide which binds (i.e. has affinity for) a desired target antigen, or more generally a desired target molecule. This 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 commonly be expressed on the surface of a cell, for example a target cell, or a cell in the vicinity of a target cell (for a bystander effect), but need not be.
  • the CAR may recognise a soluble molecule, for example where the antigen-binding domain is based on, or derived from, a cellular receptor.
  • the antigen-binding domain is most commonly derived from antibody variable chains (for example it commonly takes the form of a scFv), but may also be generated from T-cell receptor variable domains or, as mentioned above, other molecules, such as receptors for ligands or other binding molecules.
  • the CAR is typically expressed as a polypeptide also comprising a signal sequence (also known as a leader sequence), and in particular a signal sequence which targets the CAR to the plasma membrane of the cell.
  • the extracellular domain, or ectodomain, of the CAR may thus comprise a signal sequence and an antigen-binding domain.
  • the antigen-binding domain provides the CAR with the ability to bind a predetermined antigen of interest.
  • the antigen-binding domain preferably targets an antigen of clinical interest or an antigen at a site of disease.
  • the antigen-binding domain may be any protein or peptide that possesses the ability to specifically recognize and bind to a biological molecule (e.g., a cell surface receptor or a component thereof).
  • the antigen-binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule of interest.
  • Illustrative antigen- specific targeting domains include antibodies or antibody fragments or derivatives, extracellular domains of receptors, ligands for cell surface molecules/receptors, or receptor binding domains thereof, and tumor binding proteins.
  • the antigen-specific targeting domain may preferably be an antibody or derived from an antibody, other antigen-specific targeting domains are encompassed, e.g. antigen-specific targeting domains formed from an antigenic peptide/MHC or HLA combination which is capable of binding to the TCRs of Tcon cells active at a site of transplantation, inflammation or disease.
  • the CAR may be directed towards any desired target antigen or molecule. This may be selected according to the intended therapy, and the condition it is desired to treat. It may for example be an antigen or molecule associated with a particular condition, or an antigen or molecule associated with a cell it is desired to target to treat the condition. Typically, the antigen or molecule is a cell-surface antigen or molecule.
  • the term “directed against” is synonymous with “specific for” or “anti”. Put another way, the CAR recognises a target molecule. Accordingly, it is meant that the CAR is capable of binding specifically to a specified or given antigen, or target.
  • the antigen-binding domain of the CAR is capable of binding specifically to the target molecule or antigen (more particularly when the CAR is expressed on the surface of a cell, notably an immune effector cell). Specific binding may be distinguished from non-specific binding to a non-target molecule or antigen.
  • a cell expressing the CAR is directed, or re-directed, to bind specifically to a target cell, expressing the target molecule or antigen, particularly a target cell expressing the target antigen or molecule on its cell surface.
  • Antigens which may be targeted by the present 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 disease).
  • the antigen may be simply present and/or expressed at the site of transplantation, inflammation or disease.
  • Antigens expressed on cells associated with neurodegenerative disease include those presented on glial cells, e.g. MOG.
  • Antigens associated with organ transplants and/or cells associated with transplanted organs include, but are not limited to, a HLA antigen present in the transplanted organ but not in the patient, or an antigen whose expression is up- regulated during transplant rejection such as CCL19, MMP9, SLC1A3, MMP7, HMMR, TOP2A, GPNMB, PLA2G7, CXCL9, FABP5, GBP2, CD74, CXCL10, UBD, CD27, CD48, CXCL11.
  • the CAR is directed against an HLA antigen, and in particular an HLA-A2 antigen.
  • Antibodies against such antigens are known in the art, and conveniently a scFv may be obtained or generated based on a known or available antibody.
  • VH and VL, and CDR sequences are publicly available to aid the preparation of such an antibody-binding domain, for example in WO 2020/044055, the disclosure of which is herein incorporated by reference. Any of the antigen binding domains, or CDR, VH, and/or VL sequences disclosed in WO 2020/044055 may be used.
  • the CAR may comprise an antigen binding domain which is capable of binding HLA-A2 (HLA-A2 may also be referred to herein as HLA-A*02, HLA-A02, and HLA-A*2).
  • HLA-A*02 is one particular class I major histocompatibility complex (MHC) allele group at the HLA-A locus.
  • the antigen recognition domain may bind, suitably specifically bind, one or more regions or epitopes within HLA-A2.
  • An epitope also known as antigenic determinant, is the part of an antigen that is recognised by an antigen recognition domain (e.g. an antibody).
  • an antigen recognition domain e.g. an antibody
  • the epitope is the specific piece of the antigen to which an antibody binds.
  • the antigen recognition domain binds, suitably specifically binds, to one region or epitope within HLA-A2.
  • Engineered cells may be generated by introducing a nucleic acid molecule, construct, or vector as defined herein, by one of many means including transduction with a viral vector, and transfection with DNA or RNA.
  • the present cell may be made by introducing to a cell (e.g. by transduction or transfection) the nucleic acid molecule, construct or vector as defined herein. Suitable cells are discussed further below, but the cell may be from a sample isolated from a subject.
  • the subject may be a donor subject, or a subject for therapy (i.e. the cell may be an autologous cell, or a donor cell, for introduction to another recipient, e.g. an allogeneic cell).
  • the cell may be generated by a method comprising the following steps: (i) isolation of a cell-containing sample from a subject or provision of a cell- containing sample; and (ii) introduction into (e.g. by transduction or transfection) the cell-containing sample of a nucleic acid molecule, construct, or vector as defined herein, to provide a population of engineered cells.
  • a cell into which a nucleic acid molecule, construct or vector is to be introduced may be referred to as a target cell.
  • a target cell-enriched sample may be isolated from, enriched, and/or generated from the cell-containing sample prior to and/or after step (ii) of the method.
  • isolation, enrichment and/or generation of Tregs may be performed prior to and/or after step (ii) to isolate, enrich or generate a Treg-enriched sample.
  • Isolation and/or enrichment from a cell-containing sample may be performed after step (ii) to enrich for cells and/or Tregs (or other target cells) comprising the CAR, the nucleic acid molecule/polynucleotide, the construct and/or the vector as described herein.
  • a Treg-enriched sample may be isolated or enriched by any method known to those of skill in the art, for example by FACS and/or magnetic bead sorting.
  • a Treg- enriched sample may be generated from the cell-containing sample by any method known to those of skill in the art, for example, from Tcon cells by introducing DNA or RNA coding for FOXP3 and/or from 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.
  • the target cell may be a Treg cell, or precursor or a progenitor therefor.
  • An “engineered cell” means a cell which has been modified to comprise or express a polynucleotide which is not naturally encoded by the cell. Methods for engineering cells are known in the art and include, but are not limited to, genetic modification of cells e.g.
  • transduction such as retroviral or lentiviral transduction, transfection (such as transient transfection – DNA or RNA based) including lipofection, polyethylene glycol, calcium phosphate and electroporation, as discussed above.
  • transduction such as retroviral or lentiviral transduction, transfection (such as transient transfection – DNA or RNA based) including lipofection, polyethylene glycol, calcium phosphate and electroporation, as discussed above.
  • Any suitable method may be used to introduce a nucleic acid sequence into a cell.
  • Non-viral technologies such as amphipathic cell penetrating peptides may be used to introduce nucleic acid.
  • the nucleic acid molecule as described herein is not naturally expressed by a corresponding, unmodified cell. Indeed, the nucleic acid molecule encoding the chimeric protein is an artificial construct, and could not occur or be expressed naturally.
  • an engineered cell is a cell which has been modified e.g.
  • an engineered cell is a cell which has been modified or whose genome has been modified e.g. by transduction or by transfection.
  • an engineered cell is a cell which has been modified or whose genome has been modified by retroviral transduction.
  • an engineered cell is a cell which has been modified or whose genome has been modified by lentiviral transduction.
  • the term “introduced” refers to methods for inserting foreign nucleic acid, e.g. DNA or RNA, into a cell.
  • the term introduced includes both transduction and transfection methods. Transfection is the process of introducing nucleic acids 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 a nucleic acid as described herein by one of many means including transduction with a viral vector, transfection with DNA or RNA.
  • Cells may be activated and/or expanded prior to, or after, the introduction of a nucleic acid as described herein, for example by treatment with an anti-CD3 monoclonal antibody or 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 be substituted with IL-15.
  • Other components which may be used in a cell e.g.
  • Treg) expansion protocol include, but are not limited to rapamycin, all-trans retinoic acid (ATRA) and TGF ⁇ .
  • activated means that a cell has been stimulated, causing the cell to proliferate.
  • expanded means that a cell or population of cells has been induced to proliferate. The expansion of a population of cells may be measured for example by counting the number of cells present in a population. The phenotype of the cells may be determined by methods known in the art such as flow cytometry.
  • the cell may be an immune cell, or a precursor therefor.
  • a precursor cell may be a progenitor cell.
  • T-cells include cytotoxic T-cells (CTLs; CD8+ T-cells), helper T-cells (HTLs; CD4+ T-cells) and regulatory T cells (Tregs).
  • CTLs cytotoxic T-cells
  • HTLs helper T-cells
  • Tregs regulatory T cells
  • Other populations of T-cells are also useful herein, for example naive T-cells and memory T-cells.
  • Other immune cells include NK cells, NKT cells, dendritic cells, MDSC, neutrophils, and macrophages.
  • Precursors of immune cells include pluripotent stem cells, e.g. induced PSC (iPSC), or more committed progenitors including multipotent stem cells, or cells which are committed to a lineage.
  • iPSC induced PSC
  • Precursor cells can be induced to differentiate into immune cells in vivo or in vitro.
  • a precursor cell may be a somatic cell which is capable of being transdifferentiated to an immune cell of interest.
  • the immune cell may be an NK cell, a dendritic cell, a MDSC, or a T cell, such as a cytotoxic T lymphocyte (CTL) or a Treg cell.
  • CTL cytotoxic T lymphocyte
  • the immune cell may be a Treg cell.
  • Regulatory T cells (Treg) or T regulatory cells” are immune cells with immunosuppressive function that control cytopathic immune responses and are essential for the maintenance of immunological tolerance.
  • Treg refers to a T cell with immunosuppressive function.
  • a T cell as used herein 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, or a Treg cell.
  • immunosuppressive function may refer to the ability of the Treg to reduce or inhibit one or more of a number of physiological and cellular effects facilitated by the immune system in response to a stimulus such as a pathogen, an alloantigen, or an autoantigen. Examples of such effects include increased proliferation of conventional T cell (Tcon) and secretion of proinflammatory cytokines. Any such effects may be used as indicators of the strength of an immune response.
  • a relatively weaker immune response by Tconv in the presence of Tregs would indicate an ability of the Treg to suppress immune responses. For example, a relative decrease in cytokine secretion would be indicative of a weaker immune response, and thus indicative of the ability of Tregs to suppress immune responses.
  • Tregs can also suppress immune responses by modulating the expression of co-stimulatory molecules on antigen presenting cells (APCs), such as B cells, dendritic cells and macrophages.
  • APCs antigen presenting cells
  • CD80 and CD86 can be used to assess suppression potency of activated Tregs in vitro after co-culture. Assays are known in the art for measuring indicators of immune response strength, and thereby the suppressive ability of Tregs.
  • antigen-specific Tconv cells may be co-cultured with Tregs, and a peptide of the corresponding antigen added to the co-culture to stimulate a response from the Tconv cells.
  • the degree of proliferation of the Tconv cells and/or the quantity of the cytokine IL-2 they secrete in response to addition of the peptide may be used as indicators of the suppressive abilities of the co-cultured Tregs.
  • Antigen-specific Tconv cells co-cultured with Tregs as referred to herein may proliferate 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95% or 99% less than the same Tconv cells cultured in the absence of the Tregs.
  • antigen-specific Tconv cells co-cultured with the present Tregs may proliferate 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95% or 99% less than the same Tconv cells cultured in the presence of non-engineered Tregs.
  • the cells comprising the nucleic acid, expression construct or vector as defined herein, e.g. Tregs may have an increased suppressive activity as compared to non-engineered Tregs (e.g. an increased suppressive activity of at least 5, 10, 20, 30, 40, 50, 60, 70, 80 or 90%).
  • Antigen-specific Tconv cells co-cultured with the Tregs herein may express at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% less effector cytokine than corresponding Tconv cells cultured in the absence of the Tregs (e.g. in the presence of non-engineered Tregs).
  • the effector cytokine may be selected from IL-2, IL-17, TNF ⁇ , GM-CSF, IFN- ⁇ , IL-4, IL-5, IL-9, IL-10 and IL-13.
  • the effector cytokine may be selected from IL-2, IL-17, TNF ⁇ , GM-CSF and IFN- ⁇ .
  • Tregs generally are T cells which express the markers CD4, CD25 and FOXP3 (CD4 + CD25 + FOXP3 + ). Tregs may also express CTLA-4 (cytotoxic T-lymphocyte associated molecule-4) or GITR (glucocorticoid-induced TNF receptor). Treg cells are present in the peripheral blood, lymph nodes, and tissues and Tregs for use herein include thymus-derived, natural Treg (nTreg) cells, peripherally generated Tregs, and induced Treg (iTreg) cells.
  • CTLA-4 cytotoxic T-lymphocyte associated molecule-4
  • GITR glucocorticoid-induced TNF receptor
  • a Treg may be identified using the cell surface markers CD4 and CD25 in the absence of or in combination with low-level expression of the surface protein CD127 (CD4 + CD25 + CD127 ⁇ or CD4 + CD25 + CD127 low ).
  • the use of such markers to identify Tregs is known in the art and described in Liu et al. (JEM; 2006; 203; 7(10); 1701- 1711), for example.
  • a Treg may be a CD4 + CD25 + FOXP3 + T cell, a CD4 + CD25 + CD127 ⁇ T cell, or a CD4 + CD25 + FOXP3 + CD127 ⁇ /low T cell.
  • the Treg may be a natural Treg (nTreg).
  • Natural T reg means a thymus-derived Treg.
  • Natural T regs are CD4 + CD25 + FOXP3 + Helios + Neuropilin 1 + .
  • nTregs have higher expression of PD-1 (programmed cell death-1, pdcd1), Neuropilin 1 (Nrp1), Helios (Ikzf2), and CD73.
  • nTregs may be distinguished from iTregs on the basis of the expression of Helios protein or Neuropilin 1 (Nrp1) individually.
  • the Treg may have a demethylated Treg-specific demethylated region (TSDR).
  • TSDR demethylated Treg-specific demethylated region
  • Tregs include, but are not limited to, Tr1 cells (which do not express FOXP3, and have high IL-10 production); CD8 + FOXP3 + T cells; and ⁇ FOXP3 + T cells.
  • Tr1 cells which do not express FOXP3, and have high IL-10 production
  • CD8 + FOXP3 + T cells CD8 + FOXP3 + T cells
  • ⁇ FOXP3 + T cells Different subpopulations of Tregs are known to exist, including na ⁇ ve Tregs (CD45RA + FOXP3 low ), effector/memory Tregs (CD45RA-FOXP3 high ) and cytokine- producing Tregs (CD45RA-FOXP3 low ).
  • “Memory Tregs” are Tregs which express CD45RO and which are considered to be CD45RO + . These cells have increased levels of CD45RO as compared to na ⁇ ve Tregs (e.g. at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% more CD45RO) and which preferably do not express or have low levels of CD45RA (mRNA and/or protein) as compared to na ⁇ ve Tregs (e.g. at least 80, 90 or 95% less CD45RA as compared to na ⁇ ve Tregs).
  • na ⁇ ve Tregs e.g. at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% more CD45RO
  • CD45RA mRNA and/or protein
  • Cytokine-producing Tregs are Tregs which do not express or have very low levels of CD45RA (mRNA and/or protein) as compared to na ⁇ ve Tregs (e.g. at least 80, 90 or 95% less CD45RA as compared to na ⁇ ve Tregs), and which have low levels of FOXP3 as compared to Memory Tregs, e.g. less than 50, 60, 70, 80 or 90% of the FOXP3 as compared to Memory Tregs. Cytokine-producing Tregs may produce interferon gamma and may be less suppressive in vitro as compared to na ⁇ ve Tregs (e.g.
  • Reference to expression levels herein may refer to mRNA or protein expression.
  • expression may refer to cell surface expression, i.e. the amount or relative amount of a marker protein that is expressed on the cell surface.
  • Expression levels may be determined by any known method of the art. For example, mRNA expression levels may be determined by Northern blotting/array analysis, and protein expression may be determined by Western blotting, or preferably by FACS using antibody staining for cell surface expression.
  • the Treg may be a na ⁇ ve Treg.
  • a na ⁇ ve regulatory T cell, a na ⁇ ve T regulatory cell, or a na ⁇ ve Treg refers to a Treg cell which expresses CD45RA (particularly which expresses CD45RA on the cell surface). Na ⁇ ve Tregs are thus described as CD45RA + . Na ⁇ ve Tregs generally represent Tregs which have not been activated through their endogenous TCRs by peptide/MHC, whereas effector/memory Tregs relate to Tregs which have been activated by stimulation through their endogenous TCRs.
  • a na ⁇ ve Treg may express at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% more CD45RA than a Treg cell which is not na ⁇ ve (e.g. a memory Treg cell).
  • a na ⁇ ve Treg cell may express at least 2, 3, 4, 5, 10, 50 or 100-fold the amount of CD45RA as compared to a non-na ⁇ ve Treg cell (e.g. a memory Treg cell).
  • the level of expression of CD45RA can be readily determined by methods of the art, e.g. by flow cytometry using commercially available antibodies.
  • non-na ⁇ ve Treg cells do not express CD45RA or low levels of CD45RA.
  • na ⁇ ve Tregs may not express CD45RO, and may be considered to be CD45RO-.
  • na ⁇ ve Tregs may express at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% less CD45RO as compared to a memory Treg, or alternatively viewed at least 2, 3, 4, 5, 10, 50 or 100 fold less CD45RO than a memory Treg cell.
  • na ⁇ ve Tregs express CD25 as discussed above, CD25 expression levels may be lower than expression levels in memory Tregs, depending on the origin of the na ⁇ ve Tregs.
  • na ⁇ ve Tregs isolated from peripheral blood expression levels of CD25 may be at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% lower than memory Tregs. Such na ⁇ ve Tregs may be considered to express intermediate to low levels of CD25. However, a skilled person will appreciate that na ⁇ ve Tregs isolated from cord blood may not show this difference.
  • a na ⁇ ve Treg as defined herein may be CD4 + , CD25 + , FOXP3 + , CD127 low , CD45RA + .
  • Low expression of CD127 as used herein refers to a lower level of expression of CD127 as compared to a CD4 + non-regulatory or Tcon cell from the same subject or donor.
  • na ⁇ ve Tregs may express less than 90, 80, 70, 60, 50, 40, 30, 20 or 10% CD127 as compared to a CD4 + non-regulatory or Tcon cell from the same subject or donor. Levels of CD127 can be assessed by methods standard in the art, including by flow cytometry of cells stained with an anti-CD127 antibody.
  • na ⁇ ve Tregs do not express, or express low levels of CCR4, HLA- DR, CXCR3 and/or CCR6.
  • na ⁇ ve Tregs may express lower levels of CCR4, HLA-DR, CXCR3 and CCR6 than memory Tregs, e.g.
  • Na ⁇ ve Tregs may further express additional markers, including CCR7 + and CD31 + Isolated na ⁇ ve Tregs may be identified by methods known in the art, including by determining the presence or absence of a panel of any one or more of the markers discussed above, on the cell surface of the isolated cells. For example, CD45RA, CD4, CD25 and CD127 low can be used to determine whether a cell is a na ⁇ ve Treg.
  • the cell such as a Treg
  • PBMCs peripheral blood mononuclear cells
  • the subject from whom the PBMCs are obtained is a mammal, preferably a human.
  • the cell is matched (e.g. HLA matched) or is autologous to the subject to whom the engineered cell is to be administered.
  • the subject to be treated is a mammal, particularly a human.
  • the cell may be generated ex vivo either from a patient’s own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2nd party), or peripheral blood from an unconnected donor (3rd party).
  • the cell is autologous to the subject to whom the engineered cell is to be administered.
  • the Treg is part of a population of cells.
  • the population of Tregs comprises 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”.
  • the Treg may be derived from ex-vivo differentiation of inducible progenitor cells (e.g. iPSCs) or embryonic progenitor cells to the Treg.
  • inducible progenitor cells e.g. iPSCs
  • a nucleic acid molecule, construct or vector as described herein may be introduced into the inducible progenitor cells or embryonic progenitor cells prior to, or after, differentiation to a Treg. Suitable methods for differentiation are known in the art and include that disclosed in Haque et al, J Vis Exp., 2016, 117, 54720 (incorporated herein by reference).
  • the term “conventional T cell” or Tcon or Tconv means a T lymphocyte cell which expresses an ⁇ T cell receptor (TCR) as well as a co-receptor which may be cluster of differentiation 4 (CD4) or cluster of differentiation 8 (CD8) and which does not have an immunosuppressive function.
  • TCR ⁇ T cell receptor
  • CD4 cluster of differentiation 4
  • CD8 cluster of differentiation 8
  • Conventional T cells are present in the peripheral blood, lymph nodes, and tissues.
  • the engineered Treg may be generated from a Tcon by introducing the nucleic acid which includes a sequence coding for FOXP3.
  • the engineered Treg may be generated from a Tcon by in vitro culture of CD4+ CD25-FOXP3- cells in the presence of IL-2 and TGF- ⁇ .
  • a Treg herein may have increased persistence as compared to a Treg cell without the chimeric protein for a constitutively active chimeric protein or without the inducer for an inducible chimeric protein (e.g. a Treg cell transduced with the inducible chimeric protein in the absence of the inducer).
  • “Persistence” as used 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).
  • a Treg as disclosed herein may have at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% increased persistence as compared to a Treg which does not express the chimeric protein herein, or a Treg which is not induced.
  • Persistence can be measured by for example, determining the amount or numbers of administered cells within a subject or patient over time, where cells expressing a chimeric protein of the invention are compared to equivalent cell types which do not express the chimeric protein, or compared to non-engineered cells. It is possible to track administered cells, for example, using a marker protein, e.g. CD34 for cells which also express a RQR8 safety switch.
  • a marker protein e.g. CD34 for cells which also express a RQR8 safety switch.
  • the target cell into which the nucleic acid molecule, construct or vector is introduced is not a cell intended for therapy.
  • the cell is a production host cell.
  • the cell may be for production of the nucleic acid, e.g. cloning, or vector, or polypeptides.
  • a cell population comprising a cell as defined or described herein. It will be appreciated that a cell population may comprise both the present cells comprising a nucleic acid molecule, construct or vector as defined herein, and cells which do not comprise the nucleic acid molecule, construct or vector, e.g. untransduced or untransfected cells.
  • all the cells in a population may comprise the nucleic acid, expression construct or vector
  • cell populations having at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 99% of cells comprising a nucleic acid, expression construct or vector are provided.
  • the chimeric protein may be co-expressed in or on a cell in conjunction with a mutant calcineurin which is resistant to at least one calcineurin inhibitor (CNI).
  • the mutant calcineurin may be resistant to one calcineurin inhibitor and sensitive to another calcineurin inhibitor.
  • Such calcineurin mutants are described in Brewin et al., 2009, Blood, 114(23), 4792-4802, incorporated herein by reference.
  • Calcineurin inhibitors notably FK506 (tacrolimus) and cyclosporine (CsA) are immunosuppressants commonly used in ACT.
  • FK506 tacrolimus
  • CsA cyclosporine
  • the cell may be rendered resistant to the immunosuppressant.
  • co-expression may advantageously allow the ACT subject (i.e. cell recipient) to continue immunosuppressant therapy, and may allow lymphodepletion of the recipient to be avoided. Thus, it may not be necessary to interrupt or stop administering an immunosuppressant to the subject.
  • co-expressing such a mutant in the cell along with the chimeric protein (and any other protein of interest, such as a CAR), may allow for selective expansion of transduced (or transfected etc.) cells.
  • the transduced or transfected cell population that is the cells resulting from the transduction/transfection protocol etc.
  • the calcineurin inhibitor to which the cells have been rendered resistant by virtue of the mutant calcineurin. Any non-transduced/non-transfected cells which are undesired in the final cell preparation for ACT will not be able to grow (proliferate) in the presence of the calcineurin inhibitor and will not be expanded.
  • mutant calcineurin is resistant to a particular inhibitor, but sensitive to another, then that provides a safety mechanism by which the cell containing the mutant calcineurin may be controlled or eliminated – by administering the inhibitor to which the mutant calcineurin is sensitive.
  • mutant calcineurin proteins are known in the art.
  • the mutant calcineurin may be resistant to FK506 but sensitive to CsA, resistant to CsA but sensitive to FK506, or resistant to both FK506 and CsA. Such mutants are described in Brewin et al., 2009 (supra).
  • the mutant calcineurin may comprise mutations in the A subunit (CNa) (SEQ ID NO: 97) or the B subunit (CNb) (SEQ ID NO: 98) of calcineurin.
  • Mutations to the A subunit may comprise a mutation at one or more of the following positions with reference to SEQ ID NO: 97; V314, Y341, M347, T351, W352, S353, L354, F356 and K360.
  • a mutated A subunit may comprise one or more of the following mutations with reference to SEQ ID NO: 97: V314K, V314R or V314F; Y341F; M347W, M347R or M347E; T351E; W352A, W352C or W352E; S353H or S353N; L354A; F356A; and/or K360A or K360F.
  • Combinations of mutations that may be made to the A subunit include with respect to SEQ ID NO: 97: L354A and K360A; L354A and K360F; T351E and K360F; W352A and S353H; T351E and L354A; W353C and K360F; W352C, L354A and K360F; V314K and Y341F; and V314R and Y341F.
  • Mutations to the B subunit may be at one or more of the following positions with reference to SEQ ID NO: 98; Q51, L116, M119, V120, G121, N122, N123, L124, K125 and K165.
  • a mutated B subunit may comprise one or more of the following mutations with reference to SEQ ID NO: 98: Q51S; L116R or L116Y; M119A, M119W or M119-F-Ins; V120L, V120S, V120D or V120F; G121-LF-Ins; N122A, N122H, N122F, or N122S; N123H, N123R, N123F, N123K, or N123W; L124T; K125A, K125E, K125W, K125-LA-Ins, K125-VQ-Ins or K125-IE-Ins; and/or K165Q.
  • Combinations of mutations that may be made to the B subunit include with respect to SEQ ID NO: 98: V120S and L124T; V120D and L124T; N123W and K125-LA-Ins; L124T and K125-LA-Ins; V120D and K125-LA-Ins; and M119-F-Ins and G121-LF-Ins
  • SEQ ID NO: 98 V120S and L124T; V120D and L124T; N123W and K125-LA-Ins; L124T and K125-LA-Ins; V120D and K125-LA-Ins; and M119-F-Ins and G121-LF-Ins
  • mutants (i) CNa12 which is resistant to FK506, but sensitive to CsA. This mutant comprises mutations T351E and L354A.
  • the sequence of CNa12 is shown in SEQ ID NO: 94; (ii) CNa18, CNa19, CNa20, CNa21 and CNa22 and CNb26 which are resistant to CsA, but sensitive to FK506. These mutants comprise, respectively, mutations M347E; V314K; V314R; Y3F, V314K and Y341F; V314R and Y341F; and V120S and L124T.
  • the sequence of CNa22 is shown in SEQ ID NO: 95; (iii) CNb21, CNb23, and CNb30 which are resistant to both CsA and FK506.
  • mutants comprise, respectively, mutations K125-VQ- Ins; K125-LA-Ins; and L124T and K125-LA-Ins.
  • the sequence of CNb30 is shown in SEQ ID NO: 96.
  • a mutant calcineurin for use herein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 94, 95 or 96 or an amino acid sequence which has at least 85, 90, 95, 97, 98 or 99% sequence identity to SEQ ID NOs: 94, 95 or 96, provided that the mutations indicated above for SEQ NOs: 94, 95 or 96 are retained.
  • Other mutants are described in Brewin (supra) and may also be used.
  • a mutant which is resistant to a given CNI means a mutant which exhibits resistance to the CNI, that is a mutant which is able to confer on a cell expressing it the ability to grow, or proliferate, or to exhibit a functional property in its presence.
  • a cell comprising the mutant may exhibit increased growth, which may be reflected in an increase in a functional property, for example increased IL-2 secretion in Jurkat cells expressing the mutant, compared to a cell which does not comprise the mutant. It is not required that a mutant exhibits 100% resistance to a given CNI. The levels of resistance may vary, and some degree of susceptibility may be tolerated, as long as there is an observable increase in growth or functionality of cells expressing the mutant in the presence of the CNI in question, e.g. as compared to cells without (not expressing) the mutant. For example, the mutant may exhibit at least 35, 40 or 45% resistance, more particularly at least 50, 55, 60 or 65% resistance.
  • a particular type of cell of interest is a Treg cell.
  • Treg cells are administered in ACT to suppress undesired immune response or immune activity.
  • the expression of mutant calcineurins in Treg cells is of particular interest.
  • a Treg cell which comprises a mutant calcineurin which is resistant to at least one calcineurin inhibitor.
  • the mutant calcineurin is resistant to at least one calcineurin inhibitor and sensitive to at least one calcineurin inhibitor.
  • the mutant is (i) resistant to FK506 and sensitive to CsA or (ii) resistant to CsA and sensitive to FK506.
  • the mutant may be any one of the mutants discussed above.
  • the Treg cell may be engineered or modified to comprise the mutant calcineurin by introducing a nucleic acid, construct or vector comprising a nucleotide sequence encoding the mutant calcineurin.
  • a Treg cell may further comprise or express another protein of interest. This may be, for example, a chimeric protein as described herein and/or a CAR or other chimeric receptor, a TCR, or any other polypeptide as referred to or described herein.
  • a cell such as a Tcell, e.g., a Treg cell, may further comprise or express a chimeric protein which is capable of providing a STAT5 mediated signal to the cell.
  • Such a chimeric protein may be one as described herein, or may be an alternative molecule, such as one described in WO2020/044055, which is incorporated by reference. Particularly, such a chimeric protein may comprise a JAK1- and/or JAK2-binding domain and a STAT5 association motif, as described in detail above.
  • a cell e.g., a Treg cell
  • a cell which comprises a nucleic acid molecule comprising a nucleotide sequence encoding a mutated calcineurin and a nucleic acid molecule comprising a nucleotide sequence encoding a chimeric protein comprising a JAK1- and/or JAK2-binding domain and a STAT5 association motif, particularly wherein said chimeric protein is capable of dimerization.
  • the JAK1- and/or JAK2-binding domain and STAT5 association motif may be present within the endodomain of a chimeric antigen receptor, as described in WO2020/044055.
  • the chimeric protein should be capable of providing a STAT5 mediated signal to the cell (e.g. upon dimerization which may be constitutively provided for or inducible, e.g. by binding of the chimeric protein to a target or to a ligand or CID as discussed herein).
  • the cell may also in this embodiment comprise a CAR as defined herein.
  • the mutant calcineurins may be used in Treg cells which have been, are being, or will be engineered for another purpose, e.g. to express another desired heterologous (i.e. non-native) protein.
  • Another aspect provided herein is a method for selectively expanding engineered Treg cells, said method comprising introducing into a Treg cell a nucleic acid molecule, construct or vector comprising a nucleotide sequence encoding a mutant calcineurin as defined herein, and culturing said cells in the presence of a calcineurin inhibitor to which the mutant calcineurin is resistant.
  • the cell may further comprise a chimeric receptor comprising a JAK1- and/or JAK2-binding domain and a STAT5 association motif.
  • the engineered Treg cell may comprise a further nucleotide sequence which is, or has been, introduced into the cell, and which encodes a protein of interest.
  • the culturing step may comprise any known or desired protocol for activating and/or expanding cells.
  • the cells may be grown in the presence of CD3 and/or CD28 as described above.
  • a pharmaceutical composition comprising a cell or cell population as defined or described herein, or a vector as defined herein.
  • the vector may be used for gene therapy.
  • a vector may be administered instead, 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.
  • a pharmaceutical composition is a composition that comprises or consists of a therapeutically effective amount of a pharmaceutically active agent i.e. the cell (e.g. Treg), cell population or vector.
  • compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilising agent(s).
  • the formulation is sterile and pyrogen free.
  • the carrier, diluent, and/or excipient must be “acceptable” in the sense of being compatible with the cell or vector and not deleterious to the recipients thereof.
  • the carriers, diluents, and excipients will be saline or infusion media which will be sterile and pyrogen free, however, other acceptable carriers, diluents, and excipients may be used.
  • Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl- cellulose, polyvinylpyrrolidone, and the like.
  • the cells, cell population or pharmaceutical compositions may be administered in a manner appropriate for treating and/or preventing the desired disease or condition.
  • the quantity and frequency of administration will be determined by such factors as the condition of the subject, and the type and severity of the subject's disease or condition, although appropriate dosages may be determined by clinical trials.
  • the pharmaceutical composition may be formulated accordingly.
  • the cell, cell population or pharmaceutical composition as described herein can be administered parenterally, for example, intravenously, or they may be administered by infusion techniques.
  • the cell, cell population or pharmaceutical composition may be administered in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the aqueous solution may be suitably buffered (preferably to a pH of from 3 to 9).
  • the pharmaceutical composition may be formulated accordingly.
  • the preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
  • the pharmaceutical compositions may comprise cells in infusion media, for example sterile isotonic solution.
  • the pharmaceutical composition may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • the cell, cell population or pharmaceutical composition may be administered in a single or in multiple doses. Particularly, the cell, cell population or pharmaceutical composition may be administered in a single, one off dose.
  • the pharmaceutical composition may be formulated accordingly.
  • 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 lympho-depletive agents (e.g.
  • thymoglobulin campath- 1H
  • anti-CD2 antibodies anti-CD3 antibodies
  • anti-CD20 antibodies cyclophosphamide, fludarabine
  • inhibitors of mTOR e.g. sirolimus, everolimus
  • drugs inhibiting costimulatory pathways e.g. anti-CD40/CD40L, CTAL4Ig
  • drugs inhibiting specific cytokines IL-6, IL-17, TNFalpha, IL18.
  • the cell, cell population or pharmaceutical composition may be administered at varying doses (e.g. measured in cells/kg or cells/subject).
  • kits, or combination products, comprising the cell, cell population and/or pharmaceutical composition herein.
  • said kits are for use in the methods and uses as described herein, e.g., the therapeutic methods as described herein.
  • kits comprise instructions for use of the kit components.
  • Kits or compositions may further comprise the inducer, e.g. rapamycin or an analogue thereof.
  • the cells, cell populations, compositions and vectors herein may be for use therapy, that is in treating or preventing a disease or condition.
  • the cell in or on which the chimeric protein is expressed is typically a cell which is modified, or engineered to express a further molecule (e.g. a further protein), notably a receptor, e.g. a CAR or TCR.
  • the therapy may be for the prevention or treatment of a disease or condition which may be treated by or with a cell expressing the receptor, e.g. the CAR.
  • the cells and compositions containing them are for adoptive cell therapy (ACT).
  • ACT adoptive cell therapy
  • Various conditions may be treated by administration of cells, including particularly Treg cells, expressing a CAR according to the present disclosure. As noted above, this may be conditions responsive to immunosuppression, and particularly the immunosuppressive effects of Treg cells.
  • the cells, cell populations, compositions and vectors described herein may thus be used for inducing, or achieving, immunosuppression in a subject.
  • the Treg cells administered, or modified in vivo may be targeted by expression of the receptor, e.g. CAR.
  • Conditions suitable for such treatment include infectious, neurodegenerative or inflammatory disease, or more broadly a condition associated with any undesired or unwanted or deleterious immune response.
  • Conditions to be treated or prevented include inflammation, or alternatively put, a condition associated with or involving inflammation.
  • Inflammation may be chronic or acute.
  • the inflammation may be low-level or systemic inflammation.
  • the inflammation may be inflammation which occurs in the context of a metabolic disorder, for example metabolic syndrome, or in the context of insulin resistance, or type II diabetes or obesity and such like.
  • the cells, cell populations, vectors and pharmaceutical compositions provide a means for inducing tolerance to a transplant; treating and/or preventing cellular and/or humoral transplant rejection; treating and/or preventing graft-versus-host disease (GvHD), an autoimmune or allergic disease; or to promote tissue repair and/or tissue regeneration; or to ameliorate inflammation.
  • GvHD graft-versus-host disease
  • the cells, cell populations, vectors and pharmaceutical compositions may be used in a method which comprises the step of administering a cell, cell populations, vector or a pharmaceutical composition as described herein to a subject.
  • inducing tolerance to a transplant refers to inducing tolerance to a transplanted organ in a recipient.
  • inducing tolerance to a transplant means to reduce the level of a recipient’s immune response to a donor transplant organ.
  • Inducing tolerance to a transplanted organ may reduce the amount of immunosuppressive drugs that a transplant recipient requires, or may enable the discontinuation of immunosuppressive drugs.
  • the engineered cells e.g.
  • Tregs may be administered to a subject with a disease in order to lessen, reduce, or improve at least one symptom of disease such as jaundice, dark urine, itching, abdominal swelling or tenderness, fatigue, nausea or vomiting, and/or loss of appetite.
  • the at least one symptom may be lessened, reduced, or improved 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.
  • the engineered cells, e.g. Tregs may be administered to a subject with a disease in order to slow down, reduce, or block the progression of the disease.
  • the progression of the disease may be slowed down, reduced, or blocked by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a subject in which the engineered cells are not administered, or progression of the disease may be completely stopped.
  • the subject is a transplant recipient undergoing immunosuppression therapy.
  • the subject is a mammal.
  • the subject is a human.
  • the transplant may be selected from a liver, kidney, heart, lung, pancreas, intestine, stomach, bone marrow, vascularized composite tissue graft, and skin transplant.
  • the cells may express a CAR which comprises an antigen binding domain which is capable of specifically binding to a HLA antigen that is present in the graft (transplant) donor but not in the graft (transplant) recipient.
  • the transplant is a liver transplant.
  • the antigen may be a HLA antigen present in the transplanted liver but not in the patient, a liver-specific antigen such as NTCP, or an antigen whose expression is up-regulated during rejection such as CCL19, MMP9, SLC1A3, MMP7, HMMR, TOP2A, GPNMB, PLA2G7, CXCL9, FABP5, GBP2, CD74, CXCL10, UBD, CD27, CD48, CXCL11.
  • the antigen is HLA-A2.
  • a method for treating a disease or condition relates to the therapeutic use of the cells herein.
  • the cells may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease or condition and/or to slow down, reduce or block the progression of the disease.
  • treating and/or preventing cellular and/or humoral transplant rejection may refer to administering an effective amount of the cells (e.g. Tregs) such that the amount of immunosuppressive drugs that a transplant recipient requires is reduced, or may enable the discontinuation of immunosuppressive drugs.
  • Preventing a disease or condition relates to the prophylactic use of the cells herein.
  • the cells may be administered to a subject who has not yet contracted or developed the disease or condition and/or who is not showing any symptoms of the disease or condition to prevent the disease or condition or to reduce or prevent development of at least one symptom associated with the disease or condition.
  • the subject may have a predisposition for, or be thought to be at risk of developing, the disease or condition.
  • the autoimmune or allergic disease may be selected from inflammatory skin diseases including psoriasis and dermatitis (e.g.
  • atopic dermatitis responses associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); dermatitis; allergic conditions such as food allergy, eczema and asthma; rheumatoid arthritis; systemic lupus erythematosus (SLE) (including lupus nephritis, cutaneous lupus); diabetes mellitus (e.g. type 1 diabetes mellitus or insulin dependent diabetes mellitus); multiple sclerosis; neurodegenerative disease, for example, Amyotrophic Lateral Sclerosis (ALS); Chronic inflammatory demyelinating polyneuropathy (CIPD) and juvenile onset diabetes.
  • SLE systemic lupus erythematosus
  • diabetes mellitus e.g. type 1 diabetes mellitus or insulin dependent diabetes mellitus
  • multiple sclerosis neurodegenerative disease, for example, Amyotrophic Lateral Sclerosis (ALS); Chronic inflammatory demyelinating polyneuropathy (CIPD
  • the chimeric protein is not limited to use in the context of immunosuppressive therapy, and the protein may be expressed in cells for the treatment of conditions such as cancer or infections. It may be desirable in such contexts to kill or ablate cancer or infected cells, and in such cases the chimeric protein may be expressed in cytotoxic cells, such as cytotoxic T cells or NK cells, or precursors therefor.
  • the receptor e.g. CAR or TCR
  • co-expressed with the chimeric protein in such cases may be directed against a cancer antigen or an antigen from a pathogen etc.
  • the medical use of or method herein may involve the steps of: (i) isolating a cell-containing sample or providing a cell-containing sample; (ii) introducing a nucleic acid molecule, construct or a vector as defined herein to the cell; and (iii) administering the cells from (ii) to a subject.
  • the cell may be a Treg as defined herein.
  • An enriched Treg population may be isolated and/or generated from the cell containing sample prior to, and/or after, step (ii) of the method. For example, isolation and/or generation may be performed prior to 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 chimeric protein, the nucleic acid molecule, construct, and/or the vector as described herein.
  • the cell may be autologous.
  • the cell may be allogeneic.
  • the cell e.g. the engineered Treg
  • the engineered cell e.g. Treg
  • Tregs may be activated and/or expanded prior to, or after, the introduction of a nucleic acid molecule as described herein, for example by treatment with an anti-CD3 monoclonal antibody or both anti-CD3 and anti-CD28 monoclonal antibodies. Expansion protocols are discussed above.
  • the cell e.g. Tregs, may be washed after each step of the method, in particular after expansion.
  • the population of engineered cells, e.g., Treg cells may be further enriched by any method known to those of skill in the art, for example by FACS or magnetic bead sorting.
  • the steps of the method of production may be performed in a closed and sterile cell culture system.
  • the invention may also provide a method for increasing the stability and/or suppressive function of a cell comprising the step of introducing a nucleic acid molecule, an expression construct or vector as provided herein into the cell.
  • An increase in suppressive function can be measured as discussed above, for example by co-culturing activated antigen-specific Tconv cells with cells of the invention, and for example measuring the levels the cytokines produced by the Tconv cells.
  • An increase in suppressive function may be an increase of at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% as compared to a non-engineered or non-induced Treg.
  • An increase in stability of a cell e.g.
  • a Treg refers to an increase in the persistence or survival of those cells or to an increase in the proportion of cells retaining a Treg phenotype over a time period (e.g. to cells retaining Treg markers such as FOXP3 and Helios) as compared to a non- engineered or non-induced Treg.
  • An increase in stability may be an increase in stability of at least 10, 20, 30, 40, 50, 60, 70, 80 or 90%, and may be measured by techniques known in the art, e.g. staining of Treg cell markers within a population of cells, and analysis by FACS.
  • a further aspect provided herein is a combination product comprising (a) a cell, cell population, vector or pharmaceutical composition as defined herein, and (b) an inducer molecule, for use in therapy, particularly ACT or gene therapy.
  • the therapy may be any therapy as defined above, and further described herein.
  • the components (a) and (b) of the combination product may be for separate, sequential or simultaneous use.
  • the components (a) and (b) of the combination product will typically be provided as separate compositions, i.e. they will be formulated separately.
  • the combination product may alternatively be defined or referred to as a kit.
  • the components (a) and (b) will be administered to the subject separately. They may be administered to a subject at the same time, or at different times, for example at spaced apart time intervals.
  • the cells etc. may be administered first, followed by the CID.
  • the CID may be administered after an appropriate time interval, or shortly after the cells etc.
  • the skilled clinician would readily be able to devise an appropriate administration regime according to principles known in the art.
  • This disclosure is not limited by the exemplary methods and materials disclosed herein, and any 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 indicated, any 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, respectively.
  • Example 1 preparation, expression and testing of chimeric proteins Materials and Methods Cloning: A representative cytoplasmic construct was designed in-house and whole sequences were codon optimized for expression in human cells. This construct was cloned into a Lentiviral backbone (pcLV) which included the GFP reporter gene, and also into a Lentiviral backbone (pcLV) which contains a GFP reporter gene as well as an A2CAR. Two constructs were therefore generated for testing (plus and minus A2CAR), as shown in Figure 5. These constructs were designated pQTX-0340 (without CAR) and pQTX-0370 (with CAR). These constructs were transformed into Stble3 bacteria with the selection agent Kanamycin.
  • pcLV Lentiviral backbone
  • pcLV Lentiviral backbone
  • HEK293T/17 cells were seeded and cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) + 10% FBS (Fetal Bovine Serum) for 24 hours. Fugene HD (Promega) transfection reagent was brought to room temperature and mixed with the construct DNA, packaging plasmid (pD8.91) and envelope plasmid (pVSV-G).
  • CD45RA + human Tregs were isolated from healthy donor blood. Tregs were transduced with various constructs using the required multiplicity of infection (MOI). Non-tissue culture treated well plates were coated with RetroNectin® (Takara Bio, Shiga, Japan) prior to addition of viral particles and Tregs followed by spinoculation.
  • Tregs were expanded using DynabeadsTM Human T-Activator CD3/CD28 for T Cell Expansion and Activation (ThermoFisher Scientific, Massachusetts, USA), X-VIVOTM 15 media (Lonza, Basel, Switzerland) supplemented with 5% heat-inactivated human serum (Merck Life Science UK Limited, Dorset, UK) and interleukin-2 (IL-2; Proleukin®, Clinigen, Burton upon Trent, UK). IL-2 was supplemented at 300 international units (IU) per 1 ml of culture volume every 2 days. The protocol is illustrated in Figure 6. Flow cytometry staining to determine transduction efficiency: Once Tregs had sufficiently expanded, cells were taken for staining to check phenotype and transduction efficiency.
  • FACS buffer phosphate buffer saline (PBS) with 2% fetal bovine serum (FBS) and 2mM EDTA). Cells were washed in PBS before LIVE/DEADTM Fixable Near-IR (ThermoFisher Scientific) was used to stain the dead cells for 15 mins at RT. Cells were washed in FACS buffer before Fc blocking with Human TruStain FcXTM (Biolegend) and stained with cell surface antibodies: Brilliant Violet 510TM anti-human CD4 (A161A1; Biolegend, California, USA), PE/Cyanine7 anti-human CD25 (BC96; Biolegend).
  • FACS buffer phosphate buffer saline
  • FBS 2% fetal bovine serum
  • 2mM EDTA 2mM EDTA
  • Washed cells were then fixed and permeabilized using the eBioscienceTM FoxP3/Transcription Factor Staining Buffer Set before addition of Alexa Fluor® 488 anti-GFP (FM264G; Biolegend) and PE anti-human FoxP3 antibodies (206D; Biolegend). Cells were acquired by flow cytometry and data was analysed on FlowJo_V10 software.
  • pSTAT5 assay set up for human Tregs Setup The protocol is shown in Figure 6. Tregs were rested for 24-36 hours prior to setting up the assay. This assay was setup in U bottom 96 well plates with 150,000-200,000 cell per cells per well.
  • pSTAT5 Staining Cells were washed in PBS and the supernatant was discarded. Cells were then incubated in 50ul Live/Dead- Near IF (ThermoFisher) and Human TruStain FcX: Fc receptor blocking solution (Biolegend), in PBS respectively.
  • CAR Tregs were co-cultured in the presence of DynabeadsTM Human T-Activator CD3/CD28 for T Cell Expansion and Activation beads or media alone for 6-10 days with or without 10 IU/ml of IL2.
  • CAR Tregs were treated with 100nM, 1000nM or 2740nM rapamycin (Sigma-Aldrich) or vehicle alone every day.
  • the vehicle used was dimethyl sulfoxide (DMSO; Sigma-Aldrich).
  • DMSO dimethyl sulfoxide
  • cells were washed in FACS buffer before Fc blocking with Human TruStain FcXTM (Biolegend) and staining with cell surface antibodies: Brilliant Violet 510TM anti-human CD4 (Biolegend), PE/Cyanine7 anti-human CD25 (Biolegend). Washed cells were then stained with Annexin V/7AAD using the PE Annexin V Apoptosis Detection Kit with 7AAD (Biolegend) according to the manufacturer’s guidelines. Cells were acquired by flow cytometry. Data was analysed on FlowJo_V10 software. Graphs were produced using GraphPad Prism 8 software.
  • a representative cytoplasmic construct was designed comprising an interaction domain comprising two dimerization domains and a signalling domain comprising a JAK1-binding domain and a STAT5 association motif to determine if constructs lacking a transmembrane domain could result in increased levels of pSTAT5 within cells.
  • Tregs were transduced with construct pQTX 0340 and were treated with different levels of Rapamycin. As can be seen from Figure 7, pSTAT5 expression was shown to be increased in transduced Tregs as compared to non-transduced cells or mock cells, or as compared to transduced cells in the absence of Rapamycin (23.4% as compared to 5.82%).
  • a survival assay further demonstrated that cells transduced with pQTX 0340 had an increase in survival after treatment with Rapamycin, both under bead stimulated conditions and media only.
  • Two different gating strategies were employed as shown in Figures 9 and 10, and both demonstrated an increase in survival for pQTX 0340 transduced cells in the presence of Rapamycin.
  • Figure 11 in particular shows an increase in survival in transduced cells after Rapamycin treatment from 4.34% to 18.4% (with beads) and from 37.9% to 59.8% (media alone). No increase in survival is seen for non-transduced cells.

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Abstract

The present invention relates to a chimeric protein, said chimeric protein being a non-membrane associated protein comprising, linked together in any order: (i) an interaction domain comprising at least one dimerization domain; (ii) a signalling domain comprising a tyrosine kinase activating domain comprising a JAK1-binding motif and a tyrosine effector domain comprising one or more tyrosine residues that can be phosphorylated by JAK1; wherein the dimerization domain of said chimeric protein is capable of dimerizing with a cognate dimerization domain of at least a second, non-membrane associated, chimeric protein providing a second component of the signalling protein, wherein the at least a second chimeric protein may be the same chimeric protein, or a different chimeric protein, and wherein dimerization of the chimeric protein with the at least a second chimeric protein provides a signal in a cell expressing the chimeric protein and the at least a second protein.

Description

Signalling protein Field The present disclosure and invention relate to a chimeric protein useful in adoptive cell therapy (ACT). In particular, the chimeric protein is a signalling protein, or a component thereof, which can provide a cell expressing the protein with a desired signal, e.g. a STAT-mediated signal. The signal may confer a desired effect or property on the cell, e.g. increased function, activity, vitality, or survival, e.g. persistence in a transplanted host subject. Also provided are nucleic acid molecules encoding such a chimeric protein, recombinant constructs, vectors and cells containing the nucleic acid molecule, methods of producing such cells, and therapeutic uses thereof. Background Adoptive cell therapy (ACT), that is the administration of functional immune cells to a subject, has become an established and evolving immunotherapeutic approach for various medical conditions, including notably malignant or infectious diseases. Tumour-infiltrating lymphocytes were initially shown to be effective in treating metastatic melanoma, and subsequently re-directed T-cells or NK cells expressing chimeric antigen receptors (CARs) or heterologous T-cell receptors (TCRs) to target different 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 deleterious cells in the body, but more recently regulatory T cells (Tregs) have been developed for ACT. Tregs have immunosuppressive function. They act to control cytopathic immune responses and are essential for the maintenance of immunological tolerance. The suppressive properties of Tregs can be exploited therapeutically, for example to improve and/or prevent immune-mediated organ damage in inflammatory disorders, autoimmune diseases and in transplantation. To be useful in ACT, the transplanted, or administered, cells need to survive and persist in the recipient (the subject of the ACT therapy) in a functional state long enough to exert a useful therapeutic effect. Further, to be prepared in sufficient numbers for therapeutic use, the cells need to be generated (e.g. engineered), cultured and expanded in vitro. The growth factor interleukin-2 (IL-2) is essential for the homeostasis of immune cells, including notably Tregs (generation, proliferation, survival), as well as for their suppressive function and phenotypic stability. Activated conventional T cells (Tcons) are the main source of IL-2 in vivo. Tregs, in contrast, cannot produce IL-2 and depend on paracrine access to IL-2 produced by Tcons present in the microenvironment. The availability of IL-2 has a critical impact on the therapeutic effects of Tregs expanded in vitro and transferred into patients. This is due to the following: 1) in vitro expansion protocols typically require high concentrations of IL-2, which renders Tregs highly dependent on this cytokine; 2) the concentration of IL-2 is often reduced in patients as a result of the administration of immunosuppressive drugs; and 3) within the inflamed tissue microenvironment access to IL-2 is often limited. Liver transplantation constitutes a particularly challenging indication, given that the levels of IL-2 in the inflamed liver are known to be reduced, which is further aggravated by the routine use of calcineurin inhibitors, which substantially decrease the capacity of Tcons to produce IL-2. The administration of low doses of exogenous IL-2 restores the Treg dysfunction induced by calcineurin inhibitors and promotes the accumulation of Tregs in the liver. However, a concern with the therapeutic use of low-dose IL-2 is the risk of simultaneously activating Tcons, which can enhance tissue damage. In WO 2020/044055 an approach is described to circumvent the need to administer exogenous IL-2. In this case Treg cells are engineered to express a CAR which has been modified such that it is capable of providing a productive IL-2 signal to the cell upon binding to its target antigen. In other words, the intracellular signalling domain of the CAR, the endodomain, includes sequences, or domains, derived from IL receptors, 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 signals through the transcription factor STAT5 (Signal Transducer and Activator of Transcription 5), which is phosphorylated in its active state by the kinases JAK1 and/or JAK2, which are normally activated when interleukins (e.g. IL-2) bind to their receptors. Accordingly, the CAR in WO 2020/044055 comprises an endodomain which comprises a STAT5 association motif and a JAK1- and/or a JAK2-binding motif. Analogously, other immune cells for ACT, e.g. cytotoxic T-cells, or other Teffector cells, including CAR-T cells, may also require or benefit from additional signalling capacity being provided to the cell to increase survival or persistence of function. Accordingly, the need for additional signalling, or more particularly engineered signalling, whether to increase survival or persistence, or to improve the functional activity or therapeutic effect of cells for ACT, is not limited to Treg cells. Whilst WO 2020/04405 provides an important advance, there is a continuing need in the field of ACT for new and improved approaches, and in particular approaches which avoid or reduce the need to develop a modified CAR for each target, and which may have a more universal application. Summary The present inventors have developed a new signalling protein which can be used to impart a desired signalling capacity to a cell, notably in which the signalling involves tyrosine kinase activity, and protein phosphorylation. Particularly, the signalling involves Janus kinase (JAK) phosphorylation and activity, and more particularly activation of the JAK-STAT signalling pathway involving JAK1. The signalling protein may be inducible. That is, the signalling provided by the protein may be induced, when desired, by exposing a cell expressing the protein to an inducer molecule. Thus, the protein may be responsive to an inducer molecule which may be provided to the cell to induce the desired signalling. However, the new signalling protein is not limited to inducible systems, and a constitutive signalling capacity may be provided to the cell. The signalling protein is based on dimerization – that is, the signalling protein is formed from monomers which are designed to dimerize, or more generally to multimerize, and in the dimerized/multimerized state to assume a signalling capacity, i.e. to become functional when dimerized to mediate signalling in a cell by which it is expressed. The monomer of the signalling protein is a chimeric protein comprising a signalling domain and a dimerization domain, that is a domain capable of multimerization (i.e. at least dimerizing) with a cognate dimerization domain in another chimeric protein. More particularly, at least one dimerization domain may be contained in an interaction domain capable of interacting with (e.g. a binding domain capable of binding to) with an interaction domain in another chimeric protein. The interaction, or dimerization, between the domains may be direct or indirect. In other words, individual dimerization domains in different molecules of the chimeric protein may interact (e.g. multimerize, or dimerize) directly with one another, or indirectly, via the intermediacy of another molecule, with which cognate dimerization domains may interact, for example an inducer molecule to which two more molecules of a chimeric protein may bind. The signalling domain is capable of transmitting a signal, for example a STAT-5 or other STAT mediated signal, when the chimeric protein is dimerized (or more generally multimerized). For example, signalling may be induced by interacting with (e.g. binding to) the inducer molecule. Accordingly, the chimeric protein is a dimerizable protein, or a protein capable of higher multimerization (oligomerization). The signalling activity of the chimeric protein is induced, or enabled, by multimerization (e.g. dimerization). Multimerization (e.g. dimerization) of the protein may be induced by an inducer, or may occur spontaneously when two molecules of the chimeric protein come into proximity. A surprising feature of the chimeric signalling protein herein is that it is a cytoplasmic cell protein, or alternatively put, a soluble protein, that is, it is not associated in any way with the surface membrane (plasma membrane) of the cell in which it is present or expressed. It is a protein that is present or expressed inside a cell. It is surprising that such a non-membrane associated protein is capable of mediating an effective signal, since effective signalling, including notably STAT- mediated signalling, is generally thought to require a membrane protein, or proximity to a membrane protein. Particularly, reports in the literature of JAK1 being predominantly located at the cell membrane, have fuelled the design of previous STAT signalling constructs as transmembrane proteins (e.g. WO2020/044055). Cytoplasmic signalling proteins as developed herein, demonstrating effective STAT signalling were therefore unexpectedly functional and are further associated with the benefit of not requiring successful transport to and through the membrane for function. Accordingly, a first aspect provides a chimeric protein, said chimeric protein being a non-membrane associated protein comprising, linked together in any order: (i) an interaction domain comprising at least one dimerization domain; (ii) a signalling domain comprising a tyrosine kinase activating domain comprising a JAK1-binding motif and a tyrosine effector domain comprising one or more tyrosine residues that can be phosphorylated by JAK1; wherein the dimerization domain of said chimeric protein is capable of dimerizing with a cognate dimerization domain of at least a second, non-membrane associated, chimeric protein, which may be the same chimeric protein, or a different chimeric protein, and wherein dimerization of the chimeric protein with the at least a second chimeric protein provides a signal in a cell expressing the chimeric protein and the at least a second protein. The chimeric protein defined above may accordingly be seen as a first chimeric protein. The first and second, and optionally any further, chimeric proteins may thus be seen as monomers, or components, of a cytoplasmic multi-component signalling protein. Said dimerization may enable, facilitate or contribute to signalling by the signalling protein. The second chimeric protein may also comprise a signalling domain. More particularly, dimerization of the dimerization domains of the two chimeric proteins (i.e. the first and the second chimeric proteins) may cause the signalling domains of the respective chimeric proteins to dimerize to enable, facilitate or contribute to signalling by the signalling protein. By “cognate” is meant the dimerization domain is able to dimerize with its cognate dimerization domain, e.g. they are designed or selected to be able to dimerize with one other. In other words, they are paired. Since the chimeric protein may comprise more than one dimerization domain, the possibility exists to create higher order oligomers (or alternatively termed, multimers). In effect, the chimeric protein may be viewed as a monomer, or subunit, of a higher order signalling protein, e.g. a dimeric signalling protein, or a trimer or 4-mer etc. In an embodiment, two molecules of the chimeric protein may dimerize together in order to constitute, or form, the signalling protein, without requiring a different, or second component (i.e. without requiring a separate component). In such an embodiment, it can be seen that the chimeric protein is in itself capable of providing a signal in a cell expressing the protein, when it is dimerized. The second chimeric protein may thus also be defined as being a non-membrane associated protein comprising, linked together in any order: (i) an interaction domain comprising at least one dimerization domain; (ii) a signalling domain comprising a tyrosine kinase activating domain comprising a JAK1-binding motif and a tyrosine effector domain comprising one or more tyrosine residues that can be phosphorylated by JAK1. However, it will be understood that in such a situation the two chimeric proteins need not necessarily be absolutely identical as long as they have domains (i) and (ii) as defined above. In another embodiment, the second chimeric protein may be a different chimeric protein. The term “different” in this context is used in the sense of a different type of protein, or in other words a separate component. In particular, the second chimeric protein may have a different signalling domain. As noted above, where the second chimeric protein is a different chimeric protein, the chimeric protein herein may be seen as a first chimeric protein. In an embodiment, the signalling domain of the chimeric protein herein (that is the first chimeric protein) further comprises a JAK3-binding motif. In another embodiment, the signalling domain of the second chimeric protein comprises a JAK3- binding motif. Accordingly, also provided herein according to the first aspect is a chimeric protein, said protein being a non-membrane associated protein comprising, linked together in any order: (i) an interaction domain comprising at least one dimerization domain; (ii) a signalling domain A comprising a tyrosine kinase activating domain comprising a JAK1-binding motif and a tyrosine effector domain comprising one or more tyrosine residues that can be phosphorylated by JAK1, or a signalling domain B comprising a tyrosine kinase activating domain comprising a JAK3-binding motif; wherein when at least two chimeric proteins are expressed in a cell, the dimerization domain of one chimeric protein is capable of dimerizing with the dimerization domain of another of the chimeric proteins, causing the signalling domains of the at least two chimeric proteins at least to dimerize (or in other words, to multimerize) to enable signalling by the chimeric protein; and wherein the chimeric proteins of the at least two chimeric proteins may be the same or different, and at least one chimeric protein of the at least two chimeric proteins comprises a signalling domain A. In a particular embodiment the chimeric protein forms a dimer. In such an embodiment the chimeric protein comprises, linked together in any order: (i) an interaction domain comprising at least one dimerization domain; (ii) a signalling domain A comprising a tyrosine kinase activating domain comprising a JAK1-binding motif and a tyrosine effector domain comprising one or more tyrosine residues that can be phosphorylated by JAK1, or a signalling domain B comprising a tyrosine kinase activating domain comprising a JAK3-binding motif; wherein the dimerization domain of one chimeric protein of a pair of chimeric proteins is capable of dimerizing with the dimerization domain of the other chimeric protein of the pair, causing the signalling domains of the pair of chimeric proteins to dimerize to enable signalling by the dimerized chimeric protein; and wherein the chimeric proteins of the pair of chimeric proteins may be the same or different, and at least one chimeric protein of the pair comprises a signalling domain A. In other embodiments, the chimeric protein may form higher multimers such as trimers, or 4-, 5- or 6-mers for example. In an embodiment, the signalling domain B does not comprise a tyrosine effector domain. Alternatively, in an embodiment the signalling domain B is not capable of providing a signal to a cell expressing the chimeric protein in the absence of signalling domain A (e.g. in the absence of a chimeric protein comprising signalling domain A). It will be understood from the above that a chimeric protein comprising a signalling domain B is not capable of dimerizing with another chimeric protein containing signalling domain B to provide a functional signalling protein. At least one of two chimeric proteins capable of dimerizing comprises a signalling domain A. A second aspect provides a pair of dimerizable chimeric proteins, which together when dimerized are capable of providing a signal in a cell expressing the proteins, each protein of the pair being a non-membrane associated protein, wherein the first chimeric protein of the pair comprises, linked together in any order: (i) an interaction domain comprising at least one dimerization domain; (ii) a signalling domain A comprising a tyrosine kinase activating domain comprising a JAK1-binding motif and a tyrosine effector domain comprising one or more tyrosine residues that can be phosphorylated by JAK1; and the second chimeric protein of the pair comprises, linked together in any order: (i) an interaction domain comprising at least one dimerization domain; and (ii) a signalling domain B comprising a tyrosine kinase activating domain comprising a JAK3-binding motif; and wherein the dimerization domain of the first chimeric protein is capable of dimerizing with the dimerization domain of the second chimeric protein causing the signalling domains of a pair of chimeric proteins to dimerize to enable signalling. In a particular embodiment the chimeric protein, when dimerized or higher multimerized is capable of providing an inducible signal in a cell expressing the protein. In this embodiment the dimerization domains (i.e. cognate dimerization domains) in two chimeric proteins are able to form a dimer in the presence of a dimerization inducer. Accordingly, in such an embodiment the chimeric protein may comprise, linked together in any order: (i) an interaction domain comprising at least one dimerization domain; (ii) a signalling domain comprising a tyrosine kinase activating domain comprising a JAK1-binding motif and a tyrosine effector domain comprising one or more tyrosine residues that can be phosphorylated by JAK1; wherein in the presence of a dimerization inducer, the dimerization domain of said chimeric protein is induced to dimerize with a cognate dimerization domain of a second, non-membrane associated, chimeric protein, wherein the second chimeric protein may be the same chimeric protein, or a different chimeric protein and wherein dimerization of the chimeric protein with the second chimeric protein provides a signal in a cell expressing the chimeric protein and the second chimeric protein In a particular embodiment, the chimeric protein comprises, linked together in any order: (i) an interaction domain comprising at least one dimerization domain; (iii) a signalling domain A comprising a tyrosine kinase activating domain comprising a JAK1-binding motif and a tyrosine effector domain comprising one or more tyrosine residues that can be phosphorylated by JAK1, or a signalling domain B comprising a tyrosine kinase activating domain comprising a JAK3-binding motif; wherein in the presence of a dimerization inducer the dimerization domain of one chimeric protein of a pair of chimeric proteins is induced to dimerize with the dimerization domain of the other chimeric protein of the pair, causing the signalling domains of the pair of chimeric proteins to dimerize to enable signalling by the dimerized chimeric protein; and wherein the chimeric proteins of the pair of chimeric proteins may be the same or different, and at least one chimeric protein of the pair comprises a signalling domain A. Analogously, the chimeric protein may alternatively multimerize to form a higher order multimer. The inducer may induce, or mediate, dimerization of the chimeric protein by binding to two separate sites, or dimerization domains, in the interaction domain, one in each of two chimeric proteins. Alternatively, the inducer may be capable of binding to two copies of the dimerization domain, one in each of two chimeric proteins. This brings the two proteins together and thereby allows the two signalling domains or a portion of a signalling domain, to dimerize, and thereby to be able to exert their function, namely transmission of a signal. Higher multimers may similarly be created, for example where two dimerization domains in the interaction domain each dimerize with a separate chimeric protein. Alternatively, an interaction domain may comprise more than two dimerization domains to achieve higher multimerization. Dimerization, or higher multimerization, of the interaction domain, by interacting with, or binding to, the inducer, allows the functional protein component of the chimeric protein, here the signalling domain, to be multimerized (e.g. dimerized) in order to exert its function. The inducer may thus be seen as an inducer of multimerization, or more particularly an inducer of dimerization, which allows a protein which is active in dimeric form or higher multimeric form to be dimerized, or multimerized, thereby to induce the activity of the protein. The inducer creates an interface between two or more binding sites, at least one in each copy of the protein, which in effect “multimerizes” the binding sites, e.g. “dimerizes” two binding sites. Such an inducer may thus be a so-called chemical inducer of dimerization (CID). A dimerization domain in the chimeric protein may be capable of homodimerization, that is two identical copies of a dimerization domain may dimerize. Such dimerization domains may be termed homodimerization domains. Alternatively, a dimerization domain may be capable of heterodimerization, that is two different dimerization domains may dimerize. Such dimerization domains may be termed heterodimerization domains. In an embodiment, the interaction domain may comprise a single dimerization domain, which may be a homodimerization domain or a heterodimerization domain. In another embodiment, the interaction domain may comprise two or more dimerization domains, which may be homodimerization and/or heterodimerization domains. In the latter case, the chimeric protein may be configured such that a dimerization domain from each one of the pair of chimeric proteins binds to an inducer (e.g. CID) molecule. The pair of chimeric proteins in a dimer is thus bound together via binding of their respective dimerization domains to an inducer molecule, as depicted in Figure 1. In a particular embodiment, the interaction domain comprises: (i) a first dimerization domain D1; (ii) a second dimerization domain D2; wherein D1 and D2 may be the same or different; and wherein, when at least two chimeric proteins are expressed in a cell, and in the presence of a dimerization inducer, the D1 and D2 domains of one chimeric protein of the at least two chimeric proteins are induced to dimerize with the D1 and D2 domains of another chimeric protein, causing the signalling domains of the at least two chimeric proteins to dimerize to enable signalling by the chimeric protein; and wherein the chimeric proteins of the at least two chimeric proteins may be the same or different and the D1 and D2 domains of one chimeric protein may each bind with the D1 or D2 domain of another chimeric protein. The configuration of the chimeric protein is such that D1 does not dimerize to any significant extent with D2 within the same chimeric protein in the presence of the inducer. Thus, particularly, D1 of a first chimeric protein dimerizes with D2 of a second chimeric protein and D2 of a first chimeric protein dimerizes with D1 of a second chimeric protein. The signal which may be induced by the chimeric protein, or the signalling protein, may be a signal which improves or increases a functional property or activity of the cell. Thus, the function or effect of a cell may be increased, which may be a function or effect in vitro or in vivo, that is during generation or expansion of a cell which is being prepared for ACT, or once the cell has been administered to a subject. This may be, for example cell survival, persistence of the cell, persistence of function of the cell, vitality, functional effect (e.g. immunosuppressive or cytotoxic effect), phenotype of the cell, including memory phenotype, proliferation capacity and/or therapeutic efficacy of the cell. The increase may be seen in a cell which comprises the protein relative to a cell which does not comprise the protein. In a particular embodiment the signal is a pro-survival signal, which helps the cell to survive and to maintain its ability to function during and after culture, and to persist and maintain its functional ability following administration to a subject in the course of therapy. It may alternatively be referred to as a persistence signal. Thus, the chimeric protein may be expressed in a cell to impart an inducible pro-survival signalling capacity to the cell. It has particular utility 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 a CAR, or any chimeric receptor. The protein thus has utility in the engineering of cells for ACT. In an embodiment the signal is a STAT-mediated signal (e.g. a STAT3 or a STAT5 mediated signal), and more particularly, a STAT5-mediated signal, which can normally be induced in a cell by interleukins such as IL-2. In such an embodiment the tyrosine effector domain comprises at least one STAT association motif. Accordingly, in the various embodiments of the first and second aspects herein, as discussed above, the tyrosine effector domain may alternatively be defined as comprising a STAT association motif. As mentioned above and discussed in more detail below, in one embodiment the dimerization domains interact with an inducer of dimerization. In particular, the dimerization domains bind to the inducer. In other words, they are, or they comprise, binding sites for the inducer. In such an embodiment, the inducer may be a ligand, or more particularly a dimerization ligand. It will be seen therefore that in such an embodiment the dimerization domains allow, or mediate, dimerization by binding to the inducer (i.e. by inducer, or ligand, binding). Alternatively, the inducer may induce the dimerization domains to bind to one another by chemical cross-linking. In the situation where the dimerization domains of two interacting chimeric proteins are the same (including e.g. in the embodiment above in which the interaction domain comprises 2 dimerization domains, D1 and D2, and these are the same), the dimerization inducer may comprise two identical interaction sites (e.g. binding sites) for the dimerization domains, one from each of separate proteins. In such a situation homodimerization between two identical dimerization domains may occur. When the dimerization domains of two interacting chimeric proteins are different (including where D1 and D2 in a single interaction domain are different), the dimerization inducer may interact with (e.g. bind to) two different binding sites, one in (or provided by) the first dimerization domain (e.g. of a first chimeric protein, or D1) and the other in (or provided by) the second dimerization domain (e.g. of a second chimeric protein, or D2). In other words, the dimerization inducer may comprise two different interaction sites (e.g. binding sites) for the dimerization domains, e.g. one for the dimerization domains of two different chimeric proteins, or one for D1 and one for D2. In such a situation heterodimerization between two different dimerization domains may occur. D1 and D2 together provide a domain for interaction with the inducer, e.g. for binding to the inducer, i.e. an interaction domain. This may in an embodiment be referred to as the binding domain of the chimeric protein. The interaction domain may be in one or in separate, e.g. two, parts. In the chimeric protein, as discussed further below, D1 and D2 may be linked together. In other words, in such a protein D1 and D2 may be provided as a unit, or put another way, within a single interaction domain (e.g. a single, inducer-binding domain). Such a single-unit interaction domain may be linked, or located, at the N- or the C-terminal of the signalling domain. Alternatively, D1 and D2 may be provided as separate sites, or domains in the chimeric protein. In other words, D1 and D2 may be provided as separate functional units in the chimeric protein. For example, they may each be linked, or located, at either end of the signalling domain. Thus, the interaction domain (e.g. an inducer-binding domain) may be provided in the chimeric protein in one or two parts. A single unit (1-part) interaction domain comprises both D1 and D2. This allows “one-side” dimerization. A 2-part binding domain comprises two separate parts, which are spaced apart in the chimeric protein, the first part comprising one of D1 or D2 and the second part comprising the other of D1 and D2. This allows “two-side dimerization”. Thus, various configurations of the chimeric protein are possible, wherein different components of the chimeric protein are linked together in various orders, as described further below. The various components of the chimeric protein may be linked to each other directly or indirectly, as described further below. Where applicable, this includes the individual components of the interaction domain (e.g. D1 and D2), and of the signalling domain. Thus, in the chimeric protein one or more linkers may be present, said linkers being between any two components of the chimeric protein. For example, there may be a linker between the interaction domain, or a part thereof, and the signalling domain. Alternatively, or additionally there may be a linker between D1 and D2 (where D1 and D2 are present in a single unit, 1-part, interaction domain), and/or there may be a linker between two or more components of the signalling domain. A third aspect provides a nucleic acid molecule (or alternatively termed, a polynucleotide) comprising a nucleotide sequence which encodes a chimeric protein as defined herein. The nucleic acid molecule may be in the form of a construct, or more particularly, a recombinant construct, comprising the nucleic acid molecule and one or more other nucleotide sequences (a nucleotide sequence of interest). For example, the construct may comprise the nucleic acid molecule and a regulatory sequence, e.g. an expression control sequence, and/or a sequence encoding another functional protein (or more generally, a protein of interest), for example a receptor, e.g. a CAR or TCR etc. Where the signalling protein comprises different chimeric proteins (i.e. where different chimeric proteins are dimerized), nucleotide sequences encoding the different chimeric proteins may be provided in the same construct. Alternatively, a separate nucleic acid molecule or construct may be provided for each separate, or different, chimeric protein. The construct may comprise one or more co-expression sequences linking the nucleic acid molecule with one or more other coding nucleotide sequences. A fourth aspect provides a vector comprising a nucleic acid molecule or construct as defined herein. Where separate chimeric proteins are encoded in separate molecules or constructs, they may each be contained in a separate vector. There may accordingly be a set of vectors, each comprising a sequence encoding a separate chimeric protein. The vector may be a viral or non-viral vector. In an embodiment the vector may comprise a nucleic acid molecule as defined herein and a further nucleotide sequence encoding a protein of interest, notably a receptor, e.g., a CAR or TCR. A fifth aspect provides a cell which expresses a chimeric protein as defined herein, or which comprises a nucleic acid molecule, construct or vector as defined herein. The cell expresses the chimeric protein intracellularly, or more particularly in the cytoplasm, or cytosol, of the cell. Thus, the chimeric protein may be said to be expressed in a cell. Also provided according to this aspect is a cell population comprising a cell as defined herein. In an embodiment, the cell is an immune cell, or a precursor thereof. The cell may be a stem cell, or more particularly a haemopoietic stem cell (HSC) or pluripotent stem cell (PSC), e.g. an induced pluripotent stem cell (iPSC). Particularly, the cell may be a lymphocyte, or more particularly a T-cell, NK cell, dendritic cell or myeloid-derived suppressor cell (MDSC). In an embodiment, the T-cell may be a Treg cell. The cell may be a primary cell or from a cell line. In another embodiment the cell may be a production host cell, that is a cell into which the nucleic acid molecule construct or vector is introduced in order to produce the chimeric protein, or a viral vector encoding the chimeric protein. A sixth aspect provides a method of preparing a cell as defined herein (i.e. a cell according to the fifth aspect), said method comprising introducing into a cell (e.g. transducing or transfecting a cell with), a nucleic acid molecule, construct or vector as defined herein. The method may include allowing the chimeric protein to be expressed in the cell. This may include, for example, culturing the cell. Such a method may further comprise a preceding step of isolating, enriching, providing or generating a cell to be used in the method. Further, a cell may be isolated or enriched or generated after the step of introducing the nucleic acid molecule. For example, the nucleic acid molecule may be introduced into a precursor or progenitor cell, e.g. a stem cell, and the cell may then be induced or caused to differentiate, or change, into a desired cell type. For example, an iPSC cell may be differentiated into an immune effector cell (e.g. a Treg or other T cell) or a Tcon cell may be converted into a Treg cell, etc. This aspect may also include a method of preparing a chimeric protein as defined herein, said method comprising introducing into a cell, a nucleic acid molecule, construct or vector as defined herein, allowing the chimeric protein to be expressed by the cell, and optionally detecting and/or collecting the chimeric protein. A seventh aspect provides a method of promoting the survival or persistence of a cell, said method comprising introducing into the cell, a nucleic acid molecule, construct or vector as defined herein. This may include the step of allowing the chimeric protein to dimerize to a second chimeric protein, optionally wherein said second chimeric protein is a separate or different chimeric protein. The step of allowing the chimeric protein to dimerize may include exposing the cell to (or contacting the cell with) an inducer. The inducer induces dimerization, and signalling by the chimeric protein, or a signalling protein containing it, and provides a pro- survival signal to the cell. This aspect may comprise administering a cell as defined herein to a subject, and administering a dimerization inducer to the subject. The inducer may be administered before, during or after administration of the cell. Thus, in this aspect, the method may be carried out in vivo. Alternatively, the method of the seventh aspect may be carried out in vitro/ex vivo. As noted above, the chimeric protein may advantageously be expressed in a cell in the context of therapy. Whilst the cell may be an unmodified cell, in the sense of not being further genetically engineered for therapeutic use, for example a T cell isolated from a subject, or a cell derived from such an isolated cell (although of course the cell would be modified by the present method to express the chimeric protein), typically the cell will be a cell which is additionally modified, or engineered to express a further molecule (i.e. a further protein), notably a receptor, e.g. a CAR or TCR. Thus, an eighth aspect provides a method of preparing a cell for use in adoptive cell transfer therapy (ACT), said method comprising providing said cell with a chimeric protein as defined herein. More particularly, this method may comprise introducing into said cell a nucleic acid molecule, construct or vector as defined herein. The method may also comprise introducing into the cell a separate nucleic acid molecule, construct or vector, for example which comprises a nucleotide sequence which encodes a separate (e.g. second) chimeric protein, or a therapeutic protein, notably a receptor, e.g. a CAR or TCR. A ninth aspect provides a pharmaceutical composition comprising a cell, cell population or a vector as defined herein, together with at least one pharmaceutically acceptable carrier or excipient. In an embodiment the cell or the vector comprises an additional nucleotide sequence encoding a further protein, notably a second chimeric protein, or a receptor, e.g. a CAR or TCR. In another embodiment the cell comprises a separate nucleic acid molecule, construct or vector which comprises a nucleotide sequence which encodes a further protein, notably a second chimeric protein, or a receptor, e.g. a CAR or TCR. A tenth aspect provides a cell or cell population as defined herein, or a pharmaceutical composition as defined herein, or a vector as defined herein for use in therapy. Particularly, the cell, cell population or a pharmaceutical composition comprising the cell or cell population may be for ACT. The vector or pharmaceutical composition comprising the vector may be for gene therapy. The ACT or gene therapy may be for the treatment or prevention of any condition which is responsive to ACT or gene therapy, in particular immunotherapy by ACT or gene therapy. An eleventh aspect provides a cell, cell population, vector or pharmaceutical composition as defined herein for use in the treatment of or prevention of cancer, or an infectious, neurodegenerative, inflammatory, autoimmune or allergic disease or any condition associated with an unwanted or deleterious immune response. In particular, where the cell is a Treg or other immunosuppressive cell, the cell may be used for inducing immunosuppression (i.e. for suppressing an unwanted or deleterious immune response), for example to improve and/or prevent immune- mediated organ damage in inflammatory disorders, autoimmune or allergic diseases or conditions, and in transplantation. This aspect also provides a method of adoptive cell transfer therapy, said method comprising administering to a subject in need of said therapy a cell, cell population, or pharmaceutical composition as defined herein, particularly an effective amount of said cell, cell population or pharmaceutical composition. Also provided is a method of treating or preventing cancer, or an infectious, neurodegenerative, inflammatory, autoimmune or allergic disease or a condition associated with an unwanted or deleterious immune response, said method comprising administering to a subject in need thereof a cell, cell population, vector or pharmaceutical composition as defined herein, particularly an effective amount of said cell, cell population, vector or pharmaceutical composition. Further, there is provided use of a cell, cell population or vector as defined herein in the manufacture of a medicament for use in treating or preventing cancer, or an infectious, neurodegenerative, inflammatory, autoimmune or allergic disease or a condition associated with an unwanted or deleterious immune response. In some embodiments of these therapeutic aspects the use may be in induction of tolerance to a transplant; treating and/or preventing cellular and/or humoral transplant rejection; treating and/or preventing graft-versus-host disease (GvHD), an autoimmune or allergic disease; to promote tissue repair and/or tissue regeneration; or to ameliorate inflammation. In particular, in such embodiments the cell may be a Treg cell. In the various therapeutic aspects set out above the cell, cell population, vector or pharmaceutical composition may be for use in combination with, or together with, a dimerization inducer. Accordingly, a twelfth aspect provides a combination product comprising (a) a cell, cell population, vector or pharmaceutical composition as defined herein, and (b) an inducer (e.g. a CID), for use in therapy, particularly ACT or gene therapy. The therapy may be any therapy as defined above, and further described herein. The components (a) and (b) of the combination product may be for separate, sequential or simultaneous use. Description of the Figures Figure 1 depict formats of the chimeric protein which comprise a single dimerization domain, showing in (A) one-sided heterodimerization and (B) one-sided homodimerization. The signalling domain comprises, N-terminal to C-terminal, JAK1- STAT5 motifs. In (A) the interaction domain, comprising a single dimerization domain, depicted as FKBP or FRB, which each bind to the inducer, depicted as rapamycin, is linked to the N-terminus of the signalling domain. In (B), the dimerization domain in each chimeric protein is the same. Figure 2 depicts various formats of the chimeric protein which comprises two dimerization domains, D1 and D2, in various formats. The signalling domain comprises, N-terminal to C-terminal, JAK1-STAT5 motifs (double construct) or JAK1- STAT5-JAK3 motifs (triple construct; JAK3 is reverse orientation), wherein: in (A), the chimeric protein comprises a 1-part interaction (inducer-binding) domain linked to the N-terminus of the signalling domain (double construct/one-sided dimerization); in (B), the chimeric protein comprises a 2 part interaction (inducer-binding) domain wherein D1 is linked to the N-terminal of the signalling domain and D2 is linked to the C-terminal of the signalling domain (double construct/two-sided dimerization); in (C), the chimeric protein comprises a 2 part interaction (inducer-binding) domain wherein D1 is linked to the N-terminal of the signalling domain and D2 is linked to the C-terminal of the signalling domain (triple construct/two-sided dimerization). Figure 3 shows Rapamycin and rapalogs. A) Rapamycin; B) C-20- methyllyrlrapamycin (MaRap); C) C16(S)-Butylsulfonamidorapamycin (C16-BS-Rap); D) C16-(S)-3- methylindolerapamycin (C16-iRap); and E) C16-(S)-7- methylindolerapamycin (AP21976/C16-AiRap). Figure 4 shows the structures of inducer molecules (A) cyclosporine (CsA); (B) FK506; (C) FKCsA; (D) Fusicoccin and (E) HaSX8. Figure 5 shows schematic diagrams of representative cytoplasmic constructs, pQTX-0340 and pQTX-0370. Both constructs contain the SFFV (Spleen focus forming virus) promoter followed by the rapamycin binding domains FRB (FKBP Rapamycin Binding) which is linked to the other rapamycin binding domain FKBP by a 5 amino acid linker. This is then linked by 12 amino acids to the truncated IL2RB signaling domain containing the Y510 amino acid. In pQTX-340 this is followed by the P2A self-cleaving peptide and the GFP reporter gene. In pQTX-0370 this is followed by the T2A self-cleaving peptide and the A2CAR, then the P2A self-cleaving peptide and the GFP reporter gene. Figure 6 shows a schematic diagram of the pSTAT5 assay using Treg cells. Figure 7 shows that Human Tregs transduced with pQTX-340 demonstrate an increased pSTAT5 expression level upon Rapamycin treatment. pQTX-340 transduced human Tregs (MOI 5) were rested for 24 hours prior to the assays, and cells were then treated with 0nM, 1nM, 10nM, 100nM, 1000nM or 2740nM of rapamycin for 48, 24 or 4 hours prior to staining. FACS plots show GFP+ cell populations and the pSTAT5 expression levels after 48 hour treatment of rapamycin with vehicle alone or with 1000nM. The bar graph shows the mean fluorescence intensity (MFI) of pSTAT5 expression levels with the various indicated rapamycin concentrations treatments. The line graph shows the percentage of pSTAT5 expression levels for pQTX-340 transduced Tregs within the GFP+ or GFP- populations. The bar graph shows the percentage of pSTAT5 expression levels of pQTX-340 transduced Tregs within the GFP+ or GFP- populations or Mock Tregs. Data normalized to vehicle. Figure 8 shows a schematic diagram of the survival assay. Figure 9 shows that human Tregs transduced with pQTX-340 demonstrate an increased survival ability upon Rapamycin treatment. pQTX-340 transduced human Tregs were co-cultured with CD3/28 beads or media alone for 10 days.1000nM rapamycin or vehicle alone treatments were added everyday to the co-cultures. At day 10, cells were stained with CD4, CD25, Annexin V and 7AAD to assess cell viability. For Gating Strategy 1- the FACS plots displayed show the lymphocytes gate, then within this population the single cells gate followed by cells which were negative for both Annexin V and 7AAD, thus the viable cells gate. Within the viable cells’ population, the CD4+CD25+ cells where selected and then within this the GFP expression was assessed. The gates for GFP+ and GFP- cells were set, and the ‘frequency of grandparent’ were reported. Graphs show the percentage of live cells within the GFP+ and GFP- populations with or without the CD3/28 beads co-culture. Figure 10 shows Gating Strategy 2- the FACS plots displayed show the lymphocytes gate, then within this population the single cells gate followed by the gates defining the GFP+ and GFP- cell populations. For each of the GFP+ or GFP- populations, the negative expression of Annexin V and 7AAD were taken as percentage of live cells and were reported. Figure 11 shows that human Tregs transduced with pQTX-0340 demonstrate an increased survival ability upon Rapamycin treatment, either stimulated with beads or in media alone. No increase in survival ability was seen in GFP negative cells (i.e. non-transduced) or in mock cells. Figure 12 shows that human Tregs transduced with pQTX-370 (pQTX-340 with a CAR) demonstrate an increased survival ability upon Rapamycin treatment. A schematic diagram of pQTX-370 which is similar to pQTX-340 but with the addition of a CAR is shown. pQTX-370 transduced human Tregs were co-cultured with CD3/28 beads for 6 days.1000nM rapamycin or vehicle alone treatments were added every day to the co-cultures. At day 6, cells were stained with CD4, CD25, Annexin V and 7AAD to assess cell viability. For Gating Strategy 1- the FACS plots displayed show the lymphocytes gate, then within this population the single cells gate followed by cells which were negative for both Annexin V and 7AAD, thus the viable cells gate. Within the viable cells’ population, the CD4+CD25+ cells where selected and then within this the GFP expression was assessed. The gates for GFP+ and GFP- cells were set, and the ‘frequency of grandparent’ were reported. Graph shows the percentage of live cells within the GFP+ and GFP- populations. Figure 13 shows cell counts under different treatment conditions (IL2, DMSO and different concentrations of Rapamycin) for cells transduced with pQTX-0340, in the presence of beads or media. Tregs transduced with pQTX-0340 were seeded in a 96 well plate for a survival assay. Cells were treated every day with varying concentrations of Rapamycin. At Day 10 of the survival assay, cells were stained and acquired by flow cytometry and cell counts were taken. The live GFP positive population of cells were counted and plotted, below the bar chart are the conditions for each bar displayed. The first chart displayed the data from the cells cultured with CD3/28 beads and the second chart shows the cells cultured with no beads and media alone. Figure 14 shows schematic diagrams of additional cytoplasmic full-length constructs, pQTX-570, pQTX-571, pQTX-572, pQTX-573 and pQTX-574. pQTX-570: FRB (FKBP Rapamycin Binding), 5aa linker, Cytoplasmic domain (beta), P2A self- cleaving peptide and the GFP reporter gene. pQTX-571: FKBP, 5aa linker, Cytoplasmic domain (beta), P2A self-cleaving peptide and the GFP reporter gene. pQTX-572: FKBP, 5aa linker, Cytoplasmic domain (gamma), P2A self-cleaving peptide and the mCherry reporter gene. pQTX-573: FRB (FKBP Rapamycin Binding), 5aa linker, FKBP, 17aa linker, Cytoplasmic domain (beta), P2A self-cleaving peptide and the GFP reporter gene. pQTX-574: FKBP, 5aa linker, FRB (FKBP Rapamycin Binding), 17aa linker, Cytoplasmic domain (gamma), P2A self-cleaving peptide and the mCherry reporter gene. Figure 15 shows schematic diagrams of additional cytoplasmic full-length constructs pQTX-0583-0587, which additionally contain a furin sequence. pQTX- 0583 FRB 100aa (SEQ ID NO.6), linker 5aa, IL2RB cytoplasmic domain 286aa (SEQ ID NO.9), furin 9aa, P2A 19aa and GFP 238 aa; pQTX-0584 FKBP 110aa (SEQ ID NO.8), linker 5aa, IL2RB cytoplasmic domain 286aa (SEQ ID NO.9), furin 9aa, P2A 19aa and GFP 238aa; pQTX-0585 FKBP 110aa (SEQ ID NO.8), linker 5aa, IL2RG cytoplasmic domain 86aa, furin 9aa, P2A 19aa, mCherry 236aa; pQTX- 0586 FRB 100aa (SEQ ID NO.6), linker 5aa, FKBP 109aa (SEQ ID NO.107), linker 17aa, IL2RB cytoplasmic domain 286aa (SEQ ID NO.9), furin 9aa, P2A 19aa, GFP 238aa; pQTX-0587 FKBP 110aa (SEQ ID NO.8), linker 5aa, FRB 99aa (SEQ ID NO. 112), linker 17aa, IL2RG cytoplasmic domain 86 aa, furin 9aa, P2A 19aa, mCherry 236aa. Detailed description The subject of the products, methods and uses herein is a chimeric protein which can be used to promote the functionality or survival, or indeed any property, of a cell by which it is expressed. The protein thus has utility in adoptive cell transfer, to assist in the preparation of cells for ACT, and/or to help keep the cells alive and functional following transfer to a subject. Therapeutic efficacy of the cell may be improved. The chimeric protein is based on a dimerization system, in which a pair of cognate dimerizable domains are comprised within the protein, and when brought together in proximity, or in the presence of an inducer, a so-called dimerization inducer, the cognate dimerization domains of two protein molecules are brought together to form a dimer. The “dimerization domain” may be any amino acid sequence that can dimerize, or even trimerize or multimerize. This brings together the signalling domains of two chimeric proteins, allowing them to dimerize and become functional, or in other words for signalling to be activated. Thus, a feature of the present chimeric protein is that the interaction domain and the signalling domain are present in the same protein chain. In other words, the chimeric protein monomer is a single chain protein. In particular, dimerization of the chimeric protein provided herein activates a signalling pathway mediated by JAK kinase activity, including notably JAK1 activity, and especially the JAK1-STAT signalling pathway. In this way the chimeric protein may mimic the signalling which is induced by activation of a natural cytokine receptor, for example an interleukin (e.g. IL-2) receptor. By “mimic”, it is meant that the signalling cascade activated by the chimeric protein of the present disclosure is similar to the signalling cascade activated by a natural cytokine receptor, while the magnitude of activation induced by the chimeric proteins of the present disclosure could be different from that of a natural cytokine receptor. A “dimerization domain” as provided herein may be capable of supporting multimerization (e.g. in the event that multiple copies of the dimerization domain are provided in the same molecule); however, such domains may also still be considered “dimerization domains” as used herein, based on the ability of such domains to dimerize. Typically, signalling can be effectively induced upon dimerization of chimeric proteins (i.e. trimerization or other multimerization is not typically required, but it is not excluded). Thus, two or more chimeric proteins dimerize to form a functional signalling protein. This includes that two chimeric proteins, which may be the same or different, form a dimer. In other embodiments, more than two chimeric proteins may associate together to form a higher order multimer, for example, where a chimeric protein contains two or more dimerization domains. The signalling protein may accordingly be a simple dimer of two molecules of the same chimeric protein, or it may be a dimer between a chimeric protein as provided herein and another chimeric protein, which may be a different chimeric protein, and may for example confer another property on the protein, or on the cell, or may augment the functionality of the cell which expresses the signalling protein (e.g. by introducing a JAK3-binding motif, as mentioned above and described further below). A functional signalling protein capable of signalling in a cell, or an activated signalling protein, is thus a multi-component protein, in that it comprises at least two chimeric proteins. “Multi” in this regard means two or more, e.g.3, 4 or more, for example, 2-6, 2-5, 2-4, 2-3 etc. The chimeric proteins, and hence the signalling protein which is formed therefrom, are cytoplasmic proteins. When expressed, they are present in the cytosol or the cytoplasm of the cell, or in the soluble compartment of the cell. In this regard, the chimeric proteins are “non-membrane associated”. This means that they are not associated with the cell surface membrane of the cell in any way, and are not expressed on or in the cell surface membrane. The chimeric proteins do not comprise a transmembrane domain or a portion thereof, or a membrane-targeting motif, e.g. a myristoyl group, or any moiety which is capable of associating with, e.g. binding to, or inserting into the cell membrane, or in any way associating with any component of a cell membrane. The chimeric proteins therefore do not comprise an exodomain (i.e. a domain which is present on the outside of the cell). The dimerization between dimerization domains in two chimeric proteins may be by any means, by which the respective domains are associated together. In the case of interaction domains which contain more than one dimerization domain, e.g. D1 and D2, this includes any means or method by which D1 and D2 in different proteins are associated together. This can be a direct or indirect association, and does not mean that the two domains need to be bound, or be linked directly, to one another, although this is not excluded. In an embodiment, the two interacting domains each bind to a dimerization inducer. The inducer thus creates an interface between two dimerization domains and brings them together as a dimer. In another embodiment the inducer is a molecule which allows chemical cross-linking between two dimerization domains. Thus, the inducer may permit the dimerization domains to each be covalently bonded to the inducer molecule. Thus, in an embodiment the dimerization between two dimerization domains is induced, and the chimeric protein, or signalling protein of which it is a component, is an inducible chimeric protein, or inducible signalling protein. In another embodiment, the dimerization between two dimerization domains is constitutive, and the chimeric protein, or signalling protein of which it is a component is a constitutive signalling protein. Thus, the dimerization may occur spontaneously when the two dimerization domains are brought together. Chemically-induced dimerization systems are known in the art, using various inducer molecules, and different protein domains for dimerization, are described further below. Direct systems of dimerization, based on moieties which are able to bind to or interact with each other when in contact or proximity, for example based on affinity binding, or other forms of interaction or association (e.g. amphipathic interaction such as occurs with leucine zippers), are also known in the art, and described further below. The interaction domain of the chimeric protein, which comprises the dimerization domain(s), which as noted above may be in 1- or 2-part form, thus allows two chimeric protein molecules to interact with an inducer molecule. A dimerization domain in one chimeric protein molecule binds to an inducer molecule which is also bound by a dimerization domain from another chimeric protein molecule, and in this way the dimerization domains in the two chimeric protein molecules become dimerized, and therefore the chimeric protein is also dimerized. Figure 1 depicts hetero- and homo-dimerization between two chimeric proteins each comprising a single dimerization domain, which dimerize together in the presence of an inducer. Since there is only one dimerization domain in each chimeric protein, there is only dimerization on one side of the proteins (so-called “one-sided” dimerization). In the embodiment in which each monomer of the chimeric protein comprises two dimerization domains D1 and D2, each protein pair will bind to two molecules of the inducer. In a more particular embodiment, D1 and D2 may heterodimerize, that is D1 and D2 may be different. In such an embodiment, D1 and D2 may be referred to as heterodimerization domains Ht1 and Ht2. Each inducer molecule is in this case bound by Ht1 from one protein and Ht2 from the other. Various configurations of chimeric proteins comprising two dimerization domains are depicted in Figure 2. As noted above, the configuration of the chimeric protein is such that D1, and in particular Ht1, does not significantly dimerize with D2, in particular Ht2, within the same chimeric protein molecule. However, when two chimeric proteins come together in the presence of a dimerization inducer, D1/Ht1 from one chimeric protein dimerizes with D2/Ht2 from the other chimeric protein, causing dimerization of the two signaling domains. For example, in a cell expressing the present chimeric protein the presence of the inducer causes a greater proportion of dimerization between two chimeric proteins, than dimerization within the same chimeric protein. The amount of chimeric proteins which are dimerized within the same molecule in a cell or cell population, or in solution, may be less than 50%, 40%, 30%, 20%, 10%, 5% or 1 % of the amount of chimeric proteins which are dimerized with a separate chimeric protein molecule, in the presence of the inducer. 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 dimerization between proteins of interest that are fused to inducer-binding domains. Such systems have been described for use in cell biology for different applications, to bring proteins into proximity, for example to investigate signalling pathways and other biological mechanisms, in medicine to degrade or inactivate pathogenic proteins, and in gene and cell therapy. A typical chemical inducer of dimerization (CID), or dimerization inducer to use the terminology herein, has the feature of being able to interact with, or bind to, two proteins or protein domains, one on either side of the molecule. It thus has two binding sites, or binding surfaces (or more generally, interaction sites). In the case of heterodimerization, the inducer is capable of interacting with, or binding to, two different proteins or dimerization domains. In the case of homodimerization, the inducer is capable of interacting with, or binding to, two copies, or molecules of the same dimerization domain (the same dimerization may be contained within two different chimeric proteins, for example where the signalling domains differ, as indicated above). The original systems were based on the macrolides FK506 and rapamycin, which are capable of binding to, and therefore inducing heterodimerization of, various different proteins or protein domains, including FK506-binding protein (FKBP), the FKBP-rapamycin domain of mTOR (FRB), calcineurin, and cyclophilin, which can be used in different combinations to achieve heterodimerization domain pairs and CID combinations, as detailed below. Such systems may include the use of cyclosporine, which binds to calcineurin or to cyclophilin, Subsequently, other CID heterodimerization systems based on different molecules have been developed and are described in the literature. Furthermore, homodimerization systems based on FK506 derivatives which are able to bind two FKBP molecules have been developed, i.e. based on symmetric or dimeric inducers, which comprise two binding sites for the same dimerization domain, as discussed further below. In an embodiment the inducer is rapamycin or an analogue thereof, and the dimerization domains are protein domains which bind thereto. Rapamycin and rapamycin analogues induce heterodimerization by generating an interface between the FRB domain of mTOR and a FK506-bindng protein (FKBP). This association results in FKBP blocking access to the mTOR active site inhibiting its function. While mTOR is a very large protein, the precise small segment of mTOR required for interaction with Rapamycin is known and can be used. The macrolides rapamycin and FK506 act by inducing the heterodimerization of cellular proteins. Each drug binds with a high affinity to the FKBP12 protein, creating a drug-protein complex that subsequently binds and inactivates mTOR/FRAP and calcineurin, respectively. The FKBP-rapamycin binding (FRB) domain of mTOR has been defined and applied as an isolated 89 amino acid protein moiety that can be fused to a protein of interest. Rapamycin can then induce the approximation of FRB fusions to FKBP12 or proteins fused with FKBP12. In the present context one of the dimerization domains (including one of Ht1 or Ht2 where there are two dimerization domains in an interaction domain) may be or comprise FRB, and the other dimerization domain (including the other of Ht2 or Ht1) may be or comprise FKBP. 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. Rapamycin has several properties of an ideal dimerizer: it has a high affinity (KD<1 nM) for FRB when bound to FKBP, and is highly specific for the FRB domain of mTOR. Rapamycin is an effective therapeutic immunosuppressant with a favourable pharmacokinetic and pharmacodynamics profile in mammals. Pharmacological analogues of Rapamycin with different pharmacokinetic and dynamic properties such as Everolimus, Temsirolimus and Deforolimus (Benjamin et al, Nature Reviews, Drug Discovery, 2011) may also be used according to the clinical setting. In order to prevent rapamycin binding and inactivating endogenous mTOR, the surface of rapamycin which contacts FRB may be modified. Compensatory mutation of the FRB domain to form a surface that accommodates the "bumped" rapamycin restores dimerizing interactions only with the FRB mutant and not to the endogenous mTOR protein. Bayle et al. (Chem Bio; 2006; 13; 99-107) describes various rapamycin analogues, or "rapalogs" and their corresponding modified FRB binding domains. For example, Bayle et al. (2006) describes the rapalogs: C-20-methyllyrlrapamycin (MaRap), C16(S)-Butylsulfonamidorapamycin (C16-BS-Rap) and C16-(S)-7- methylindolerapamycin (AP21976/C16-AiRap), as shown in Figure 3, in combination with the respective complementary binding domains for each. Other rapamycins/rapalogs include sirolimus and tacrolimus (FK506). Thus, in such an embodiment, a dimerization domain in one chimeric protein comprises FKBP and the cognate dimerization domain in another chimeric protein comprises FRB. In the case of an interaction domain with 2 dimerization domains Ht1 and Ht2, one of Ht1 and Ht2 comprises FKBP and the other comprises FRB. In particular, the FKBP domain may comprise FKBP12. Heterodimerization between the FRB domain of one chimeric protein molecule and the FKBP domain of another chimeric protein molecule causes dimerization of the signalling domains. Rapamycin is a standard pharmaceutical with well understood properties, excellent bioavailability and volume of distribution and which is widely available. Rapamycin also does not aggravate the condition being treated, in fact, as it is an immunosuppressant it is likely to have a beneficial effect on unwanted toxicity. Furthermore, in cases where the cells expressing the protein are being used to achieve immunosuppression, notably with Treg cells expressing a CAR or TCR, the immunosuppressive properties of rapamycin and its analogues may be beneficial. As indicated above, sequences for FRB and FKBP domains are known in the art. For example, FKBP/FRB may have or may comprise a sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 5, or a variant thereof. SEQ ID NO: 1 is the native (wild-type) human FKBP12 domain; SEQ ID NO: 2 is the wild-type FRB segment of mTOR; SEQ ID NO: 3 is FRB with T to L substitution at 2098 which allows binding to AP21967; SEQ ID NO: 4 is a FRB segment of mTOR with T to H substitution at 2098 and W to F substitution at residue 2101 of the full mTOR which binds Rapamycin with reduced affinity to wild type; SEQ ID NO: 5 is a FRB segment of mTOR with K to P substitution at residue 2095 of the full mTOR which binds Rapamycin with reduced affinity. Additional FRB and FKBP domain sequences include the following SEQ ID NOs: and variants thereof: SEQ ID NO: 6 represents a FRB sequence as shown as part of the protein of SEQ ID NO.1 of WO 2016/135470; SEQ ID NO: 7 represents a FKBP sequence which is contained in the protein of SEQ ID NO.4 of WO 2016/135470; SEQ ID NO: 8 is a so-called “codon-wobbled” FKBP sequence, in which the codons encoding the FKBP domain have been altered to prevent recombination; SEQ ID NO: 88 represents a FKBP sequence as shown as part of the protein of SEQ ID NO.1 of WO 2016/135470; SEQ ID NO: 89 represents a FKBP sequence as shown as part of the protein of SEQ ID NO.2 of WO 2016/135470. SEQ ID NO: 107 represents a 109 amino acid FKBP sequence. SEQ ID NOs: 112 and 113 represent 99 and 94 amino acid FRB sequences respectively. Variant sequences may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 1 to 8, 88, 89, 107, 112 or 113 provided that the sequences provide an effective dimerization system. That is, provided that the sequences facilitate sufficient co-localization of the two chimeric proteins to allow dimerization of the two signalling domains. Particularly, FRB and/or FKBP variants may retain the ability to bind to rapamycin or an analogue thereof, e.g. have at least 30, 40, 50, 60, 70, 80 or 90% of the binding affinity of FRB or FKBP to rapamycin/rapamycin analogue. The "wild-type" FRB domain shown as SEQ ID NO: 2 comprises amino acids 2025-2114 of human mTOR. Using the amino acid numbering system of human mTOR, the FRB sequence of the chimeric protein may comprise an amino acid substitution at one of more of the following positions: 2095, 2098 and 2101. The variant FRB used in the chimeric protein of the invention may comprise one of the following amino acids at positions 2095, 2098 and 2101: 2095: K, P, T or A; 2098: T, L, H or F; 2101: W or F. Bayle et al (as above) describe the following FRB variants, annotated according to the amino acids at positions 2095, 2098 and 2101 (see Table 1 of Bayle): KTW, PLF, KLW, PLW, TLW, ALW, PTF, ATF, TTF, KLF, PLF, TLF, ALF, KTF, KHF, KFF, KLF. These variants are capable of binding rapamycin and rapalogs to varying extents, as shown in Table 1 and Figure 5A of Bayle et al. The chimeric protein herein may comprise any one of these FRB variants. Whilst in many cases, rapamycin is an attractive inducer, there may nonetheless be situations where it is desired or preferable to use another inducer or other heterodimerization domains, for example in certain clinical situations. Thus, systems based on other inducers and other heterodimerization domains are available and may be used. For example, FK506 is an inhibitor of the protein calcineurin. Thus, calcineurin also binds to FK506 (when FK506 is bound by FKBP) and may also be used to provide, or is the basis for, a heterodimerization domain which binds to FK506 as inducer, along with FKBP as the cognate heterodimerization domain. Thus, in another embodiment the inducer is FK506, and the cognate dimerization domains of two chimeric proteins, or Ht1 and Ht2 in a single chimeric protein, are protein domains which bind thereto. In such an embodiment, one of the dimerization domains (including one of Ht1 or Ht2) may be or comprise calcineurin or a FK506- binding fragment thereof, and the dimerization domain (including Ht2 or Ht1) may be or comprise FKBP (which may be as defined above, and includes for example FKBP12). The term “calcineurin” includes variants and derivatives 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. In this context a variant or derivative of calcineurin retains the ability of the parent molecule to bind to FK506. Calcineurin A is the 59 kDa catalytic subunit of the calcineurin protein and the dimerization domain may be or may comprise calcineurin A or a FK506-binding fragment thereof. By way of representative example, the sequence of an isoform of human calcineurin A (UniprotKB –Q08209) is shown in SEQ ID NO.90. In another embodiment, the inducer may be the calcineurin inhibitor cyclosporine, also known as cyclosporine A (CsA), and the cognate dimerization domains of two chimeric proteins, or Ht1 and Ht2 in a single chimeric protein, may be protein domains which bind thereto. In this regard, cyclophilins (CyPs) are a family of proteins characterised by their ability to bind to CsA. Calcineurin activity is inhibited by CsA when bound to CyP (in other words, calcineurin binds to CsA when it is bound to CyP). Thus, cyclophilins may be used to provide a heterodimerization domain capable of binding to CsA as inducer. In such an embodiment, one of the dimerization domains (including Ht1 or Ht2) may be or comprise calcineurin or a CsA-binding fragment thereof (more particularly a CsA-CyP binding fragment), and the other dimerization domain (including Ht2 or Ht1) may be or comprise cyclophilin or a CsA-binding fragment thereof. As noted above the calcineurin may be a variant or derivative of a native, or wild-type calcineurin molecule. In this context a variant or derivative of calcineurin retains the ability of the parent molecule to bind to CsA. The dimerization domain may be or may comprise calcineurin A or a CsA- (or CsA-CyP)- binding fragment thereof. Various isoforms of cyclophilin exist and the term “cyclophylin” (CyP) includes all such isoforms, e.g. cyclophilin A, B, C and D, and variants and derivatives thereof. In particular the cyclophilin may be CyPA. Variants of CyP include amino acid sequences having one or more amino acid modifications (e.g. substitutions, additions and/or deletions) relative to the native sequence. In this context a variant or derivative of cyclophilin retains the ability of the parent molecule to bind to CsA. For use in CID systems, a cyclophilin heterodimerization domain may be provided as a fusion protein with Fas (CyP-Fas). A representative sequence of cyclophilin, isoform 1 (UniprotKB P62937-1) is shown in SEQ ID NO.91. Based on the CsA and FK506 systems discussed above, a “heterodimeric inducer” molecule has been developed which combines a protein-binding interface from FK506 with a protein-binding interface from CsA, to form the molecule FKCsA (Belshaw, et al., 1996, PNAS, 93, 4604-4607). Accordingly, in a further embodiment, the dimerization inducer (CID) is FKCsA, and the cognate dimerization domains of two chimeric proteins, or Ht1 and Ht2 in a single chimeric protein, are protein domains which bind thereto. In such an embodiment, one of the dimerization domains (including Ht1 or Ht2) may be or comprise cyclophilin or a CsA-binding fragment thereof, and the other dimerization domain (including Ht2 or Ht1) may be or comprise FKBP (Cyclophilin and FKBP and their respective fragments and variants may be as defined above, and include FKBP12 and CyP-Fas). Further CID dimerization systems which may be used include those based on the self-labelling proteins SNAP-tag, a mutant of the DNA Repair protein O6- alkylguanine-DNA-alkyltransferase (available from New England Biolabs; and see Gautier et al., 2008, Chem. Biol.15, 128-136), and HALO-Tag, a haloalkane dehalogenase derivative designed to covalently bind to synthetic ligands (available from Promega; and see Los, et al., 2008. ACS Chem. Biol.3, 373-382). CIDs for SNAP-tag and HALO-tag have been developed, collectively termed HaXS, which function as chemical cross-linkers to bind SNAP-tag and HALO-tag fusion proteins together. Thus, in another embodiment the inducer is a HaXS molecule, e.g. HaXS8, and the cognate dimerization domains of two chimeric proteins, or Ht1 and Ht2 in a single chimeric protein, are protein domains which bind thereto. In such an embodiment, one of the dimerization domains (including Ht1 or Ht2) may be or comprise a SNAP-tag and the other dimerization domain (including Ht2 or Ht1) may be or comprise a HALO-tag. A still further heterodimerization system which may be used is based on fusicoccin (FC) as CID, which binds simultaneously to a 14-3-3 protein and the C- terminal 52 amino acids (CT52) of a plant plasma membrane H+-ATPase (PMA) to stabilize the interaction between these two proteins. Accordingly, in another embodiment, the inducer is fusicoccin, and the cognate dimerization domains of two chimeric proteins, or Ht1 and Ht2 in a single chimeric protein, are protein domains which bind thereto. In such an embodiment, one of the dimerization domains (including Ht1 or Ht2) may be or comprise a 14-3-3 protein or a FC-binding fragment thereof, and the other dimerization domain (including Ht2 or Ht1) may be or comprise a C-terminal peptide of PMA, e.g. a CT52 peptide. The 14-3-3 protein may for example be tobacco 14-3-3 (T14-3-3c) or a truncation thereof (T14-3-3cΔC, residues 1-242). The C-terminal peptide of PMA may be the CT52 fragment of tobacco H+ ATPase PMA2. The terms “14-3-3 protein”, “C-terminal peptide of PMA” and “CT52” include variants and derivatives 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. In this context a variant or derivative retains the ability of the parent molecule to bind to fusicoccin. One such variant of CT52 of tobacco H+ ATPase PMA2 comprises three amino acid substitutions S939A, T955D and V956I. The tobacco 14-3-3 and CT52 interaction partners been described in the literature (Ottman et al., 2007, Mol. Cell 25(3), 427- 440, and Truong et al., 2002, Proteins 49(3), 321-325). The full T14-3-3-3c sequence (UniProtKB- Q5KTN5) is shown in SEQ ID NO: 92. The CT52 sequence of Tobacco H+ ATPase PMA2 as derived from UniProtKB-Q40409 is shown in SEQ ID NO: 93. 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. to a reference SEQ ID NO: as specified herein), for example to any one of SEQ ID NOs: 90, 91, 92 or 93 as referred to above. In particular, such a variant retains the desired or required property of the parent molecule from which it is derived, i.e. the reference sequence. Thus, the variant sequence may have the stated % sequence identity provided that the variant sequence provides an effective dimerization system. That is, provided that the sequences facilitate sufficient co-localization of the two chimeric proteins to allow dimerization of the two signalling domains. Particularly, the variants may retain the ability to bind to their respective inducer, e.g. have at least 30, 40, 50, 60, 70, 80 or 90% of the binding affinity of the parent molecule to calcineurin, cyclophilin or fusicoccin. Inducer-based homodimerization systems are also available, most notably based on FK1012, a homodimer of FK506, capable of binding to two molecules of FKBP. Various derivatives of such dimeric inducer molecules have been developed, including various dimeric FK506-like analogues, including for example the molecules AP1903 (CAS 195514-63-7) and AP20187 (CAS 195514-80-8) which are commercially available from various sources. Other multimeric inducer molecules capable of binding to FKBP have been developed. Accordingly, in an embodiment the dimerization domain in a chimeric protein may be or may comprise FKBP, and homodimerization between two chimeric proteins each comprising a FKBP domain may be achieved using a dimeric inducer such as FK1012, AP1903 and AP20187. It will be noted that AP1903 and AQP21087 may bind with high affinity to a FKBP12 variant sequence comprising the amino acid substitution F36V, as shown in SEQ ID NO: 35, which represents an exemplary homodimerization domain sequence for use herein. In this regard FKBP12 F35V binds to AP1903 with higher affinity than wild-type FKBP12 binding to AP1903. In some embodiments, a dimerization domain may contain an amino acid sequence of an FKBP comprising a modification selected from the group consisting of: (i) a FKBP polypeptide containing one or more amino acid substitutions, (ii) two or three tandem repeats of an unmodified (naturally occurring) FKBP amino acid sequence, and (iii) two or three tandem repeats of a FKBP polypeptide containing one or more amino acid substitutions in some embodiments, the FKBP protein is a human FKBP protein (GenBank: CAG46965.1) and the modifications to FKBP described herein are made to the human FKBP protein in some embodiments, the one or more amino acid substitutions in FKBP include one or more of: F36V, L106P, E31G, R71G, and K105E, residues in reference to the human FKBP protein (GenBank: CAG46965.1). In the embodiments, where the dimerization domain comprises two or three tandem repeats of the dimerization domain sequences disclosed herein, each repeat may comprise a different mutation of that sequence. For example, in an exemplary embodiment, the dimerization domain comprises three tandem repeats of an FKBP sequence, where one of the repeats comprises a natural FKBP sequence, a second repeat comprises FKBP containing F36V substitution, and a third repeat comprises FKBP containing F36V and L106P substitutions, in any order. In some embodiments, a dimerization domain may contain an amino acid sequence of an FKBP comprising a modification selected from the group consisting of: (i) a FKBP polypeptide containing F38V substitution, (ii) a FKBP polypeptide containing F36V and L106P substitutions, (iii) a FKBP polypeptide containing E31G, F38V, R71G, and K105E substitutions, and (iv) two or three tandem repeats of any of these FKBP polypeptides. Representative dimerization domains may comprise a FKBP dimerization domain sequence selected from SEQ ID NOs: 36-40. Other inducible homodimerization systems which may be used include dimerization domains based on the amino-terminal subdomain of the B subunit of bacterial DNA gyrase (GyrB), which may be induced to homodimerize by the inducer courmermycin (Mohi et al., 1998 Mol. Biol. Cell 9, 3299-3308). Homodimerizable dimerization domains may also be obtained from the extracellular binding regions of various receptors, such as cytokine receptors or TNFR-family receptors, or from binding proteins for various molecules. This may include for example steroid binding protein, estrogen binding protein, glucocorticoid binding protein, or tetracycline binding protein. Such dimerization domains may be induced to dimerize in the presence of the ligand (e.g. cytokine etc.) or the binding target for the receptor/binding protein in question. Since the administration of a cytokine ligand to induce dimerization may have other effects, this may in some circumstances be less preferred. However, dimerization domains based on TNFR- family receptors such as TNFR2 or OX40, for example, may be used. Various such homodimerization systems which may be used are described in WO2019/169290 of Allogene Therapeutics Inc., the disclosure of which is incorporated herein by reference. By way of representative example, the sequence of a dimerization domain based on OX40 (1-214) is given in SEQ ID NO: 73. Other homodimerization domains described in WO2019/169290 which may be used include those based on or derived from the extracellular domains of immune co-receptors or ligands such as CD80 and PD-1. It will thus be seen that the dimerization inducer may be any molecule which induces dimerization between dimerization domains on separate chimeric molecules (including D1 and D2 on separate chimeric molecules having the same D1 and D2 domains). It may be a molecule to which both dimerization domains may bind in any way. This may be for example affinity-based binding (in other words the inducer may be a ligand), or the inducer may be a molecule which allows interaction between the dimerization domains by other means, such as chemical cross-linking. Thus, the binding may include covalent bonding, and the inducer may be a chemical cross- linker (i.e. a bifunctional molecule which comprises a first group reactive with a first dimerization domain (e.g. D1) and a second group reactive with a second dimerization domain (e.g. D2). The inducer may be rapamycin or a rapamycin analogue ("rapalog") which has improved or differing pharmacodynamic or pharmacokinetic properties to rapamycin but have the same broad mechanism of action. The inducer may be an altered rapamycin with engineered specificity for complementary FKBP12 or FRB - for example as shown in Figure 3. Bayle et al (2006, as above) describes various rapalogs functionalized at C16 and/or C20. Examples of such rapalogs in the first category include Sirolimus, Everolimus, Temsirolimus and Deforolimus. Examples of rapalogs in the second category include C-20-methyllyrlrapamycin (MaRap); C16(S)-Butylsulfonamidorapamycin (C16-BS- Rap); C16-(S)-3-methylindolerapamycin (C16-iRap); and C16-(S)-7- methylindolerapamycin (AP21976/C16-AiRap). Rapamycin is a potent immunosuppressive agent. Analogues of rapamycin are in every day clinical use. Modern rapalogs have excellent bioavailability and volumes of distribution. Although they are potent immunosuppressive agents, a short dose (to activate the signalling protein) would be expected to have minimal side- effects when treating conditions requiring an increased immune response, such as cancer (although long term dosing of rapamycin may not be desirable in such subjects/patients). As discussed above, the immunosuppressive effects would be beneficial when treating an inflammatory condition or disease, e.g. an autoimmune disease or transplant rejection, e.g. using a cell of the invention. Other examples of inducers include FK506, CsA, FKCsA and Fusicoccin, as discussed above and depicted in Figure 4A-D. HaSX inducers may be represented by HaSX8, also depicted in Figure 4E. Other inducers include dimeric FK506 molecules or derivatives or analogues thereof, including FK1012, AP1903 and AP21087. Still further inducers include ligands and binding partners for binding proteins as discussed above. In other embodiments, an inducer for dimerization is not required, and dimerization systems can be based on cognate dimerization domains which associate, e.g. bind or interact in any way, when in proximity. Again, such dimerization systems are known in the art, and any such system may be adopted, or adapted, for use herein. Particular mention may be made in this regard 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 leucine residues evenly spaced through an alpha-helix. Thus, in an embodiment a dimerization domain herein is or comprises a leucine zipper sequence. These may be used for hetero- or homodimerization, according to the leucine zipper sequence which is used. 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: 99 (this can include GG at N terminus) and a Fos leucine zipper sequence is shown in SEQ ID NO: 100. Heterodimerization domains comprising Jun and Fos leucine zippers respectively may be used. Alternatively, homodimerization domains comprising Jun leucine zippers may be used. Other leucine zipper dimerization domains known in the art include those based on ZIP proteins, a class of transcription factors. A ZIP domain is a region of alpha-helix containing leucines which line up to form the leucine zipper motif. A ZIP domain can interact with leucines on other ZIP domains to reversibly hold the alpha- helices together (i.e. to dimerize them). Thus, a dimerization domain herein can comprise a BZip or AZip leucine zipper domain. For example, a heterodimerization domain can be or comprise a BZip (RR) domain which heterodimerizes with an AZip (EE) domain. Leucine zippers are an example of a coiled coil structural protein motif which may 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 and any of these could be used. For example, suitable leucine zipper domains can include SYNZIP 1 to SYNZIP 48. Other examples of leucine zipper domains include BATF, ATF4, ATF3, BACH1, JUND, NFE2L3, and HEPTAD. The sequence of a BZip (RR) leucine zipper domain is shown in SEQ ID NO: 101. The sequence of a AZip (EE) leucine zipper domain is shown in SEQ ID NO: 102. In some embodiments, a suitable pair of leucine zipper domains has a dissociation constant (Kd) of 1000 nM or less, for example 100 nM or less, 10 nM or less, or 1 nM or less. Further exemplary pairs of dimerization domains can include PSD95- Dlgl-zo-1 (PDZ) domains, or a streptavidin domain and a streptavidin binding protein (SBP) domain. Other dimerization domains may be obtained or derived from other proteins known to interact or bind to each other. For example, a heterodimerization domain pair can comprise CD80 and PDL-1. A still further example of a homodimerization domain is the Fc region of immunoglobulin G. Fc regions have widely been used in fusion proteins, including to provide dimerization domains, and various fragments and mutants of dimerizable Fc regions have been described in the literature, for example a fragment lacking the first 5 amino acids of the Fc region. The present chimeric protein comprises a signalling domain, also referred to as an endodomain, which comprises a tyrosine kinase activating domain comprising at least a JAK1-binding motif, and a tyrosine effector domain which can be phosphorylated by the JAK1 kinase. Phosphorylation of the tyrosine effector domain allows the signalling cascade to be effected, for example to allow other proteins in the signalling cascade to bind to the effector domain and/or become activated to transmit the signal in the cell. In other words, upon phosphorylation, the tyrosine effector domain can recruit a signal transduction factor. The tyrosine kinase activating domain may in some embodiments also include a JAK2-binding motif and/or a JAK3-binding motif. In particular, it may include a JAK1- and a JAK3-binding motif. In an embodiment, for example where the chimeric protein does not include a JAK3-binding motif in the signalling domain, the chimeric protein may be used in conjunction with a second chimeric protein which comprises a signalling domain comprising a JAK3-binding motif. The signalling domain of the chimeric protein may signal through the JAK- STAT signalling pathway, or in other words, the signal may be mediated by activation of the JAK-STAT signalling pathway. STAT proteins are transcription factors which are recruited to an activated receptor, and accordingly, in particular the tyrosine effector domain may comprise a STAT association motif, that is a binding site for a STAT. The 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 (RTK). The tyrosine effector domain may contain one or more, e.g. two or more, for example, 3, 4, 5 or more STAT association motifs, which may be the same or different. By way of example, STAT5 is a transcription factor involved in the IL-2 signalling pathway that plays a key role in Treg function, stability and survival by promoting the expression of genes such as FOXP3, IL2RA and BCLXL. In order to be functional and translocate into the nucleus, STAT5 needs to be phosphorylated. IL-2 ligation results in STAT5 phosphorylation by activating the JAK1/JAK2 and JAK3 kinases via specific signalling domains present in the IL-2Rβ and IL-2Rγ chain, respectively. Although JAK1 (or JAK2) can phosphorylate STAT5 without the need of JAK3, STAT5 activity is increased by the transphosphorylation of both JAK1/JAK2 and JAK3, which stabilizes their activity. “STAT association motif” as used herein refers to an amino acid motif which comprises a tyrosine and, upon phosphorylation of the tyrosine, is capable of binding a STAT polypeptide. Any method known in the art for determining protein:protein interactions may be used to determine whether an association motif is capable of binding to a STAT. For example, co-immunoprecipitation followed by western blot. The STAT association motif may for example be a STAT5 association motif which is capable, upon phosphorylation, of binding a STAT5 polypeptide (and similarly for the other STAT polypeptides). In one embodiment, the STAT association motif is a STAT5 association motif. Suitably, the signalling domain may comprise two (e.g. at least two) or more 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 an embodiment, the signalling domain may comprise two or three STAT5 association motifs as defined herein. Suitably, the STAT5 association motif may exist endogenously in a cytoplasmic domain of a transmembrane protein which may be used to provide the signalling domain of the chimeric protein herein. For example, the STAT5 association motif may be from an interleukin receptor (IL) receptor endodomain or a hormone receptor. The signalling domain may comprise an amino acid sequence selected from any chain of the interleukin receptors where STAT5 is a downstream component, for example, the cytoplasmic domain comprising amino acid numbers 266 to 551 of IL-2 receptor β chain (NCBI REFSEQ: NP_000869.1, SEQ ID NO: 9), amino acid numbers 265 to 459 of IL- 7R α chain (NCBI REFSEQ: NP_002176.2, SEQ ID NO: 10), amino acid numbers 292 to 521 of IL-9R chain (NCBI REFSEQ: NP_002177.2, SEQ ID NO: 11), amino acid numbers 257 to 825 of IL-4R α chain (NCBI REFSEQ: NP_000409.1, SEQ ID NO: 12), amino acid numbers 461 to 897 of IL-3R β chain (NCBI REFSEQ: NP_000386.1, SEQ ID NO: 13) and/or amino acid numbers 314 to 502 of IL-17R β chain (NCBI REFSEQ: NP_061195.2, SEQ ID NO: 14) may be used. It will be appreciated by a skilled person that any one or more of these sequences can be used. The entire region of the cytoplasmic domain of an interleukin receptor chain may be used. SEQ ID NO: 15 represents an IL7RA 2Y truncated sequence, which may also be used. The signalling domain may comprise one or more STAT5 association motifs that comprise an amino acid sequence shown as SEQ ID NOs: 9-15 or a variant which is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NOs: 9-15. For example, the variant may be capable of binding STAT5 to 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 an amino acid sequence shown as one of SEQ ID NOs: 9-15. The variant or derivative may be capable of binding STAT5 to a similar or the same level as one of SEQ ID NOs: 9-15 or may be capable of binding STAT5 to a greater level than an amino acid sequence shown as one of SEQ ID NOs: 9-15 (e.g. increased by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%). For example, the STAT5 association motif may be from any one or more of IL2Rβ, IL7Rα, IL-3Rβ (CSF2RB), IL-9R, IL-17Rβ, erythropoietin receptor, thrombopoietin receptor, growth hormone receptor and prolactin receptor. A chimeric protein may, for example, comprise STAT association motifs from both IL2Rβ and IL7Rα. The STAT5 association motif may comprise the amino acid motif YXXF/L (SEQ ID NO: 16); wherein X is any amino acid. Suitably, the STAT5 association motif may comprise the amino acid motif YCTF (SEQ ID NO: 17), YFFF (SEQ ID NO: 18), YLSL (SEQ ID NO: 19), or YLSLQ (SEQ ID NO: 20). The signalling domain may comprise one or more STAT5 association motifs comprising the amino acid motif YCTF (SEQ ID NO: 17), YFFF (SEQ ID NO: 18), YLSL (SEQ ID NO: 19), and/or YLSLQ (SEQ ID NO: 20). The signalling domain may comprise a first STAT5 association motif comprising the amino acid motif YLSLQ (SEQ ID NO: 20) and a second STAT5 association motif comprising the amino acid motif YCTF (SEQ ID NO: 17) or YFFF (SEQ ID NO: 18). The signalling domain may comprise the following STAT5 association motifs: YLSLQ (SEQ ID NO: 20), YCTF (SEQ ID NO: 17) and YFFF (SEQ ID NO: 18). Association motifs for other STAT polypeptides are known in the art, and may be used. For example, to provide a STAT3 signal to a cell (particularly a Tcon cell), the tyrosine effector domain of the signalling domain may comprise YXXQ (SEQ ID NO: 84), where X is any amino acid, for example YRHQ (SEQ ID NO: 85). The STAT3 association motif is present in signalling proteins for example IL-6R, IL10R and IL21R. In one embodiment, the signalling domain of a chimeric protein may comprise the cytoplasmic domain of the IL21R alpha chain, e.g. comprising amino acid numbers 256-538 of the IL-21R alpha chain (NCBI RefSeq: NP_068570.1), or a truncated fragment thereof comprising a box 1 motif (amino acid numbers 266 to 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 flanking 3 residues at the C-terminal side of tyrosine residue 500, i.e. YLRQ (SEQ ID NO: 114), required for STAT1/3 association. Alternatively, STAT1 or STAT4 signalling 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.105) “JAK1-binding motif” as used herein refers to a BOX motif which allows for tyrosine kinase JAK1 association. Analogously, “JAK2-binding motif” as used herein refers to a BOX motif which allows for tyrosine kinase JAK2 association. Suitable JAK1- and JAK2-binding motifs are described, for example, by Ferrao & Lupardus (Frontiers in Endocrinology; 2017; 8(71); which is incorporated herein by reference). As noted above, the JAK1- and/or JAK2-binding motif may occur endogenously in a cytoplasmic domain of a transmembrane protein. For example, the JAK1- and/or JAK2-binding motif may be 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. The JAK1-binding motif may comprise or consist of an amino acid motif shown as SEQ ID NOs: 21-27 or a variant thereof which is capable of binding JAK1. KVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERD K (SEQ ID NO: 21) NPWFQRAKMPRALDFSGHTHPVATFQPSRPESVNDLFLCPQKELT (SEQ ID NO: 22) GYICLRNSLPKVLNFHNFLAWPFPNLPPLEAMDMVEVIYINR (SEQ ID NO: 23) PLKEKSIILPKSLISVVRSATLETKPESKYVSLITSYQPFSL (SEQ ID NO: 24) RRRKKLPSVLLFKKPSPFIFISQRPSPETQDTIHPLDEEAFLK (SEQ ID NO: 25) YIHVGKEKHPANLILIYGNEFDKRFFVPAEKIVINFITLNISDDS (SEQ ID NO: 26) RYVTKPPAPPNSLNVQRVLTFQPLRFIQEHVLIPVFDLSGP (SEQ ID NO: 27) The variant of SEQ ID NOs: 21-27 may comprise one, two or three amino acid differences compared to any of SEQ ID NOs: 21-27 and retain the ability to bind JAK1. The variant may be at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to any one of SEQ ID NOs: 21-27 and retain the ability to bind JAK1. In a preferred embodiment, the JAK1-binding domain comprises or consists of SEQ ID NO: 21 or a variant thereof which is capable of binding JAK1. For example, the variant may be capable of binding JAK1 to 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 a corresponding, reference sequence. The variant or derivative may be capable of binding JAK1 to a similar or the same level as a corresponding, reference sequence or may be capable of binding JAK1 to a greater level than a corresponding, reference sequence (e.g. increased by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%). A JAK2-binding motif may comprise or consist of an amino acid motif shown as SEQ ID NO: 28-30 or a variant therefore which is capable of binding JAK2. NYVFFPSLKPSSSIDEYFSEQPLKNLLLSTSEEQIEKCFIIEN (SEQ ID NO: 28) YWFHTPPSIPLQIEEYLKDPTQPILEALDKDSSPKDDVWDSVSIISFPE (SEQ ID NO: 29) YAFSPRNSLPQHLKEFLGHPHHNTLLFFSFPLSDENDVFDKLSVIAEDSES (SEQ ID NO: 30) The variant of SEQ ID NOs: 28-30 may comprise one, two or three amino acid differences compared to any of SEQ ID NOs: 28-30 and retain the ability to bind JAK2. For example, the variant may be capable of binding JAK2 to 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 a corresponding, reference sequence. The variant or derivative may be capable of binding JAK2 to a similar or the same level as a corresponding, reference sequence or may be capable of binding JAK2 to a greater level than a corresponding, reference sequence (e.g. increased by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%). Any method known in the art for determining protein:protein interactions may be used to determine whether a JAK1- or JAK2-binding motif is capable of binding to a JAK1 or JAK2. For example, co-immunoprecipitation followed by western blot. Suitably, the signalling domain may comprise an IL2Rβ endodomain shown as SEQ ID NO: 9; or a variant which has at least 80% sequence identity to SEQ ID NO: 9. The variant may be at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 9. Suitably, the signalling domain may comprise a truncated IL2Rβ endodomain shown as any one of SEQ ID NO: 31 or 32 or a variant of any one of SEQ ID NO: 31 or 32 which has at least 80% sequence identity thereto. SEQ ID NO: 31 represents a IL2RB truncated variant with a Y510 mutation. SEQ ID NO: 32 represents a IL2RB truncated variant with Y510 and Y392 mutations. The variant may be at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 31 or 32. STAT, e.g. STAT5, activity is increased by the transphosphorylation of both a JAK1/2 and JAK3, as this stabilizes their activity. As noted above, the signalling domain, or more particularly the tyrosine kinase activating domain thereof, may further comprise a JAK3-binding motif. “JAK3-binding motif” as used herein refers to a BOX motif which allows for tyrosine kinase JAK3. Suitable JAK3-binding motifs are described, for example, by Ferrao & Lupardus (Frontiers in Endocrinology; 2017; 8(71); which is incorporated herein by reference). Any method known in the art for determining protein:protein interactions may be used to determine whether a motif is capable of binding to JAK3. For example, co- immunoprecipitation followed by western blot. The JAK3-binding motif may occur endogenously in a cytoplasmic domain of a transmembrane protein. For example, the JAK3-binding motif may be from an IL-2Rγ polypeptide. A functional truncated or variant IL2Rγ polypeptide may be used within the signalling domain of the chimeric protein, wherein 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 binding activity of IL2Rγ). Particularly, a truncated IL2Rγ comprising a JAK3-binding motif and a truncated IL2Rβ comprising a STAT5 association motif, and a JAK1-binding motif may be comprised in the signalling domain of a chimeric protein as defined herein. Functional truncations may provide an advantage of reducing construct size for expression. The JAK3-binding motif may comprise or consist of an amino acid motif sequence shown as SEQ ID NO: 33 or SEQ ID NO: 34 or a variant thereof which is capable of binding JAK3 (e.g. a functional variant or fragment having at least 80, 85, 90, 95 or 99% identity to SEQ ID NOs: 33 or 34). The variant may be at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 33 or SEQ ID NO: 34. In a particular embodiment, the signalling domain comprises one or more JAK1-binding domains and at least one JAK3-binding domain/motif (e.g. at least 2 or 3 JAK3-binding domains/motifs). It will be appreciated by a skilled person that the polynucleotide sequence encoding the JAK3-binding domain may be positioned upstream or downstream (5’ or 3’) of the polynucleotide sequence encoding the tyrosine effector domain, for example, the STAT, e.g. STAT5, association motif and JAK1-binding motif. Typically, the JAK1- (and if present additionally the JAK2-) binding motif would be upstream (5’) of the tyrosine effector domain, e.g. STAT/STAT5, but this may be varied. Particularly, the polynucleotide encoding the JAK3-binding domain may be positioned downstream (3’) of the polynucleotide encoding the STAT association motif and the JAK1, and optionally JAK2, binding motifs. Thus, alternatively viewed, in the chimeric protein as described herein, the JAK3-binding domain may be N- or C-terminal to the tyrosine effector domain (e.g. STAT association motif) and the JAK1-binding domain, preferably C-terminal. In one embodiment, the JAK3-binding domain and the STAT association motif/JAK1-binding domains are positioned directly adjacent to one another (i.e. are not separated distally by sequence). In a particular embodiment, the JAK3-binding domain is translated in reverse orientation, thus the JAK3-binding motif may comprise a sequence in the reverse orientation to SEQ ID NOs: 33 or 34 (e.g. as shown in SEQ ID NO: 87). The polynucleotide encoding the signalling domain may thus comprise nucleotide sequences in the following order: 5’-3’ JAK1, 5’-3’ STAT association motif, 3’-5’ JAK3. The interaction domain may be linked to the N- or C-terminal of the signalling domain. Furthermore, where the interaction domain is a 2-part domain, comprising two separate parts each comprising a dimerization domain, the two parts may be separately linked to the N- and C-terminals of the signalling domain. Thus, various configurations of the interaction domain, and individual dimerization domains (e.g. Ht1, Ht2), and signalling domains of the chimeric protein are possible. This may depend upon whether there is one or more than one dimerization domain, and when there are 2 or more dimerization domains, whether the interaction domain is a 1-part or 2-part domain. In representative embodiments the signalling domain may comprise, from N- to C-terminal: (i) JAK1 motif – STAT motif; (ii) JAK1 motif – STAT motif – JAK3 motif, particularly in reverse orientation; (iii) JAK1 motif – JAK2 motif – STAT motif; or (iv) JAK1 motif – JAK2 motif – STAT motif – JAK3 motif, particularly in reverse orientation; wherein the motifs are linked directly or indirectly to one another. Where the motifs are linked indirectly, they may be linked by a linker. The STAT motif may in particular be a STAT5 motif. In a particular embodiment, a linker or a hinge may be present between the JAK3-binding motif and the STAT association motif/JAK1-, or JAK1- and JAK2-, binding motifs. The 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, e.g. at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 glycine residues. In a most particular embodiment, the signalling domain comprises a first amino acid sequence derived from IL2Rγ comprising a JAK3-binding domain (e.g. SEQ ID NOs: 33 or 34) and a second amino acid sequence derived from IL2Rβ comprising a STAT5 association motif and a JAK1-binding motif (e.g. SEQ ID NOs: 31 or 32), where the first and second amino acid sequences are connected or joined by a linker or hinge. For example, the signalling domain may comprise SEQ ID NO: 33 and SEQ ID NO: 31 wherein SEQ ID NOs: 33 and 31 are connected by a linker or hinge. As indicated above, the interaction domain or parts thereof and the signalling domain may be joined to each other via a linker. Where the interaction domain comprises D1 and D2 (e.g. Ht1 and Ht2) together in a single 1-part domain, these may also be joined together directly, or by a linker. Further, if the chimeric protein comprises other domains or sequences, for example an additional stabilising or functional domain at either end of the molecule, then such domains or sequences may be linked directly or indirectly via a linker. Linker sequences are discussed in more detail below. A 1-part interaction domain may be linked to the N- or the C-terminal of the signalling domain. A 1-part interaction domain may occur where there is a single dimerization domain (as shown in Figure 1), or where there are 2 or more dimerization domains. Figure 2 shows various configurations for interaction domains which comprise 2 dimerization domains D1 and D2. As noted above, D1 and D2 may be homo- or heterodimerization domains, but typically they will be heterodimerization domains Ht1 and Ht2. Figure 2A depicts a configuration wherein the binding domain is at the N-terminus of the signalling domain. As explained above, a 1-part interaction domain, including a domain such as depicted here, allows a one-sided dimerization. The signalling domain is depicted as comprising, N- to C-terminal JAK1-STAT5, but this may be modified to introduce a JAK3 domain linked at the C-terminus of STAT5, particularly in reverse orientation. Where the binding domain is a 2-part domain, in which Ht1 and Ht2 are separated from one another, Ht1 and Ht2 may each be linked to an end of the signalling domain, i.e. one of Ht1 or Ht2 may be linked at the N- terminus of the signalling domain, and the other of Ht1 and Ht2 may be linked at the C-terminus of the signalling domain. Such a configuration is depicted in Figure 2B. Figure 2C shows a version in which a JAK3 domain is added to signalling domain, particularly in reverse orientation. A 2-part binding domain allows a 2-sided dimerization. It will be understood that the configurations depicted in Figure 2 are exemplary only, and may be modified or altered. For example, the STAT5 association motif may be replaced or supplemented by one or more other STAT association motifs, e.g. STAT3. D1 and D2 are shown as Ht1 and Ht2 in Figure 2 but could be homodimerization domains. In representative embodiments, the chimeric protein may have the following configurations: (i) the JAK1-, or JAK1- and JAK2-, binding motifs in the signalling domain may be linked N- or C-terminal to the STAT motif(s); (ii) where the signalling domain of the chimeric protein comprises a JAK3- binding motif, this is linked to the other end of the STAT motif than the JAK-, or the JAK1- and JAK2-, binding motifs. The skilled person would be able to design an appropriate configuration for the chimeric protein, depending on the nature of the dimerization domains and the signalling domain, for example whether or not the signalling domain includes a JAK3- binding motif. Generally speaking, for 2-sided dimerization, where the interaction domain is in 2 parts and D1 and D2 are separated, the signalling domain includes a JAK3-binding motif. The purpose of the dimerization of the interaction domain is to bring together 2 chimeric proteins each comprising a signalling domain, whether this be 2 molecules of the same chimeric protein or 2 different chimeric proteins with different interaction and/or signalling domains, and to allow them to dimerize to form a functional signalling protein. The dimerization between the signalling domains does not require a physical association between the two signalling domain monomers, in the sense of the 2 domains becoming physically linked or joined to one another – it suffices that they are brought into proximity together in such a manner that they can act together to mediate a signal in the cell. The dimerization between the two signalling domains thus includes a functional association between them. The skilled person is well aware of the requirements for dimerization of signalling domains, or more particularly the individual motifs thereof. Particular motifs may homo-dimerize with one another, but this is not necessarily the case. In the case of a 1-part binding domain, where there is 1-sided dimerization, the signalling domain may be designed such that individual motifs homo-dimerize. Thus, as depicted in Figure 2A, for example, the STAT5 association motif may homodimerize, as may the JAK1-binding motif. However, the motifs do not need to homodimerize with one another, and whilst typically in a chimeric protein, STAT motifs will be designed to homo-dimerize with one another, STAT5 association motifs may for example heterodimerize with JAK1 or JAK2 (as depicted for example in Figure 2B). Further, JAK1 may heterodimerize with JAK3. JAK1 dimerization with JAK3 is depicted in Figure 2C for example). To achieve functional dimerization of JAK1 and JAK3, from separate chimeric protein chains, JAK3 is typically translated in reverse orientation. As noted above, in certain embodiments a chimeric protein comprising a signalling domain comprising a tyrosine kinase activating domain and a tyrosine effector domain as defined herein may dimerize with a second chimeric protein which is different, and in particular which comprises a different signalling domain. In particular embodiment, the second chimeric protein may comprise a signalling domain comprising a JAK3-binding motif. In such an embodiment, the first chimeric protein may not comprise a JAK3-binding motif. Accordingly, in such an embodiment a first chimeric protein as provided herein may comprise a signalling domain A comprising a tyrosine kinase activating domain comprising a JAK1-binding motif and a tyrosine effector domain comprising one or more tyrosine residues that can be phosphorylated by JAK1 (e.g. comprising one or more STAT association motifs). The first chimeric protein may dimerize with a second chimeric protein comprising a signalling domain B comprising a tyrosine kinase activating domain comprising a JAK3-binding motif. The first and second chimeric proteins may be provided as a pair, as indicated above. In a particular embodiment the signalling domain B does not comprise a tyrosine effector domain. Thus, the signalling domain B may not be capable on its own, when dimerized to another signalling domain B, of transmitting or mediating a signal to the cell. The signalling domain here allows a chimeric protein comprising a signalling domain A to become dimerized to effect signalling activity, for example by dimerization of the JAK1-binding motif in signalling domain A with the JAK3-binding motif in signalling domain B. The presence of the JAK3-binding motif, and recruitment and activation of JAK3 may augment the signalling by the dimerized signalling protein, as discussed above. It will thus be seen that chimeric proteins herein may provide a so-called “split” signalling protein, wherein different functional parts of the signalling domain are provided on separate chains, or by separate chimeric proteins. Examples of such constructs, or chimeric proteins, are depicted in Figure 14. Chimeric proteins A, B, and C have an interaction domain comprising a single dimerization domain; as depicted this is a heterodimerization domain, being either FKBP or FRB. Thus, chimeric protein A comprises FRB and dimerizes either with construct B or construct C comprising FKBP. Chimeric protein B comprises a signalling domain which is identical to that of protein A, based on the IL2RB chain, which comprises a JAK1- binding motif and a STAT5 association motif. Chimeric protein C comprises a different signalling domain based on the IL2RG chain, which comprises a JAK3- binding motif. Chimeric proteins D and E comprising a 1-part interaction domain comprising 2 dimerization domains, Ht1 and Ht2, here represented by FRB and FKBP. Protein D has a signalling domain comprising an IL2RB sequence and hence may dimerize with itself (i.e. it works alone) or with protein E, which comprises an IL2RG sequence. Protein E is only functional when dimerized with protein D. Similar constructs are shown in Figure 15 (these comprise an additional sequence which comprises a furin sequence, adjacent to the P2A peptide sequence). The signalling domain may provide other signalling functions (e.g. those capable of providing a pro-survival or persistence signal, a signal which maintains cell phenotype or induces activation or function in addition to providing a STAT signal), and thus may comprise further domains which are capable of providing such signalling functions. The signalling domain however may not comprise any suicide or safety switch moiety or domain, and thus particularly does not comprise any killing or suicide function. The signalling domain for example, may additionally comprise an intracellular signalling domain such as ζ chain endodomain of the T-cell receptor or any of its homologs (e.g., η chain, FcεR1γ and β chains, MB1 (Igα) chain, B29 (Igβ) chain, etc.), CD3 polypeptide domains (∆, 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 human CD3 zeta chain endodomain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors or combinations thereof. Thus, the additional intracellular signaling domain may comprise the intracellular signaling domain of a human CD3 zeta chain, which in one embodiment comprises or consists of the following sequence: UNIPROT: P20963, CD3Z_HUMAN, position 31-143 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR (SEQ ID NO: 81) In one embodiment, the intracellular signaling domain comprises at least 85, 90, 95, 97, 98 or 99% identity to SEQ ID NO: 81. The intracellular signaling domain of the chimeric protein may comprise the following CD28 signaling domain: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 82) In one embodiment, the intracellular signaling domain comprises a signaling motif which has at least 85, 90, 95, 97, 98 or 99% identity to SEQ ID NO: 82. The intracellular signaling domain of the chimeric protein may comprise the following CD27 signaling domain: QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 83). In one embodiment, the intracellular signaling domain comprises a signaling motif which has at least 85, 90, 95, 97, 98 or 99% identity to SEQ ID NO: 83. Additional intracellular signalling domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. In this aspect, the signalling domain may comprise additional domains or sequences which provide transcription factor activity to the cell in which it is expressed, e.g. a transcription factor which has importance for phenotype or function of the cell. For Tregs for example, the signalling domain may additionally be capable of providing the cell with FOXP3, c-Rel, Runx, Ets-1, CREB, NFAT and/or JunB (directly or indirectly). Particularly, the signalling domain may be capable of providing a FOXP3 activating or inducing signal to the cell. In one aspect, the signalling domain may comprise FOXP3 (or any functional variant, truncation or isoform thereof), wherein the FOXP3 may be cleavable from the chimeric protein upon induction with a CID (for example, using a Notch system). In this instance, the signalling domain may be present at the C terminus of the chimeric protein, and any cleavable portion (e.g. FOXP3) would be present at the C-terminus of the signalling domain. The chimeric protein may if desired comprise a further domain, for example at either or both ends of the protein. Such an additional domain may be any desired domain, but since chimeric proteins and signalling proteins herein are cytoplasmic, non-membrane associated, proteins, it will not be a transmembrane domain, or any domain capable of associating with the cell membrane in any way (as discussed above). It may be a functional domain, or it may be non-functional. That is, the domain may be included simply to provide a structural element to the protein, without necessarily imparting any functional property or activity to it. As noted above the various domains, and individual parts of the domains (e.g. the motifs in the signalling domain) may be linked to one another by linkers. Thus, the chimeric protein may contain one or more linkers. Typically, it will contain at least one linker. Notably the protein may contain a linker linking the interaction domain, or a part thereof, to the signalling domain. For example, the chimeric protein may contain a linker linking a 1-part binding domain to the signalling domain, and a linker linking D1 to D2. In another example, the chimeric protein may comprise a 2-part binding domain wherein each of D1 and D2 (e.g. Ht1 and Ht2) are linked by a linker to the signalling domain. Any additional domains may also be linked by linkers. A linker as referred to herein is an amino acid sequence which links one domain or part of the protein to another. The linker sequence may be any amino acid sequence which functions to link, or connect, two domains or parts thereof together, such that they may perform their function. Thus, a linker may space apart the elements which are linked, for example to allow them to bind to their target. For example, in the case of a chimeric protein comprising D1 and D2, the linker may allow D1 and D2 from separate chimeric protein molecules to bind to an inducer molecule. Depending on the configuration of the protein, different linkers may be required, for example to connect the interaction domain, or D1 and D2 individually to the signalling domain, or to one another, and/or to other domains. The nature of the linker, in terms of its amino acid composition and/or sequence of amino acids may be varied and is not limited. However, the linker may be a flexible linker. It may thus comprise or consist of amino acids known to confer a flexible character to the linker (as opposed to a rigid linker). Flexible linkers are a category of linker sequences well known and described in the art. Linker sequences are generally known as sequences which may be used to link, or join together, proteins or protein domains, to create for example fusion proteins or chimeric proteins, or multifunctional proteins or polypeptides. They can have different characteristics, and for example may be flexible, rigid or cleavable. Protein linkers are reviewed for example in Chen et al., 2013, Advanced Drug Delivery Reviews 65, 1357-1369, which compares the category of flexible linkers with those of rigid and cleavable linkers. 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. A flexible linker is a linker which allows a degree of movement between the domains, or components, which are linked. They are generally composed of small non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acid residues. The small size of the amino acids provides flexibility and allows for mobility of the connected parts (domains or components). The incorporation of polar amino acids can maintain the stability of the linker in aqueous environments by forming hydrogen bonds with water molecules. The most commonly used flexible linkers have sequences primarily composed of Ser and Gly residues (so-called “GS linkers”). However, many other flexible linkers have also been described (see Chen et al, 2013, supra, for example), which may contain additional amino acids such as Thr and/or Ala, and/or Lys and/or Glu which may improve solubility. Any flexible linker known and reported in the art may be used. The use of GS linkers, or more particularly GS (“Gly-Ser”) domains in linkers, may allow the length of the linker readily to be varied by varying the number of GS domain repeats, and so such linkers represent one suitable class of linkers. However, flexible linkers are not limited to those based on “GS” repeats, and other linkers comprising Ser and Gly residues dispersed throughout the linker sequence have been reported, including in Chen et al., supra. In one embodiment, the linker sequence comprises at least one Gly-Ser domain composed solely of Ser and Gly residues. In such an embodiment, the linker may contain no more than 15 other amino acid residues, e.g. no more than 14, 13, 12, 11, 10, 9, 8, 6, 7, 5, or 4 other amino acid residues. The Gly-Ser domain may have the formula: (S)q-[(G)m-(S)m]n-(G)p wherein q is 0 or 1; m is an integer from 1-8; n is an integer of at least 1 (e.g. from 1 to 8, or more particularly 1 to 6); and p is 0 or an integer from 1 to 3. More particularly, the Gly-Ser domain may have the formula: (i) S-[(G)m-S]n; (ii) [(G)m-S]n; or (iii) [(G)m-S]n-(G)p wherein m is an integer from 2-8 (for example 3-4); n is an integer of at least 1 (for example from 1 to 8, or more particularly 1 to 6); and p is 0 or an integer from 1 to 3. In a representative example, the Gly-Ser domain may have the formula: S-[G-G-G-G-S]n wherein n is an integer of at least one (preferably 1 to 8, or 1-6, 1-5, 1-4, or 1- 3). In the formula above, the sequence GGGGS is SEQ ID NO.46. For example, S(GGGGS)2 is SEQ ID NO.42 and S(GGGGS)4 is SEQ ID NO. 44. However, it is not required for all linkers to be flexible, and in some cases the linker sequence is not a flexible linker sequence. Where the linker connects an interaction domain, or D1/Ht1 or D2/Ht2, to a signalling domain, it is preferably a flexible linker. Although the length of the linker may not be critical, it may in some cases be desirable to have a shorter linker sequence, or a longer linker sequence, depending on what domains etc. are being linked. For example, in some cases, for example where the linker connects D1/Ht1 and D2/Ht2, or where in a 2-part interaction domain configuration the linker connects D1/Ht1 and D2/Ht2 individually to the signalling domain, or when linking the motifs within a signalling domain, the linker sequence may have a length of no more than 30, e.g. no more than 25, 24, 23, 22, 21 or 20 amino acids. Particularly, the linker connecting D1/Ht1 and D2/Ht2 may be less than 15, or 10 amino acids or alternatively viewed may be between 5-15 or 5-10 amino acids in length (e.g. for chimeric proteins having a one-part binding domain, particularly for chimeric proteins having a one-part binding domain C terminal to the signalling domain). In other cases, for example where the linker connects an interaction domain, or D1/Ht1 or D2/Ht2, to a signalling domain, or to an additional domain a longer linker sequence may be desired. This may particularly be the case where the interaction domain is included internally, between two other domains. For example, a longer linker may be composed of, or may comprise, multiple repeats of a GS domain. In some cases, the linker may be from any one of 2, 3, 4, 5 or 6 to any one of 24, 23, 22 or 21 amino acids in length. In other cases, it may be from any one of 2, 3, 4, 5 or 6 to any one of 21, 20, 19, 18, 17, 16, or 15 amino acids in length. In other cases, it may be intermediate between these ranges, from example from 6 to 21, 6 to 20, 7 to 20, 8-20, 9-20, 10-20, 8-18, 9-18, 10-18, 9-17, 10-17, 9-16, 10-16 etc. It may accordingly be within a range made up from any of the integers listed above. In other cases, the linker may be of longer length, for example, from any one of 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 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 indicated above. It may accordingly be within a range made up from any of the integers listed above. As noted above, linkers may be designed or selected to allow the various domains and parts of the chimeric protein to function or to exert their effects, and so that the dimerizations may take place as set out above, particularly in the case of a chimeric protein having an interaction domain with 2 or more dimerization domains, so that D1/Ht1 from one protein molecule may dimerize to D2/Ht2 from another protein molecule, and that the signalling domain monomers of the respective proteins may dimerize with one another. It is well within the routine skill of the person skilled in the art to design or select appropriate linkers. For example, a linker should provide sufficient flexibility so the signalling domains can dimerize, but not so much flexibility so that the energic barrier to such dimerization is not overcome. The use of GS linkers, or more particularly GS (“Gly-Ser”) domains in linkers, may allow the length of the linker readily to be varied by varying the number of GS domain repeats, and so such linkers represent an advantageous type of linker to use. However, flexible linkers are not limited to those based on “GS” repeats, and other linkers comprising Ser and Gly residues dispersed throughout the linker sequence have been reported, including in Chen et al., supra. A linker sequence may be composed solely of, or may consist of, one or more Gly-Ser domains as described or defined above. However, as noted above, the linker sequence may comprise one or more Gly-Ser domains, and additional amino acids. The additional amino acids may be at one or both ends of a Gly-Ser domain, or at one or both ends of a stretch of repeating Gly-Ser domains. Thus, the additional amino acids, which may be other amino acids, may lie at one or both ends of the linker sequence, e.g. they may flank the Gly-Ser domain(s). In other embodiments, the additional amino acids may lie between Gly-Ser domains. For example, two Gly- Ser domains may flank a stretch of other amino acids in the linker sequence. Further, as also noted above, in other linkers, GS domains need not be repeated, and G and/or S residues, or a short domain such as GS, may simply be distributed along the length or the sequence. Representative exemplary linker sequences are listed below: ETSGGGGSRL (SEQ ID NO: 43) SGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:44) SGGGGSGGGGSGGGGS (SEQ ID NO:45) S(GGGGS)1-5 (where GGGGS is SEQ ID NO: 46) (GGGGS)1-5 (where GGGGS is SEQ ID NO:46) S(GGGS)1-5 (where GGGS is SEQ ID NO: 47) (GGGS)1-5 (where GGGS is SEQ ID NO: 47) S(GGGGGS)1-5 (where GGGGGS is SEQ ID NO: 48) (GGGGGS)1-5 (where GGGGGS is SEQ ID NO: 48 S(GGGGGGS)1-5 (where GGGGGGS is SEQ ID NO:49) (GGGGGGS)1-5 (where GGGGGGS is SEQ ID NO: 49) G6 (SEQ ID NO: 50) G8 (SEQ ID NO: 51) KESGSVSSEQLAQFRSLD (SEQ ID NO: 52) EGKSSGSGSESKST (SEQ ID NO: 53) GSAGSAAGSGEF (SEQ ID NO: 54) SGGGGSAGSAAGSGEF (SEQ ID NO: 55) SGGGLLLLLLLLGGGS (SEQ ID NO: 56) SGGGAAAAAAAAGGGS (SEQ ID NO: 57) SGGGAAAAAAAAAAAAAAAAGGGS (SEQ ID NO: 58) SGALGGLALAGLLLAGLGLGAAGS (SEQ ID NO: 59) SLSLSPGGGGGPAR (SEQ ID NO: 60) SLSLSPGGGGGPARSLSLSPGGGGG (SEQ ID NO: 61) GSSGSS (SEQ ID NO: 62) GSSSSSS (SEQ ID NO: 63) GGSSSS (SEQ ID NO: 64) GSSSSS (SEQ ID NO: 65) SGGGGS (SEQ ID NO: 66). For linking motifs within a signalling domain, the following linkers can be mentioned: GGGGS (SEQ ID NO.46) GGGGSGGGGSGGGGS (SEQ ID NO: 67) GGGGG (SEQ ID NO: 68) GGGGSGGGGS (SEQ ID NO: 69) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO 70) GGGGGG (SEQ ID NO: 50) GGGGGGGG (SEQ ID NO: 51). GGGGGGG (SEQ ID NO: 71) GGGGGGGGG (SEQ ID NO: 72). Illustrative constructs are represented by SEQ ID NO: 86, or more particularly by SEQ ID NO: 103 or SEQ ID NO: 104 as used in the Examples herein and shown in Figure 5. These constructs comprise an interaction domain comprising two dimerization domains FRB (94 amino acids) and FKBP (107 amino acids) separated from each other by a 5 amino acid linker. The interaction domain is joined by a 12 amino acid linker to a signalling domain comprising an IL2RB truncated variant with a Y510 mutation (SEQ ID NO.31). The depicted construct pQTX-0340 (SEQ ID NO: 103) is linked at the C-terminal to a P2A self-cleavage sequence and GFP for the purposes of expressing the construct and detecting it in the experiments described. Construct pQTX-0370 (SEQ ID NO: 104) is based on construct pQTX-0340 but additionally comprises an anti-HLA-A2 CAR linked by a T2A sequence to the N- terminal end of the IL2RB sequence (i.e. between the signalling domain and the P2A- GFP sequence). Representative constructs for use herein may be based on the constructs of SEQ ID NOs:103 or 104, but omitting or substituting the GFP, for example for another protein of interest, as described in more detail below. Other illustrative constructs which may be used as the basis for chimeric proteins as provided herein are shown in Figures 14 and 15 (see SEQ ID NOs:106, 108, 109, 110 and 111). A third aspect herein provides a nucleic acid molecule comprising a nucleotide sequence which encodes the chimeric protein. As used herein, the terms "polynucleotide" and "nucleic acid" are intended to be synonymous with each other. It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described here to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed. Nucleotide sequences encoding the various domains and motifs etc. described herein are known and available in the art, and any of these may be used or modified for use herein. Nucleic acids according to the second aspect may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest. The terms "variant", "homologue" or "derivative" in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence. Nucleic acid molecules/polynucleotides/nucleotide 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 by standard techniques. Longer nucleic acid molecules/polynucleotides/nucleotide sequences will generally be produced 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 to 30 nucleotides) flanking the target sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture with 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. A nucleic acid construct may comprise the nucleic acid molecule together with one or more other nucleotide sequences, for example, regulatory sequences, e.g. expression control sequences, and/or other coding sequences. In particular, the other coding sequence may encode a protein of interest. This may be a therapeutic protein. As noted above, a chimeric protein may be co-expressed with another protein of interest, for example a receptor, particularly an antigen receptor, for example, a CAR or a TCR or a derivative thereof (e.g. a TCR-CAR construct, or single chain TCR construct etc.). The coding sequence for such a further protein, e.g. receptor, may be comprised within a construct as referred to above. The chimeric protein may also be co-expressed with a safety switch polypeptide. The additional domain of the chimeric protein may comprise a safety switch polypeptide. A safety switch polypeptide provides a cell in or on which it is expressed with a suicide moiety. This is useful as a safety mechanism which allows a cell which has been administered to a subject to be deleted should the need arise, or indeed more generally, according to desire or need, for example once a cell has performed or completed its therapeutic effect. A suicide moiety possesses an inducible capacity to lead to cellular death, or more generally to elimination or deletion of a cell. An example of a suicide moiety is a suicide protein, encoded by a suicide gene, which may be expressed in or on a cell alongside a desired transgene, in this case the chimeric protein (and optionally a CAR or other receptor which is co-expressed by the cell along with the present chimeric protein), which when expressed allows the cell to be deleted to turn off expression of the transgene (CAR). A suicide moiety herein is a suicide polypeptide that is a polypeptide that under permissive conditions, namely conditions that are induced or turned on, is able to cause the cell to be deleted. The suicide moiety may be a polypeptide, or amino acid sequence, which may be activated to perform a cell-deleting activity by an activating agent which is administered to the subject, or which is active to perform a cell-deleting activity in the presence of a substrate which may be administered to a subject. In a particular embodiment, the suicide moiety may represent a target for a separate cell-deleting agent which is administered to the subject. By binding to the suicide moiety, the cell- deleting agent may be targeted to the cell to be deleted. In particular, the suicide moiety may be recognised by an antibody, and binding of the antibody to the safety switch polypeptide, when expressed on the surface of a cell, causes the cell to be eliminated, or deleted. The suicide moiety may be HSV-TK or iCasp9 as is known in the art. However, in other examples the suicide moiety may be, or may comprise an epitope which is recognized by a cell-deleting antibody or other binding molecule capable of eliciting deletion of the cell. The term “delete” as used herein in the context of cell deletion is synonymous with “remove” or “ablate” or “eliminate”. The term is used to encompass cell killing, or inhibition of cell proliferation, such that the number of cells in the subject may be reduced.100% complete removal may be desirable but may not necessarily be achieved. Reducing the number of cells, or inhibiting their proliferation, in the subject may be sufficient to have a beneficial effect. In particular, the suicide moiety may be a CD20 epitope which is recognised by the antibody Rituximab. Thus, in the safety switch polypeptide the suicide moiety may comprise a minimal epitope based on the epitope from CD20 that is recognized by the antibody Rituximab. More particularly, the polypeptide may comprise two CD20 epitopes R1 and R2 that are spaced apart by a linker L. Safety switches based on Rituximab epitopes are described in WO2013/15339. Peptides which mimic the epitope recognised by Rituximab (so- called mimotopes) have been developed, and these were used in WO2013/15339 as a suicide moiety in a combined suicide-marker polypeptide construct also comprising a CD34 minimal epitope as a marker moiety. Specifically, WO2013/15339 discloses a polypeptide termed RQR8, having the sequence set forth in SEQ ID NO:41, which comprises two CD20 minimal epitopes, separated from one another by spacer sequences and an intervening CD34 marker sequence, and further linked to a stalk sequence which allows the polypeptide to project from the surface of a cell on which it is expressed. The safety switch polypeptide may be RQR8 or a variant thereof having at least 80% sequence identity thereto, e.g. at least 85, 88, 90, 95, 96, 97, 98, or 99% sequence identity thereto. Other safety switch polypeptides which may be used as the basis of safety switch domains include those described in our co-pending PCT patent application No. WO 2021/239812. Other polypeptides which may be co-expressed with the chimeric protein include transcription factors, growth factors or other factors which may assist in enhancing functionality of survival of the cell. For example, the transcription factor FOXP3 may be used to maintain the suppressive phenotype of Treg cells. “FOXP3” is the abbreviated name of the forkhead box P3 protein. FOXP3 is a member of the FOX protein family of transcription factors and functions as a master regulator of the regulatory pathway in the development and function of regulatory T cells. “FOXP3” as used herein encompasses variants, isoforms, and functional fragments of FOXP3. A “FOXP3 polypeptide” is a polypeptide having FOXP3 activity i.e. a polypeptide able to bind FOXP3, target DNA and function as a transcription factor regulating development and function of Tregs. The present nucleic acid molecule or construct may further comprise a nucleic acid sequence encoding a selectable marker. Suitably 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, for example GFP, YFP, RFP, tdTomato, dsRed, or variants thereof. In some embodiments the fluorescent protein is GFP or a GFP variant. 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)). The use of a selectable marker is advantageous as it allows cells (e.g. Tregs) in which a nucleic acid molecule, construct or vector has been successfully introduced (such that the encoded chimeric protein and any other encoded proteins or polypeptides are expressed) to be selected and isolated from a starting cell population using common methods, e.g. flow cytometry. In a still further embodiment, the chimeric protein may be co-expressed with a mutant calcineurin protein which is resistant to at least one calcineurin inhibitor, and in particular a mutant calcineurin protein which is resistant to at least one calcineurin inhibitor and sensitive to at least one calcineurin inhibitor. Such calcineurin mutants are discussed further below. In such an embodiment the nucleic acid molecule or construct may further comprise a nucleotide sequence encoding such a mutant calcineurin. Where two or more coding sequences are expressed from a single nucleic acid molecule or construct, they may be linked by a sequence allowing co-expression of the two or more coding sequences. In particular, the co-expression sequence, or alternatively termed, the co-expression site, may enable expression of an encoded protein or polypeptide as a discrete entity. For example, the construct may comprise an internal promoter, an internal ribosome entry sequence (IRES) sequence or a sequence encoding a cleavage site. In particular the co-expression sequence may encode a self-cleavage sequence in between encoded polypeptides. Particularly, the self-cleaving sequence may be a self-cleaving peptide. Such sequences auto-cleave during protein production. Self-cleaving peptides which may be used are 2A peptides or 2A-like peptides which are known and described in the art, for example in Donnelly et al., Journal of General Virology, 2001, 82, 1027-1041, herein incorporated by reference. 2A and 2A-like peptides are believed to cause ribosome skipping, and result in a form of cleavage in which a ribosome skips the formation of peptide bond between the end of a 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 meaning the upstream cistron will have a few additional residues added to the end, while the downstream cistron will start with the Proline. Suitable self-cleaving domains include P2A, T2A, E2A, and F2A sequences as shown in SEQ ID NOs: 73-77 respectively. The sequences may be modified to include the amino acids GSG at the N-terminus of the 2A peptides. Thus, also included as possible options are sequences corresponding to SEQ ID NOs: 73-77, but with GSG at the N-termini thereof. Such modified alternative 2A sequences are known and reported in the art. Alternative 2A-like sequences which may be used are shown in Donnelly et al (supra), for example a TaV sequence. The self-cleaving sequences included in the nucleic acid molecule may be the same or different. The self-cleaving sequence may include an additional cleavage site, which may be cleaved by common enzymes present in the cell. This may assist in achieving complete removal of the 2A sequences after translation. Such an additional cleavage site may for example comprise a Furin cleavage site. Such cleavage sites are known in the art, and may include for example RXXR (SEQ ID NO: 78), for example RRKR (SEQ ID NO: 79). A furin sequence is comprised within the constructs shown in Figure 15 – as shown in this case, the furin sequence may be linked via a linker sequence, for example at its C-terminal end. It has been shown that the inclusion of such a linker may enhance cleavage. The nucleic acid molecule/polynucleotides used herein may be codon- optimised. Codon optimisation has previously been described in WO 1999/41397 and WO 2001/79518. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. By the same token, it is possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in the particular cell type. Thus, an additional degree of translational control is available. The nucleotide sequence encoding the chimeric protein, and any other coding nucleotide sequences may be provided in a construct in which they are operably linked to a promoter. In some cases, different nucleotide sequences may be operably linked to the same promoter. A “promoter” is a region of DNA that leads to initiation of transcription of a gene. Promoters are located near the transcription start sites of genes, upstream on the DNA (towards the 5’ region of the sense strand).Any suitable promoter may be used, the selection of which may be readily made by the skilled person. The promoter may be from any source, and may be a viral promoter, or a eukaryotic promoter, including mammalian or human promoters (i.e. a physiological promoter). In an embodiment the promoter is a viral promoter. Particular promoters include LTR promoters, EFS (or functional truncations thereof), SFFV, PGK, and CMV. In an embodiment the promoter is SFFV or a viral LTR promoter. “Operably linked to the same promoter” means that transcription of the polynucleotide sequences may be initiated from the same promoter and that the nucleotide sequences are positioned and oriented for transcription to be initiated from the promoter. Polynucleotides operably linked to a promoter are under transcriptional regulation of the promoter. A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. As used herein, and by way of example, some vectors used in recombinant nucleic acid techniques allow entities, such as a segment of nucleic acid (e.g. a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell. Vectors may be non-viral or viral. Examples of vectors used in recombinant nucleic acid techniques include, but are not limited to, plasmids, mRNA molecules (e.g. in vitro transcribed mRNAs), chromosomes, artificial chromosomes and viruses. The vector may also be, for example, a naked nucleic acid (e.g. DNA). In its simplest form, the vector may itself be a nucleotide sequence of interest. The vectors used herein may be, for example, plasmid, mRNA or virus vectors and may include a promoter (as described above) for the expression of a nucleic acid molecule/polynucleotide and optionally a regulator of the promoter. In an embodiment the vector is a viral vector, for example a retroviral, e.g. a lentiviral vector or a gamma retroviral vector. The vectors may further comprise additional promoters, for example, in one embodiment, the promoter may be a LTR, for example, a retroviral LTR or a lentiviral LTR. Long terminal repeats (LTRs) are identical sequences of DNA that repeat hundreds or thousands of times found at either end of retrotransposons or proviral DNA formed by reverse transcription of retroviral RNA. They are used by viruses to insert their genetic material into the host genomes. Signals of gene expression are found in LTRs: enhancer, promoter (can have both transcriptional enhancers or regulatory elements), transcription initiation (such as capping), transcription terminator and polyadenylation signal. Suitably, the vector may include a 5’LTR and a 3’LTR. The vector may comprise one or more additional regulatory sequences which may act pre- or post-transcriptionally. “Regulatory sequences” are any sequences which facilitate expression of the polypeptides, e.g. act to increase expression of a transcript or to enhance mRNA stability. Suitable regulatory sequences include for example enhancer elements, post-transcriptional regulatory elements and polyadenylation sites. Suitably, the additional regulatory sequences may be present in the LTR(s). Suitably, the vector may comprise a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), e.g. operably linked to the promoter. Vectors comprising the present nucleic acid molecules/polynucleotides may be introduced into cells using a variety of techniques known in the art, such as transformation and transduction. Several techniques are known in the art, for example infection with recombinant viral vectors, such as retroviral, lentiviral, adenoviral, adeno-associated viral, baculoviral and herpes simplex viral vectors; direct injection of nucleic acids and biolistic transformation. Non-viral delivery systems include but are not limited to DNA transfection methods. Here, transfection includes a process using a non-viral vector to deliver a gene to a target cell. Non-viral delivery systems can include liposomal or amphipathic cell penetrating peptides, preferably complexed with a nucleic acid molecule or construct. Typical transfection methods include electroporation, DNA biolistics, lipid- mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated transfection, cationic facial amphiphiles (CFAs) (Nat. Biotechnol. (1996) 14: 556) and combinations thereof. In some cases, the present nucleic acid molecules may be designed to be used as single constructs which encode the chimeric protein and any other polypeptide (e.g. receptor or marker or other functional polypeptide or protein of interest) and this would be contained in a single vector, it is not precluded that they are introduced into a cell in conjunction with other vectors, for example encoding other polypeptides it may be desired also to introduce into the cell. As is clear from the above description in addition to the specific polypeptide and nucleotide sequences mentioned herein, also encompassed is the use of variants, or derivatives and fragments thereof. The term “derivative” or “variant” as used interchangeably herein, in relation to the present proteins or polypeptides includes any substitution of, variation of, modification of, replacement of, deletion of and/or addition of one (or more) amino acid residues from or to the sequence providing that the resultant protein or polypeptide retains the desired function. For example, where the derivative or variant is a signalling domain, the desired function may be the ability of that domain to signal (e.g. activate or inactivate a downstream molecule, where the derivative or variant is a dimerization domain, the desired function is interaction with the inducer molecule, e.g. binding to rapamycin or an analogue thereof etc., or interaction directly with another cognate dimerization domain. Alternatively viewed, the variants or derivatives referred to herein are functional variants or derivatives. For example, 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% function compared to the corresponding, reference sequence. The variant or derivative may have a similar or the same level of function as compared to the corresponding, reference sequence or may have an increased level of function (e.g. increased by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%). Typically, amino acid substitutions may be made, for example from 1, 2 or 3 to 10 or 20 substitutions provided that the modified sequence retains the required activity or ability. 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% activity or ability compared to the corresponding, reference sequence. The variant or derivative may have a similar or the same level of activity or ability as compared to the corresponding, reference sequence or may have an increased level of activity or ability (e.g. increased by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%). Proteins or peptides may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues as long as the endogenous function is retained. 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 having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine. Conservative substitutions may be made, for example according to Table 1 below. Table 1
Figure imgf000060_0001
The derivative may be a homologue. The term “homologue” as used herein means an entity having a certain homology with the wild type amino acid sequence and the wild type nucleotide sequence. The term “homology” can be equated with “identity”. A homologous or variant sequence may include an amino acid sequence which may be at least 70%, 75%, 85% or 90% identical, preferably at least 95%, 96%, 97%, 98% or 99% identical to the subject sequence. Typically, the variants will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context herein it is preferred to express homology in terms of sequence identity. 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 percentage homology or identity between two or more sequences. Percentage homology or sequence identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues. Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the nucleotide sequence may cause the following codons to be put out of alignment, thus potentially resulting in a large reduction in percent homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local homology. However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence 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 will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. 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. Calculation of maximum percentage homology/sequence identity therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al. (1984) Nucleic Acids Res.12: 387). Examples of other software that can perform 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 suite of comparison tools. 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 BLAST 2 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). 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 pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix – the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see the user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62. Suitably, the percentage identity is determined across the entirety of the reference and/or the query sequence. 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. “Fragment” typically refers to a selected region of the polypeptide or polynucleotide that is of interest functionally, e.g. is functional or encodes a functional fragment. “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion (or part) of a full-length polypeptide or polynucleotide. Such variants, derivatives and fragments may be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5' and 3' flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used. As noted above, the chimeric protein may be co-expressed in or on a cell in conjunction with a CAR. The term “chimeric antigen receptor" or "CAR" as used herein refers to engineered receptors which can confer an antigen specificity onto cells (for example Tregs). CARs are also known as artificial T-cell receptors, chimeric T-cell receptors or chimeric immunoreceptors. A CAR typically comprises an extracellular domain comprising an antigen-specific targeting region, termed herein an antigen-binding domain, a transmembrane domain, and an endodomain comprising optionally one or more co-stimulatory domains, and an intracellular signaling domain. The antigen-binding domain is typically joined to the transmembrane domain by a hinge domain. The design of CARs, and the various domains that they may contain, is well known in the art. When the CAR binds its target antigen, this results in the transmission of an activating signal to the cell in which it is expressed. Thus, the CAR directs the specificity of the engineered cells towards the target antigen, particularly towards cells expressing the targeted antigen. The antigen-binding domain of a CAR may be derived or obtained from any protein or polypeptide which binds (i.e. has affinity for) a desired target antigen, or more generally a desired target molecule. This 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 commonly be expressed on the surface of a cell, for example a target cell, or a cell in the vicinity of a target cell (for a bystander effect), but need not be. Depending on the nature and specificity of the antigen binding domain, the CAR may recognise a soluble molecule, for example where the antigen-binding domain is based on, or derived from, a cellular receptor. The antigen-binding domain is most commonly derived from antibody variable chains (for example it commonly takes the form of a scFv), but may also be generated from T-cell receptor variable domains or, as mentioned above, other molecules, such as receptors for ligands or other binding molecules. The CAR is typically expressed as a polypeptide also comprising a signal sequence (also known as a leader sequence), and in particular a signal sequence which targets the CAR to the plasma membrane of the cell. This will generally be positioned next to or close to the antigen-binding domain, generally upstream of the antigen-binding domain. The extracellular domain, or ectodomain, of the CAR may thus comprise a signal sequence and an antigen-binding domain. The antigen-binding domain provides the CAR with the ability to bind a predetermined antigen of interest. The antigen-binding domain preferably targets an antigen of clinical interest or an antigen at a site of disease. As noted above, the antigen-binding domain may be any protein or peptide that possesses the ability to specifically recognize and bind to a biological molecule (e.g., a cell surface receptor or a component thereof). The antigen-binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule of interest. Illustrative antigen- specific targeting domains include antibodies or antibody fragments or derivatives, extracellular domains of receptors, ligands for cell surface molecules/receptors, or receptor binding domains thereof, and tumor binding proteins. Although as discussed below, the antigen-specific targeting domain may preferably be an antibody or derived from an antibody, other antigen-specific targeting domains are encompassed, e.g. antigen-specific targeting domains formed from an antigenic peptide/MHC or HLA combination which is capable of binding to the TCRs of Tcon cells active at a site of transplantation, inflammation or disease. The CAR may be directed towards any desired target antigen or molecule. This may be selected according to the intended therapy, and the condition it is desired to treat. It may for example be an antigen or molecule associated with a particular condition, or an antigen or molecule associated with a cell it is desired to target to treat the condition. Typically, the antigen or molecule is a cell-surface antigen or molecule. The term “directed against” is synonymous with “specific for” or “anti”. Put another way, the CAR recognises a target molecule. Accordingly, it is meant that the CAR is capable of binding specifically to a specified or given antigen, or target. In particular, the antigen-binding domain of the CAR is capable of binding specifically to the target molecule or antigen (more particularly when the CAR is expressed on the surface of a cell, notably an immune effector cell). Specific binding may be distinguished from non-specific binding to a non-target molecule or antigen. Thus, a cell expressing the CAR is directed, or re-directed, to bind specifically to a target cell, expressing the target molecule or antigen, particularly a target cell expressing the target antigen or molecule on its cell surface. Antigens which may be targeted by the present 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 disease). It will be understood by a skilled person that where the cell engineered to express the CAR is a Treg cell, or a precursor therefor, due to the bystander effect of Treg cells, the antigen may be simply present and/or expressed at the site of transplantation, inflammation or disease. Antigens expressed on cells associated with neurodegenerative disease include those presented on glial cells, e.g. MOG. Antigens associated with organ transplants and/or cells associated with transplanted organs include, but are not limited to, a HLA antigen present in the transplanted organ but not in the patient, or an antigen whose expression is up- regulated during transplant rejection such as CCL19, MMP9, SLC1A3, MMP7, HMMR, TOP2A, GPNMB, PLA2G7, CXCL9, FABP5, GBP2, CD74, CXCL10, UBD, CD27, CD48, CXCL11. In an embodiment the CAR is directed against an HLA antigen, and in particular an HLA-A2 antigen. Antibodies against such antigens are known in the art, and conveniently a scFv may be obtained or generated based on a known or available antibody. In this regard VH and VL, and CDR sequences are publicly available to aid the preparation of such an antibody-binding domain, for example in WO 2020/044055, the disclosure of which is herein incorporated by reference. Any of the antigen binding domains, or CDR, VH, and/or VL sequences disclosed in WO 2020/044055 may be used. By way of example, the CAR may comprise an antigen binding domain which is capable of binding HLA-A2 (HLA-A2 may also be referred to herein as HLA-A*02, HLA-A02, and HLA-A*2). HLA-A*02 is one particular class I major histocompatibility complex (MHC) allele group at the HLA-A locus. The antigen recognition domain may bind, suitably specifically bind, one or more regions or epitopes within HLA-A2. An epitope, also known as antigenic determinant, is the part of an antigen that is recognised by an antigen recognition domain (e.g. an antibody). In other words, the epitope is the specific piece of the antigen to which an antibody binds. Suitably, the antigen recognition domain binds, suitably specifically binds, to one region or epitope within HLA-A2. Engineered cells may be generated by introducing a nucleic acid molecule, construct, or vector as defined herein, by one of many means including transduction with a viral vector, and transfection with DNA or RNA. The present cell may be made by introducing to a cell (e.g. by transduction or transfection) the nucleic acid molecule, construct or vector as defined herein. Suitable cells are discussed further below, but the cell may be from a sample isolated from a subject. The subject may be a donor subject, or a subject for therapy (i.e. the cell may be an autologous cell, or a donor cell, for introduction to another recipient, e.g. an allogeneic cell). The cell may be generated by a method comprising the following steps: (i) isolation of a cell-containing sample from a subject or provision of a cell- containing sample; and (ii) introduction into (e.g. by transduction or transfection) the cell-containing sample of a nucleic acid molecule, construct, or vector as defined herein, to provide a population of engineered cells. A cell into which a nucleic acid molecule, construct or vector is to be introduced may be referred to as a target cell. A target cell-enriched sample may be isolated from, enriched, and/or generated from the cell-containing sample prior to and/or after step (ii) of the method. For example, isolation, enrichment and/or generation of Tregs (or other target cells) may be performed prior to and/or after step (ii) to isolate, enrich or generate a Treg-enriched sample. Isolation and/or enrichment from a cell-containing sample may be performed after step (ii) to enrich for cells and/or Tregs (or other target cells) comprising the CAR, the nucleic acid molecule/polynucleotide, the construct and/or the vector as described herein. A Treg-enriched sample may be isolated or enriched by any method known to those of skill in the art, for example by FACS and/or magnetic bead sorting. A Treg- enriched sample may be generated from the cell-containing sample by any method known to those of skill in the art, for example, from Tcon cells by introducing DNA or RNA coding for FOXP3 and/or from 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. The target cell may be a Treg cell, or precursor or a progenitor therefor. An “engineered cell” means a cell which has been modified to comprise or express a polynucleotide which is not naturally encoded by the cell. Methods for engineering cells are known in the art and include, but are not limited to, genetic modification of cells e.g. by transduction such as retroviral or lentiviral transduction, transfection (such as transient transfection – DNA or RNA based) including lipofection, polyethylene glycol, calcium phosphate and electroporation, as discussed above. Any suitable method may be used to introduce a nucleic acid sequence into a cell. Non-viral technologies such as amphipathic cell penetrating peptides may be used to introduce nucleic acid. Accordingly, the nucleic acid molecule as described herein is not naturally expressed by a corresponding, unmodified cell. Indeed, the nucleic acid molecule encoding the chimeric protein is an artificial construct, and could not occur or be expressed naturally. Suitably, an engineered cell is a cell which has been modified e.g. by transduction or by transfection. Suitably, an engineered cell is a cell which has been modified or whose genome has been modified e.g. by transduction or by transfection. Suitably, an engineered cell is a cell which has been modified or whose genome has been modified by retroviral transduction. Suitably, an engineered cell is a cell which has been modified or whose genome has been modified by lentiviral transduction. As used herein, the term “introduced” refers to methods for inserting foreign nucleic acid, e.g. DNA or RNA, into a cell. As used herein the term introduced includes both transduction and transfection methods. Transfection is the process of introducing nucleic acids 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 a nucleic acid as described herein by one of many means including transduction with a viral vector, transfection with DNA or RNA. Cells may be activated and/or expanded prior to, or after, the introduction of a nucleic acid as described herein, for example by treatment with an anti-CD3 monoclonal antibody or 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. Suitably, IL-2 may be substituted with IL-15. Other components which may be used in a cell (e.g. Treg) expansion protocol include, but are not limited to rapamycin, all-trans retinoic acid (ATRA) and TGFβ. As used herein “activated” means that a cell has been stimulated, causing the cell to proliferate. As used herein “expanded” means that a cell or population of cells has been induced to proliferate. The expansion of a population of cells may be measured for example by counting the number of cells present in a population. The phenotype of the cells may be determined by methods known in the art such as flow cytometry. The cell may be an immune cell, or a precursor therefor. A precursor cell may be a progenitor cell. Representative immune cells thus include T-cells, in particular, cytotoxic T-cells (CTLs; CD8+ T-cells), helper T-cells (HTLs; CD4+ T-cells) and regulatory T cells (Tregs). Other populations of T-cells are also useful herein, for example naive T-cells and memory T-cells. Other immune cells include NK cells, NKT cells, dendritic cells, MDSC, neutrophils, and macrophages. Precursors of immune cells include pluripotent stem cells, e.g. induced PSC (iPSC), or more committed progenitors including multipotent stem cells, or cells which are committed to a lineage. Precursor cells can be induced to differentiate into immune cells in vivo or in vitro. In one aspect, a precursor cell may be a somatic cell which is capable of being transdifferentiated to an immune cell of interest. Most notably, the immune cell may be an NK cell, a dendritic cell, a MDSC, or a T cell, such as a cytotoxic T lymphocyte (CTL) or a Treg cell. In particular, the immune cell may be a Treg cell. “Regulatory T cells (Treg) or T regulatory cells” are immune cells with immunosuppressive function that control cytopathic immune responses and are essential for the maintenance of immunological tolerance. As used herein, the term Treg refers to a T cell with immunosuppressive function. A T cell as used herein 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, or a Treg cell. Suitably, immunosuppressive function may refer to the ability of the Treg to reduce or inhibit one or more of a number of physiological and cellular effects facilitated by the immune system in response to a stimulus such as a pathogen, an alloantigen, or an autoantigen. Examples of such effects include increased proliferation of conventional T cell (Tcon) and secretion of proinflammatory cytokines. Any such effects may be used as indicators of the strength of an immune response. A relatively weaker immune response by Tconv in the presence of Tregs would indicate an ability of the Treg to suppress immune responses. For example, a relative decrease in cytokine secretion would be indicative of a weaker immune response, and thus indicative of the ability of Tregs to suppress immune responses. Tregs can also suppress immune responses by modulating the expression of co-stimulatory molecules on antigen presenting cells (APCs), such as B cells, dendritic cells and macrophages. Expression levels of CD80 and CD86 can be used to assess suppression potency of activated Tregs in vitro after co-culture. Assays are known in the art for measuring indicators of immune response strength, and thereby the suppressive ability of Tregs. In particular, antigen-specific Tconv cells may be co-cultured with Tregs, and a peptide of the corresponding antigen added to the co-culture to stimulate a response from the Tconv cells. The degree of proliferation of the Tconv cells and/or the quantity of the cytokine IL-2 they secrete in response to addition of the peptide may be used as indicators of the suppressive abilities of the co-cultured Tregs. Antigen-specific Tconv cells co-cultured with Tregs as referred to herein may proliferate 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95% or 99% less than the same Tconv cells cultured in the absence of the Tregs. For example, antigen- specific Tconv cells co-cultured with the present Tregs may proliferate 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95% or 99% less than the same Tconv cells cultured in the presence of non-engineered Tregs. The cells comprising the nucleic acid, expression construct or vector as defined herein, e.g. Tregs may have an increased suppressive activity as compared to non-engineered Tregs (e.g. an increased suppressive activity of at least 5, 10, 20, 30, 40, 50, 60, 70, 80 or 90%). Antigen-specific Tconv cells co-cultured with the Tregs herein may express at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% less effector cytokine than corresponding Tconv cells cultured in the absence of the Tregs (e.g. in the presence of non-engineered Tregs). The effector cytokine 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 cytokine may be selected from IL-2, IL-17, TNFα, GM-CSF and IFN-γ. Several different subpopulations of Tregs have been identified which may express different or different levels of particular markers. Tregs generally are T cells which express the markers CD4, CD25 and FOXP3 (CD4+CD25+FOXP3+). Tregs may also express CTLA-4 (cytotoxic T-lymphocyte associated molecule-4) or GITR (glucocorticoid-induced TNF receptor). Treg cells are present in the peripheral blood, lymph nodes, and tissues and Tregs for use herein include thymus-derived, natural Treg (nTreg) cells, peripherally generated Tregs, and induced Treg (iTreg) cells. A Treg may be identified using the cell surface markers CD4 and CD25 in the absence of or in combination with low-level expression of the surface protein CD127 (CD4+CD25+CD127− or CD4+CD25+CD127low). The use of such markers to identify Tregs is known in the art and described in Liu et al. (JEM; 2006; 203; 7(10); 1701- 1711), for example. A Treg may be a CD4+CD25+FOXP3+ T cell, a CD4+CD25+CD127− T cell, or a CD4+CD25+FOXP3+CD127−/low T cell. Suitably, the Treg may be a natural Treg (nTreg). As used herein, the term “natural T reg” means a thymus-derived Treg. Natural T regs are CD4+CD25+FOXP3+ Helios+ Neuropilin 1+. Compared with iTregs, nTregs have higher expression of PD-1 (programmed cell death-1, pdcd1), Neuropilin 1 (Nrp1), Helios (Ikzf2), and CD73. nTregs may be distinguished from iTregs on the basis of the expression of Helios protein or Neuropilin 1 (Nrp1) individually. The Treg may have a demethylated Treg-specific demethylated region (TSDR). The TSDR is an important methylation-sensitive element regulating FOXP3 expression (Polansky, J.K., et al., 2008. European journal of immunology, 38(6), pp.1654-1663). Further suitable Tregs include, but are not limited to, Tr1 cells (which do not express FOXP3, and have high IL-10 production); CD8+FOXP3+ T cells; and γδ FOXP3+ T cells. Different subpopulations of Tregs are known to exist, including naïve Tregs (CD45RA+FOXP3low), effector/memory Tregs (CD45RA-FOXP3high) and cytokine- producing Tregs (CD45RA-FOXP3low). “Memory Tregs” are Tregs which express CD45RO and which are considered to be CD45RO+. These cells have increased levels of CD45RO as compared to naïve Tregs (e.g. at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% more CD45RO) and which preferably do not express or have low levels of CD45RA (mRNA and/or protein) as compared to naïve Tregs (e.g. at least 80, 90 or 95% less CD45RA as compared to naïve Tregs). “Cytokine-producing Tregs” are Tregs which do not express or have very low levels of CD45RA (mRNA and/or protein) as compared to naïve Tregs (e.g. at least 80, 90 or 95% less CD45RA as compared to naïve Tregs), and which have low levels of FOXP3 as compared to Memory Tregs, e.g. less than 50, 60, 70, 80 or 90% of the FOXP3 as compared to Memory Tregs. Cytokine-producing Tregs may produce interferon gamma and may be less suppressive in vitro as compared to naïve Tregs (e.g. less than 50, 60, 70, 80 or 90% suppressive than naïve Tregs. Reference to expression levels herein may refer to mRNA or protein expression. Particularly, for cell surface markers such as CD45RA, CD25, CD4, CD45RO etc., expression may refer to cell surface expression, i.e. the amount or relative amount of a marker protein that is expressed on the cell surface. Expression levels may be determined by any known method of the art. For example, mRNA expression levels may be determined by Northern blotting/array analysis, and protein expression may be determined by Western blotting, or preferably by FACS using antibody staining for cell surface expression. Particularly, the Treg may be a naïve Treg. “A naïve regulatory T cell, a naïve T regulatory cell, or a naïve Treg” as used interchangeably herein refers to a Treg cell which expresses CD45RA (particularly which expresses CD45RA on the cell surface). Naïve Tregs are thus described as CD45RA+. Naïve Tregs generally represent Tregs which have not been activated through their endogenous TCRs by peptide/MHC, whereas effector/memory Tregs relate to Tregs which have been activated by stimulation through their endogenous TCRs. Typically, a naïve Treg may express at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% more CD45RA than a Treg cell which is not naïve (e.g. a memory Treg cell). Alternatively viewed, a naïve Treg cell may express at least 2, 3, 4, 5, 10, 50 or 100-fold the amount of CD45RA as compared to a non-naïve Treg cell (e.g. a memory Treg cell). The level of expression of CD45RA can be readily determined by methods of the art, e.g. by flow cytometry using commercially available antibodies. Typically, non-naïve Treg cells do not express CD45RA or low levels of CD45RA. Particularly, naïve Tregs may not express CD45RO, and may be considered to be CD45RO-. Thus, naïve Tregs may express at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% less CD45RO as compared to a memory Treg, or alternatively viewed at least 2, 3, 4, 5, 10, 50 or 100 fold less CD45RO than a memory Treg cell. Although naïve Tregs express CD25 as discussed above, CD25 expression levels may be lower than expression levels in memory Tregs, depending on the origin of the naïve Tregs. For example, for naïve Tregs isolated from peripheral blood, expression levels of CD25 may be at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% lower than memory Tregs. Such naïve Tregs may be considered to express intermediate to low levels of CD25. However, a skilled person will appreciate that naïve Tregs isolated from cord blood may not show this difference. Typically, a naïve Treg as defined herein may be CD4+, CD25+, FOXP3+, CD127low, CD45RA+. Low expression of CD127 as used herein refers to a lower level of expression of CD127 as compared to a CD4+ non-regulatory or Tcon cell from the same subject or donor. Particularly, naïve Tregs may express less than 90, 80, 70, 60, 50, 40, 30, 20 or 10% CD127 as compared to a CD4+ non-regulatory or Tcon cell from the same subject or donor. Levels of CD127 can be assessed by methods standard in the art, including by flow cytometry of cells stained with an anti-CD127 antibody. Typically, naïve Tregs do not express, or express low levels of CCR4, HLA- DR, CXCR3 and/or CCR6. Particularly, naïve 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 level of expression. Naïve Tregs may further express additional markers, including CCR7+ and CD31+ Isolated naïve Tregs may be identified by methods known in the art, including by determining the presence or absence of a panel of any one or more of the markers discussed above, on the cell surface of the isolated cells. For example, CD45RA, CD4, CD25 and CD127low can be used to determine whether a cell is a naïve Treg. Methods of determining whether isolated cells are naïve Tregs or have a desired phenotype can be carried out as discussed below in relation to additional steps which may be carried out, and methods for determining the presence and/or levels of expression of cell markers are well-known in the art and include, for example, flow cytometry, using commercially available antibodies. Suitably, the cell, such as a Treg, is isolated from peripheral blood mononuclear cells (PBMCs) obtained from a subject. Suitably the subject from whom the PBMCs are obtained is a mammal, preferably a human. Suitably the cell is matched (e.g. HLA matched) or is autologous to the subject to whom the engineered cell is to be administered. Suitably, the subject to be treated is a mammal, particularly a human. The cell may be generated ex vivo either from a patient’s own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2nd party), or peripheral blood from an unconnected donor (3rd party). Suitably the cell is autologous to the subject to whom the engineered cell is to be administered. Suitably, the Treg is part of a population of cells. Suitably, the population of Tregs comprises 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”. In some aspects, the Treg may be derived from ex-vivo differentiation of inducible progenitor cells (e.g. iPSCs) or embryonic progenitor cells to the Treg. A nucleic acid molecule, construct or vector as described herein may be introduced into the inducible progenitor cells or embryonic progenitor cells prior to, or after, differentiation to a Treg. Suitable methods for differentiation are known in the art and include that disclosed in Haque et al, J Vis Exp., 2016, 117, 54720 (incorporated herein by reference). As used herein, the term “conventional T cell” or Tcon or Tconv (used interchangeably herein) means a T lymphocyte cell which expresses an αβ T cell receptor (TCR) as well as a co-receptor which may be cluster of differentiation 4 (CD4) or cluster of differentiation 8 (CD8) and which does not have an immunosuppressive function. Conventional T cells are present in the peripheral blood, lymph nodes, and tissues. Suitably, the engineered Treg may be generated from a Tcon by introducing the nucleic acid which includes a sequence coding for FOXP3. Alternatively, the engineered Treg may be generated from a Tcon by in vitro culture of CD4+ CD25-FOXP3- cells in the presence of IL-2 and TGF-β. When the chimeric protein is induced, a Treg herein may have increased persistence as compared to a Treg cell without the chimeric protein for a constitutively active chimeric protein or without the inducer for an inducible chimeric protein (e.g. a Treg cell transduced with the inducible chimeric protein in the absence of the inducer). “Persistence” as used 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). A Treg as disclosed herein may have at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% increased persistence as compared to a Treg which does not express the chimeric protein herein, or a Treg which is not induced. Persistence can be measured by for example, determining the amount or numbers of administered cells within a subject or patient over time, where cells expressing a chimeric protein of the invention are compared to equivalent cell types which do not express the chimeric protein, or compared to non-engineered cells. It is possible to track administered cells, for example, using a marker protein, e.g. CD34 for cells which also express a RQR8 safety switch. 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 an embodiment the cell is a production host cell. The cell may be for production of the nucleic acid, e.g. cloning, or vector, or polypeptides. Also provided herein is a cell population comprising a cell as defined or described herein. It will be appreciated that a cell population may comprise both the present cells comprising a nucleic acid molecule, construct or vector as defined herein, and cells which do not comprise the nucleic acid molecule, construct or vector, e.g. untransduced or untransfected cells. Although in a particular embodiment, all the cells in a population may comprise the nucleic acid, expression construct or vector, cell populations having at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 99% of cells comprising a nucleic acid, expression construct or vector are provided. As indicated above, the chimeric protein may be co-expressed in or on a cell in conjunction with a mutant calcineurin which is resistant to at least one calcineurin inhibitor (CNI). In particular, the mutant calcineurin may be resistant to one calcineurin inhibitor and sensitive to another calcineurin inhibitor. Such calcineurin mutants are described in Brewin et al., 2009, Blood, 114(23), 4792-4802, incorporated herein by reference. Calcineurin inhibitors, notably FK506 (tacrolimus) and cyclosporine (CsA) are immunosuppressants commonly used in ACT. Thus, by co-expressing such a calcineurin mutant in a cell for ACT, the cell may be rendered resistant to the immunosuppressant. This may confer various advantages on the cell. For example, such co-expression may advantageously allow the ACT subject (i.e. cell recipient) to continue immunosuppressant therapy, and may allow lymphodepletion of the recipient to be avoided. Thus, it may not be necessary to interrupt or stop administering an immunosuppressant to the subject. Further, advantageously, co-expressing such a mutant in the cell along with the chimeric protein (and any other protein of interest, such as a CAR), may allow for selective expansion of transduced (or transfected etc.) cells. In this regard, during in vitro expansion protocols the transduced or transfected cell population (that is the cells resulting from the transduction/transfection protocol etc.), may be cultured in the presence of the calcineurin inhibitor to which the cells have been rendered resistant by virtue of the mutant calcineurin. Any non-transduced/non-transfected cells which are undesired in the final cell preparation for ACT will not be able to grow (proliferate) in the presence of the calcineurin inhibitor and will not be expanded. Further, if the mutant calcineurin is resistant to a particular inhibitor, but sensitive to another, then that provides a safety mechanism by which the cell containing the mutant calcineurin may be controlled or eliminated – by administering the inhibitor to which the mutant calcineurin is sensitive. As indicated above, such mutant calcineurin proteins are known in the art. In particular, the mutant calcineurin may be resistant to FK506 but sensitive to CsA, resistant to CsA but sensitive to FK506, or resistant to both FK506 and CsA. Such mutants are described in Brewin et al., 2009 (supra). The mutant calcineurin may comprise mutations in the A subunit (CNa) (SEQ ID NO: 97) or the B subunit (CNb) (SEQ ID NO: 98) of calcineurin. Mutations to the A subunit may comprise a mutation at one or more of the following positions with reference to SEQ ID NO: 97; V314, Y341, M347, T351, W352, S353, L354, F356 and K360. Particularly, a mutated A subunit may comprise one or more of the following mutations with reference to SEQ ID NO: 97: V314K, V314R or V314F; Y341F; M347W, M347R or M347E; T351E; W352A, W352C or W352E; S353H or S353N; L354A; F356A; and/or K360A or K360F. Combinations of mutations that may be made to the A subunit include with respect to SEQ ID NO: 97: L354A and K360A; L354A and K360F; T351E and K360F; W352A and S353H; T351E and L354A; W353C and K360F; W352C, L354A and K360F; V314K and Y341F; and V314R and Y341F. Mutations to the B subunit may be at one or more of the following positions with reference to SEQ ID NO: 98; Q51, L116, M119, V120, G121, N122, N123, L124, K125 and K165. Particularly, a mutated B subunit may comprise one or more of the following mutations with reference to SEQ ID NO: 98: Q51S; L116R or L116Y; M119A, M119W or M119-F-Ins; V120L, V120S, V120D or V120F; G121-LF-Ins; N122A, N122H, N122F, or N122S; N123H, N123R, N123F, N123K, or N123W; L124T; K125A, K125E, K125W, K125-LA-Ins, K125-VQ-Ins or K125-IE-Ins; and/or K165Q. Combinations of mutations that may be made to the B subunit include with respect to SEQ ID NO: 98: V120S and L124T; V120D and L124T; N123W and K125-LA-Ins; L124T and K125-LA-Ins; V120D and K125-LA-Ins; and M119-F-Ins and G121-LF-Ins Particular mention may be made of the following mutants: (i) CNa12 which is resistant to FK506, but sensitive to CsA. This mutant comprises mutations T351E and L354A. The sequence of CNa12 is shown in SEQ ID NO: 94; (ii) CNa18, CNa19, CNa20, CNa21 and CNa22 and CNb26 which are resistant to CsA, but sensitive to FK506. These mutants comprise, respectively, mutations M347E; V314K; V314R; Y3F, V314K and Y341F; V314R and Y341F; and V120S and L124T. The sequence of CNa22 is shown in SEQ ID NO: 95; (iii) CNb21, CNb23, and CNb30 which are resistant to both CsA and FK506. These mutants comprise, respectively, mutations K125-VQ- Ins; K125-LA-Ins; and L124T and K125-LA-Ins. The sequence of CNb30 is shown in SEQ ID NO: 96. Accordingly, in an embodiment, a mutant calcineurin for use herein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 94, 95 or 96 or an amino acid sequence which has at least 85, 90, 95, 97, 98 or 99% sequence identity to SEQ ID NOs: 94, 95 or 96, provided that the mutations indicated above for SEQ NOs: 94, 95 or 96 are retained. Other mutants are described in Brewin (supra) and may also be used. The skilled person would be able to derive new mutants comprising other mutations in addition or in place of those disclosed in Tables 1 and 2 of Brewin, e.g. different substitutions, or different combinations of the mutations which are disclosed. Also disclosed in Brewin 2009 are assays for screening for resistance or susceptibility to FK506 or CsA which may be used to select mutants. The terms “sensitive” and “susceptible” are used interchangeably herein. A mutant which is resistant to a given CNI means a mutant which exhibits resistance to the CNI, that is a mutant which is able to confer on a cell expressing it the ability to grow, or proliferate, or to exhibit a functional property in its presence. In particular, a cell comprising the mutant may exhibit increased growth, which may be reflected in an increase in a functional property, for example increased IL-2 secretion in Jurkat cells expressing the mutant, compared to a cell which does not comprise the mutant. It is not required that a mutant exhibits 100% resistance to a given CNI. The levels of resistance may vary, and some degree of susceptibility may be tolerated, as long as there is an observable increase in growth or functionality of cells expressing the mutant in the presence of the CNI in question, e.g. as compared to cells without (not expressing) the mutant. For example, the mutant may exhibit at least 35, 40 or 45% resistance, more particularly at least 50, 55, 60 or 65% resistance. As discussed above, a particular type of cell of interest is a Treg cell. Treg cells are administered in ACT to suppress undesired immune response or immune activity. The expression of mutant calcineurins in Treg cells is of particular interest. Accordingly, in a further aspect, there is more generally provided herein a Treg cell which comprises a mutant calcineurin which is resistant to at least one calcineurin inhibitor. In an embodiment of this aspect, the mutant calcineurin is resistant to at least one calcineurin inhibitor and sensitive to at least one calcineurin inhibitor. In particular the mutant is (i) resistant to FK506 and sensitive to CsA or (ii) resistant to CsA and sensitive to FK506. The mutant may be any one of the mutants discussed above. The Treg cell may be engineered or modified to comprise the mutant calcineurin by introducing a nucleic acid, construct or vector comprising a nucleotide sequence encoding the mutant calcineurin. Such a Treg cell may further comprise or express another protein of interest. This may be, for example, a chimeric protein as described herein and/or a CAR or other chimeric receptor, a TCR, or any other polypeptide as referred to or described herein. Particularly, a cell, such as a Tcell, e.g., a Treg cell, may further comprise or express a chimeric protein which is capable of providing a STAT5 mediated signal to the cell. Such a chimeric protein may be one as described herein, or may be an alternative molecule, such as one described in WO2020/044055, which is incorporated by reference. Particularly, such a chimeric protein may comprise a JAK1- and/or JAK2-binding domain and a STAT5 association motif, as described in detail above. Thus, in this regard, a cell, e.g., a Treg cell is provided which comprises a nucleic acid molecule comprising a nucleotide sequence encoding a mutated calcineurin and a nucleic acid molecule comprising a nucleotide sequence encoding a chimeric protein comprising a JAK1- and/or JAK2-binding domain and a STAT5 association motif, particularly wherein said chimeric protein is capable of dimerization. In one embodiment, the JAK1- and/or JAK2-binding domain and STAT5 association motif may be present within the endodomain of a chimeric antigen receptor, as described in WO2020/044055. As discussed above, the chimeric protein should be capable of providing a STAT5 mediated signal to the cell (e.g. upon dimerization which may be constitutively provided for or inducible, e.g. by binding of the chimeric protein to a target or to a ligand or CID as discussed herein). The cell may also in this embodiment comprise a CAR as defined herein. Thus, the mutant calcineurins may be used in Treg cells which have been, are being, or will be engineered for another purpose, e.g. to express another desired heterologous (i.e. non-native) protein. Another aspect provided herein is a method for selectively expanding engineered Treg cells, said method comprising introducing into a Treg cell a nucleic acid molecule, construct or vector comprising a nucleotide sequence encoding a mutant calcineurin as defined herein, and culturing said cells in the presence of a calcineurin inhibitor to which the mutant calcineurin is resistant. Particularly, the cell may further comprise a chimeric receptor comprising a JAK1- and/or JAK2-binding domain and a STAT5 association motif. The engineered Treg cell may comprise a further nucleotide sequence which is, or has been, introduced into the cell, and which encodes a protein of interest. The culturing step may comprise any known or desired protocol for activating and/or expanding cells. For example, the cells may be grown in the presence of CD3 and/or CD28 as described above. There is also provided a pharmaceutical composition comprising a cell or cell population as defined or described herein, or a vector as defined herein. The vector may be used for gene therapy. Thus, rather than administering a cell, a vector may be administered instead, 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. A pharmaceutical composition is a composition that comprises or consists of a therapeutically effective amount of a pharmaceutically active agent i.e. the cell (e.g. Treg), cell population or vector. It preferably includes a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof). Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit.1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilising agent(s). By “pharmaceutically acceptable” is included 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 cell or vector and not deleterious to the recipients thereof. Typically, the carriers, diluents, and excipients will be saline or infusion media which will be sterile and pyrogen free, however, other acceptable carriers, diluents, and excipients may be used. Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl- cellulose, polyvinylpyrrolidone, and the like. The cells, cell population or pharmaceutical compositions may be administered in a manner appropriate for treating and/or preventing the desired disease or condition. The quantity and frequency of administration will be determined by such factors as the condition of the subject, and the type and severity of the subject's disease or condition, although appropriate dosages may be determined by clinical trials. The pharmaceutical composition may be formulated accordingly. The cell, cell population or pharmaceutical composition as described herein can be administered parenterally, for example, intravenously, or they may be administered by infusion techniques. The cell, cell population or pharmaceutical composition may be administered in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solution may be suitably buffered (preferably to a pH of from 3 to 9). The pharmaceutical composition may be formulated accordingly. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art. The pharmaceutical compositions may comprise cells in infusion media, for example sterile isotonic solution. The pharmaceutical composition may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. The cell, cell population or pharmaceutical composition may be administered in a single or in multiple doses. Particularly, the cell, cell population or pharmaceutical composition may be administered in a single, one off dose. The pharmaceutical composition may be formulated accordingly. 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 lympho-depletive agents (e.g. thymoglobulin, campath- 1H, anti-CD2 antibodies, anti-CD3 antibodies, anti-CD20 antibodies, cyclophosphamide, fludarabine), inhibitors of mTOR (e.g. sirolimus, everolimus), drugs inhibiting costimulatory pathways (e.g. anti-CD40/CD40L, CTAL4Ig), and/or drugs inhibiting specific cytokines (IL-6, IL-17, TNFalpha, IL18). Depending upon the disease/condition and subject to be treated, as well as the route of administration, the cell, cell population or pharmaceutical composition may be administered at varying doses (e.g. measured in cells/kg or cells/subject). The physician in any event will determine the actual dosage which will be most suitable for any individual subject and it will vary with the age, weight and response of the particular subject. Typically, however, for the cells herein, doses of 5x107 to 3x109 cells, or 108 to 2x109 cells per subject may be administered. The cell may be appropriately modified for use in a pharmaceutical composition. For example, cells may be cryopreserved and thawed at an appropriate time, before being infused into a subject. Further provided herein is the use of kits, or combination products, comprising the cell, cell population and/or pharmaceutical composition herein. Preferably said kits are for use in the methods and uses as described herein, e.g., the therapeutic methods as described herein. Preferably said kits comprise instructions for use of the kit components. Kits or compositions may further comprise the inducer, e.g. rapamycin or an analogue thereof. The cells, cell populations, compositions and vectors herein may be for use therapy, that is in treating or preventing a disease or condition. As noted above, the cell in or on which the chimeric protein is expressed is typically a cell which is modified, or engineered to express a further molecule (e.g. a further protein), notably a receptor, e.g. a CAR or TCR. Accordingly, the therapy may be for the prevention or treatment of a disease or condition which may be treated by or with a cell expressing the receptor, e.g. the CAR. The cells and compositions containing them are for adoptive cell therapy (ACT). Various conditions may be treated by administration of cells, including particularly Treg cells, expressing a CAR according to the present disclosure. As noted above, this may be conditions responsive to immunosuppression, and particularly the immunosuppressive effects of Treg cells. The cells, cell populations, compositions and vectors described herein may thus be used for inducing, or achieving, immunosuppression in a subject. The Treg cells administered, or modified in vivo, may be targeted by expression of the receptor, e.g. CAR. Conditions suitable for such treatment include infectious, neurodegenerative or inflammatory disease, or more broadly a condition associated with any undesired or unwanted or deleterious immune response. Conditions to be treated or prevented include inflammation, or alternatively put, a condition associated with or involving inflammation. Inflammation may be chronic or acute. Furthermore, the inflammation may be low-level or systemic inflammation. For example the inflammation may be inflammation which occurs in the context of a metabolic disorder, for example metabolic syndrome, or in the context of insulin resistance, or type II diabetes or obesity and such like. In particular, the cells, cell populations, vectors and pharmaceutical compositions provide a means for inducing tolerance to a transplant; treating and/or preventing cellular and/or humoral transplant rejection; treating and/or preventing graft-versus-host disease (GvHD), an autoimmune or allergic disease; or to promote tissue repair and/or tissue regeneration; or to ameliorate inflammation. The cells, cell populations, vectors and pharmaceutical compositions may be used in a method which comprises the step of administering a cell, cell populations, vector or a pharmaceutical composition as described herein to a subject. As used herein, “inducing tolerance to a transplant” refers to inducing tolerance to a transplanted organ in a recipient. In other words, inducing tolerance to a transplant means to reduce the level of a recipient’s immune response to a donor transplant organ. Inducing tolerance to a transplanted organ may reduce the amount of immunosuppressive drugs that a transplant recipient requires, or may enable the discontinuation of immunosuppressive drugs. For example, the engineered cells, e.g. Tregs, may be administered to a subject with a disease in order to lessen, reduce, or improve at least one symptom of disease such as jaundice, dark urine, itching, abdominal swelling or tenderness, fatigue, nausea or vomiting, and/or loss of appetite. The at least one symptom may be lessened, reduced, or improved 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. The engineered cells, e.g. Tregs may be administered to a subject with a disease in order to slow down, reduce, or block the progression of the disease. The progression of the disease may be slowed down, reduced, or blocked by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a subject in which the engineered cells are not administered, or progression of the disease may be completely stopped. In one embodiment, the subject is a transplant recipient undergoing immunosuppression therapy. Suitably, the subject is a mammal. Suitably, the subject is a human. The transplant may be selected from a liver, kidney, heart, lung, pancreas, intestine, stomach, bone marrow, vascularized composite tissue graft, and skin transplant. Suitably, the cells may express a CAR which comprises an antigen binding domain which is capable of specifically binding to a HLA antigen that is present in the graft (transplant) donor but not in the graft (transplant) recipient. Suitably, the transplant is a liver transplant. In embodiments where the transplant is a liver transplant, the antigen may be a HLA antigen present in the transplanted liver but not in the patient, a liver-specific antigen such as NTCP, or an antigen whose expression is up-regulated during rejection such as CCL19, MMP9, SLC1A3, MMP7, HMMR, TOP2A, GPNMB, PLA2G7, CXCL9, FABP5, GBP2, CD74, CXCL10, UBD, CD27, CD48, CXCL11. As discussed above, in one representative and preferred embodiment the antigen is HLA-A2. A method for treating a disease or condition relates to the therapeutic use of the cells herein. In this respect, the cells may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease or condition and/or to slow down, reduce or block the progression of the disease. Suitably, treating and/or preventing cellular and/or humoral transplant rejection may refer to administering an effective amount of the cells (e.g. Tregs) such that the amount of immunosuppressive drugs that a transplant recipient requires is reduced, or may enable the discontinuation of immunosuppressive drugs. Preventing a disease or condition relates to the prophylactic use of the cells herein. In this respect, the cells may be administered to a subject who has not yet contracted or developed the disease or condition and/or who is not showing any symptoms of the disease or condition to prevent the disease or condition or to reduce or prevent development of at least one symptom associated with the disease or condition. The subject may have a predisposition for, or be thought to be at risk of developing, the disease or condition. The autoimmune or allergic disease may be selected from inflammatory skin diseases including psoriasis and dermatitis (e.g. atopic dermatitis); responses associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); dermatitis; allergic conditions such as food allergy, eczema and asthma; rheumatoid arthritis; systemic lupus erythematosus (SLE) (including lupus nephritis, cutaneous lupus); diabetes mellitus (e.g. type 1 diabetes mellitus or insulin dependent diabetes mellitus); multiple sclerosis; neurodegenerative disease, for example, Amyotrophic Lateral Sclerosis (ALS); Chronic inflammatory demyelinating polyneuropathy (CIPD) and juvenile onset diabetes. As indicated above, the chimeric protein is not limited to use in the context of immunosuppressive therapy, and the protein may be expressed in cells for the treatment of conditions such as cancer or infections. It may be desirable in such contexts to kill or ablate cancer or infected cells, and in such cases the chimeric protein may be expressed in cytotoxic cells, such as cytotoxic T cells or NK cells, or precursors therefor. The receptor (e.g. CAR or TCR) co-expressed with the chimeric protein in such cases may be directed against a cancer antigen or an antigen from a pathogen etc. The medical use of or method herein may involve the steps of: (i) isolating a cell-containing sample or providing a cell-containing sample; (ii) introducing a nucleic acid molecule, construct or a vector as defined herein to the cell; and (iii) administering the cells from (ii) to a subject. The cell may be a Treg as defined herein. An enriched Treg population may be isolated and/or generated from the cell containing sample prior to, and/or after, step (ii) of the method. For example, isolation and/or generation may be performed prior to 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 chimeric protein, the nucleic acid molecule, construct, and/or the vector as described herein. Suitably, the cell may be autologous. Suitably, the cell may be allogeneic. Suitably, the cell (e.g. the engineered Treg) may be administered in combination with one or more other therapeutic agents, such as lympho-depletive agents. The engineered cell, e.g. Treg, may be administered simultaneously with or sequentially with (i.e. prior to or after) the one or more other therapeutic agents. Cells, e.g. Tregs, may be activated and/or expanded prior to, or after, the introduction of a nucleic acid molecule as described herein, for example by treatment with an anti-CD3 monoclonal antibody or both anti-CD3 and anti-CD28 monoclonal antibodies. Expansion protocols are discussed above. The cell, e.g. Tregs, may be washed after each step of the method, in particular after expansion. The population of engineered cells, e.g., Treg cells may be further enriched by any method known to those of skill in the art, for example by FACS or magnetic bead sorting. The steps of the method of production may be performed in a closed and sterile cell culture system. The invention may also provide a method for increasing the stability and/or suppressive function of a cell comprising the step of introducing a nucleic acid molecule, an expression construct or vector as provided herein into the cell. An increase in suppressive function can be measured as discussed above, for example by co-culturing activated antigen-specific Tconv cells with cells of the invention, and for example measuring the levels the cytokines produced by the Tconv cells. An increase in suppressive function may be an increase of at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% as compared to a non-engineered or non-induced Treg. An increase in stability of a cell, e.g. a Treg as defined herein, refers to an increase in the persistence or survival of those cells or to an increase in the proportion of cells retaining a Treg phenotype over a time period (e.g. to cells retaining Treg markers such as FOXP3 and Helios) as compared to a non- engineered or non-induced Treg. An increase in stability may be an increase in stability of at least 10, 20, 30, 40, 50, 60, 70, 80 or 90%, and may be measured by techniques known in the art, e.g. staining of Treg cell markers within a population of cells, and analysis by FACS. A further aspect provided herein is a combination product comprising (a) a cell, cell population, vector or pharmaceutical composition as defined herein, and (b) an inducer molecule, for use in therapy, particularly ACT or gene therapy. The therapy may be any therapy as defined above, and further described herein. The components (a) and (b) of the combination product may be for separate, sequential or simultaneous use. The components (a) and (b) of the combination product will typically be provided as separate compositions, i.e. they will be formulated separately. Thus, the combination product may alternatively be defined or referred to as a kit. The components (a) and (b) will be administered to the subject separately. They may be administered to a subject at the same time, or at different times, for example at spaced apart time intervals. For example, the cells etc. may be administered first, followed by the CID. The CID may be administered after an appropriate time interval, or shortly after the cells etc. The skilled clinician would readily be able to devise an appropriate administration regime according to principles known in the art. This disclosure is not limited by the exemplary methods and materials disclosed herein, and any 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 indicated, any 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, respectively. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure. It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The terms "comprising", "comprises" and "comprised of' as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of' also include the term "consisting of'. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. All publications mentioned herein are incorporated herein by reference. Description of Sequences: SEQ ID NO: 1 – the native FKBP12 domain MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQ E VIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE SEQ ID NO: 2 – wild-type FRB segment of mTOR MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSF N QAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKLES SEQ ID NO: 3 – FRB with T to L substitution at 2098 MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSF N QAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKLES SEQ ID NO: 4 – FRB segment of mTOR with T to H substitution at 2098 and W to F substitution at residue 2101 MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSF N QAYGRDLMEAQEWCRKYMKSGNVKDLHQAFDLYYHVFRRISKLES SEQ ID NO: 5 – FRB segment of mTOR with K to P substitution at residue 2095 MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSF N QAYGRDLMEAQEWCRKYMKSGNVPDLTQAWDLYYHVFRRISKLES SEQ ID NO: 6 FRB sequence from SEQ ID NO.1 protein of WO2016/135470 (100aa) MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSF NQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKLEY SEQ ID NO: 7 – FKBP sequence from SEQ ID NO.4 protein of WO2016/135470 GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEV IRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGI IPPHATLVFDVELLKLE SEQ ID NO: 8 – codon-wobbled FKBP sequence from SEQ ID NO.4 protein of WO2016/135470 (FKBPw) MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGK QEVIR GWEEGVAQMSVGQRAKLTISPDYAYGATGHPGI IPPHATLVFDVELLKLES SEQ ID NO: 9 – amino acid numbers 266 to 551 of IL-2 receptor β chain (NCBI REFSEQ: NP_000869.1) NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAP EISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEAC QVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSL LGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPP PELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQ GQDPTHLV SEQ ID NO: 10 – IL7RA (AA 265 to 459 of NP_002176.2) KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVE GFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSA CDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQ GQPILTSLGSNQEEAYVTMSSFYQNQ SEQ ID NO: 11 – IL9R (AA 292 to 521of NP_002177.2) KLSPRVKRIFYQNVPSPAMFFQPLYSVHNGNFQTWMGAHGAGVLLSQDCAGTPQ GALEPCVQEATALLTCGPARPWKSVALEEEQEGPGTRLPGNLSSEDVLPAGCTEW RVQTLAYLPQEDWAPTSLTRPAPPDSEGSRSSSSSSSSNNNNYCALGCYGGWHL SALPGNTQSSGPIPALACGLSCDHQGLETQQGVAWVLAGHCQRPGLHEDLQGMLL PSVLSKARSWTF SEQ ID NO: 12 – IL4RA (AA 257 to 825 of NPJD00409.1) KIKKEWWDQIPNPARSRLVAIIIQDAQGSQWEKRSRGQEPAKCPHWKNCLTKLLPC FLEHNMKRDEDPHKAAKEMPFQGSGKSAWCPVEISKTVLWPESISVVRCVELFEAP VECEEEEEVEEEKGSFCASPESSRDDFQEGREGIVARLTESLFLDLLGEENGGFCQ QDMGESCLLPPSGSTSAHMPWDEFPSAGPKEAPPWGKEQPLHLEPSPPASPTQS PDNLTCTETPLVIAGNPAYRSFSNSLSQSPCPRELGPDPLLARHLEEVEPEMPCVP QLSEPTTVPQPEPETWEQILRRNVLQHGAAAAPVSAPTSGYQEFVHAVEQGGTQA SAVVGLGPPGEAGYKAFSSLLASSAVSPEKCGFGASSGEEGYKPFQDLIPGCPGD PAPVPVPLFTFGLDREPPRSPQSSHLPSSSPEHLGLEPGEKVEDMPKPPLPQEQAT DPLVDSLGSGIVYSALTCHLCGHLKQCHGQEDGGQTPVMASPCCGCCCGDRSSP PTTPLRAPDPSPGGVPLEASLCPASLAPSGISEKSKSSSSFHPAPGNAQSSSQTPKI VNFVSVGPTYMRVS SEQ ID NO: 13 – IL3RB (AA 461 to 897 of NP_000386.1) RFCGIYGYRLRRKWEEKIPNPSKSHLFQNGSAELWPPGSMSAFTSGSPPHQGPW GSRFPELEGVFPVGFGDSEVSPLTIEDPKHVCDPPSGPDTTPAASDLPTEQPPSPQ PGPPAASHTPEKQASSFDFNGPYLGPPHSRSLPDILGQPEPPQEGGSQKSPPPGS LEYLCLPAGGQVQLVPLAQAMGPGQAVEVERRPSQGAAGSPSLESGGGPAPPAL GPRVGGQDQKDSPVAIPMSSGDTEDPGVASGYVSSADLVFTPNSGASSVSLVPSL GLPSDQTPSLCPGLASGPPGAPGPVKSGFEGYVELPPIEGRSPRSPRNNPVPPEA KSPVLNPGERPADVSPTSPQPEGLLVLQQVGDYCFLPGLGPGPLSLRSKPSSPGP GPEIKNLDQAFQVKKPPGQAVPQVPVIQLFKALKQQDYLSLPPWEVNKPGEVC SEQ ID NO: 14 – IL17RB (AA 314 to 502 of NP_061195.2) RHERIKKTSFSTTTLLPPIKVLVVYPSEICFHHTICYFTEFLQNHCRSEVILEKWQKKKI AEMGPVQWLATQKKAADKVVFLLSNDVNSVCDGTCGKSEGSPSENSQDLFPLAFN LFCSDLRSQIHLHKYVVVYFREIDTKDDYNALSVCPKYHLMKDATAFCAELLHVKQQ VSAGKRSQACHDGCCSL SEQ ID NO: 15 - IL7RA 2Y truncated: KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVE GFLQDTFPQQPILTSLGSNQEEAYVTMSSFYQNQ SEQ ID NO: 16 – STAT5 association motif YXXF/L SEQ ID NO: 17 – STAT5 association motif YCTF SEQ ID NO: 18 – STAT5 association motif YFFF SEQ ID NO: 19 – STAT5 association motif YLSL SEQ ID NO: 20 – STAT5 association motif YLSLQ SEQ ID NO: 21 – JAK1-binding motif KVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERD K SEQ ID NO: 22 – JAK1-binding motif NPWFQRAKMPRALDFSGHTHPVATFQPSRPESVNDLFLCPQKELT SEQ ID NO: 23 – JAK1-binding motif GYICLRNSLPKVLNFHNFLAWPFPNLPPLEAMDMVEVIYINR SEQ ID NO: 24 – JAK1-binding motif PLKEKSIILPKSLISVVRSATLETKPESKYVSLITSYQPFSL SEQ ID NO: 25 – JAK1-binding motif RRRKKLPSVLLFKKPSPFIFISQRPSPETQDTIHPLDEEAFLK SEQ ID NO: 26 – JAK1-binding motif YIHVGKEKHPANLILIYGNEFDKRFFVPAEKIVINFITLNISDDS SEQ ID NO: 27 – JAK1-binding motif RYVTKPPAPPNSLNVQRVLTFQPLRFIQEHVLIPVFDLSGP SEQ ID NO: 28 – JAK2-binding motif NYVFFPSLKPSSSIDEYFSEQPLKNLLLSTSEEQIEKCFIIEN SEQ ID NO: 29 – JAK2-binding motif YWFHTPPSIPLQIEEYLKDPTQPILEALDKDSSPKDDVWDSVSIISFPE SEQ ID NO: 30 – JAK2-binding motif YAFSPRNSLPQHLKEFLGHPHHNTLLFFSFPLSDENDVFDKLSVIAEDSES SEQ ID NO: 31 – IL2RB truncated – Y510 NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAP EISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLV SEQ ID NO: 32 – IL2RB truncated – Y510 & Y392 NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAP EISPLEVLERDKVTQLLDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERM PPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVS FPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV SEQ ID NO: 33 – JAK3-binding motif ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEI SEQ ID NO: 34 – JAK3-binding motif ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKG GALGEGPGASPCNQHSPYWAPPCYTLKPET SEQ ID NO: 35 – FKBP12 F36V GVGVETISPGDGRTFPKRGQTGVVHYTGMLEDGKKVDSSRDRNKPFKFM LGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVF DVELLKLES SEQ ID NO: 36 – FKBP(F36V) MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQ EVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE SEQ ID NO: 37 – FKBP(F36V)-FKBP(F36V) MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQ EVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE SEQ ID NO: 38 – FKBP(F36V,L106P) MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQ EVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKP SEQ ID NO: 39 – FKBP(F36V,L106P)-FKBP(F36V,L106P) MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQ EVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKPGVQV ETISPGDGRTFPKRGQTCVVHYTGMLEDKKVDSSRDRNKPFKFMLGKQEVIRGWE EGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKPE SEQ ID NO: 40 – FKBP(E31G,36V,R71G,K105E)-FKBP(E31G,36V,R71G,K105E) MGVQVETISPGDGRTFPKRGQTCVVHYTGMLGDGKKVDSSRDRNKPFKFMLGKQ EVIRGWEEGVAQMSVGQGAKLTISPDYAYGATGHPGIIPPHATLVFDVELLELGVQV ETISPGDGRTFPKRGQTCVVHYTGMLGDGKKVDSSRDRNKPFKFMLGKQEVIRGW EEGVAQMSVGQGAKLTISPDYAYGATGHPGIIPPHATLVFDVELLELE SEQ ID NO:41 – RQR8 ACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGS PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL LSLVITLYCNHRNRRRVCKCPRPVV SEQ ID NO: 42 – linker sequence S(GGGGS)2 SEQ ID NOs: 43 – 72 – other linker sequences ETSGGGGSRL (SEQ ID NO: 43) S(GGGGS)4 (SEQ ID NO: 44) SGGGGSGGGGSGGGGS ((SEQ ID NO: 45) S(GGGGS)1-5 (where GGGGS is SEQ ID NO: 46) (GGGGS)1-5 (where GGGGS is SEQ ID NO: 46) S(GGGS)1-5 (where GGGS is SEQ ID NO: 47) (GGGS)1-5 (where GGGS is SEQ ID NO: 47) S(GGGGGS)1-5 (where GGGGGS is SEQ ID NO: 48) (GGGGGS)1-5 (where GGGGGS is SEQ ID NO: 48 S(GGGGGGS)1-5 (where GGGGGGS is SEQ ID NO:49) (GGGGGGS)1-5 (where GGGGGGS is SEQ ID NO: 49) G6 (SEQ ID NO: 50) G8 (SEQ ID NO: 51) KESGSVSSEQLAQFRSLD (SEQ ID NO: 52) EGKSSGSGSESKST (SEQ ID NO: 53) GSAGSAAGSGEF (SEQ ID NO: 54) SGGGGSAGSAAGSGEF (SEQ ID NO: 55) SGGGLLLLLLLLGGGS (SEQ ID NO: 56) SGGGAAAAAAAAGGGS (SEQ ID NO: 57) SGGGAAAAAAAAAAAAAAAAGGGS (SEQ ID NO: 58) SGALGGLALAGLLLAGLGLGAAGS (SEQ ID NO: 59) SLSLSPGGGGGPAR (SEQ ID NO: 60) SLSLSPGGGGGPARSLSLSPGGGGG (SEQ ID NO: 61) GSSGSS (SEQ ID NO: 62) GSSSSSS (SEQ ID NO: 63) GGSSSS (SEQ ID NO: 64) GSSSSS (SEQ ID NO: 65) SGGGGS (SEQ ID NO: 66) GGGGSGGGGSGGGGS (SEQ ID NO: 67) GGGGG (SEQ ID NO: 68) GGGGSGGGGS (SEQ ID NO: 69) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 70) GGGGGGG (SEQ ID NO: 71) GGGGGGGGG (SEQ ID NO: 72) SEQ ID NO: 73 – OX40 MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVS RCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCR CRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASN SSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRA SEQ ID NO: 74 – P2A peptide – cleavage domain ATNFSLLKQAGDVEENPGP SEQ ID NO: 75 – T2A peptide – cleavage domain EGRGSLLTCGDVEENPGP SEQ ID NO: 76 – E2A peptide – cleavage domain QCTNYALLKLAGDVESNPGP SEQ ID NO: 77 – F2A peptide – cleavage domain VKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 78 – Furin cleavage site RXXR SEQ ID NO: 79 – Furin cleavage site RRKR SEQ ID NO: 80 – IL2Rβ endodomain NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAP EISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEAC QVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSL LGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPP PELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQ GQDPTHLV SEQ ID NO: 81 – CD3zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR SEQ ID NO: 82 – CD28 intracellular signalling RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 83 – CD27 intracellular signalling QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP SEQ ID NO: 84 – STAT 3 signal YXXQ SEQ ID NO: 85 – STAT 3 signal YRHQ SEQ ID NO: 86 – example construct (frb-fkbp12-IL2RB truncated) MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMWEGPQTLKETSF NQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKLEYSGGGSLEG VQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGG SGGGGSGGGGSNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSP FPSSSFSPGGLAPEISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLV SEQ ID NO: 87 – JAK3 reverse orientation IESVLCLRESYDPQLSEALGKSVGSWASFNGHYETVLDELNKLTPIRPMTRE SEQ ID NO: 88 – FKBP Sequence in SEQ ID NO.1 of WO2016/135470 LEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQ EVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGI IPPHATLVFDVELLKLE SEQ ID NO: 89 – FKBP Sequence in SEQ ID NO.2 of WO2016/135470 MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGK QEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGI IPPHATLVFDVELLKLE SEQ ID NO: 90 – human calcineurin A isoform 1 MSEPKAIDPKLSTTDRVVKAVPFPPSHRLTAKEVFDNDGKPRVDILKAHLMKEGRLE ESV ALRIITEGAS ILRQEKNLLD IDAPVTVCGD IHGQFFDLMK LFEVGGSPAN TRYLFLGDYV DRGYFSIECV LYLWALKILY PKTLFLLRGN HECRHLTEYF TFKQECKIKY SERVYDACMD AFDCLPLAAL MNQQFLCVHG GLSPEINTLD DIRKLDRFKE PPAYGPMCDI LWSDPLEDFG NEKTQEHFTH NTVRGCSYFY SYPAVCEFLQ HNNLLSILRA HEAQDAGYRM YRKSQTTGFP SLITIFSAPN YLDVYNNKAA VLKYENNVMN IRQFNCSPHP YWLPNFMDVF TWSLPFVGEK VTEMLVNVLN ICSDDELGSE EDGFDGATAA ARKEVIRNKI RAIGKMARVF SVLREESESV LTLKGLTPTG MLPSGVLSGG KQTLQSATVE AIEADEAIKG FSPQHKITSF EEAKGLDRIN ERMPPRRDAM PSDANLNSIN KALTSETNGT DSNGSNSSNI Q SEQ ID NO: 91 – Cyclophilin isoform 1 MVNPTVFFDI AVDGEPLGRV SFELFADKVP KTAENFRALS TGEKGFGYKG SCFHRIIPGF MCQGGDFTRH NGTGGKSIYG EKFEDENFIL KHTGPGILSM ANAGPNTNGS QFFICTAKTE WLDGKHVVFG KVKEGMNIVE AMERFGSRNG KTSKKITIAD CGQLE SEQ ID NO: 92 – T-14-3-3c MAVAPTAREE NVYMAKLAEQ AERYEEMVEF MEKVSNSLGS EELTVEERNL LSVAYKNVIG ARRASWRIIS SIEQKEESRG NEEHVNSIRE YRSKIENELS KICDGILKLL DAKLIPSAAS GDSKVFYLKM KGDYHRYLAE FKTGAERKEA AESTLTAYKA AQDIATTELA PTHPIRLGLA LNFSVFYYEI LNSPDRACNL AKQAFDEAIA ELDTLGEESY KDSTLIMQLL RDNLTLWTSD MQDDGADEIK EDPKPDEAKN SEQ ID NO: 93 – C terminal 52 amino acids of Tobacco plasma membrane H+ ATPase (PMA2) TN FNELNQLAEE AKRRAEIARQ RELHTLKGHV ESVVKLKGLD IETIQQSYTV SEQ ID NO: 94 – CNa12 GQ463594 MSEPKAIDPKLSTTDRVVKAVPFPPSHRLTAKEVFDNDGKPRVDILKAHLMKEGRLE ESVALRIITEGASILRQEKNLLDIDAPVTVCGDIHGQFFDLMKLFEVGGSPANTRYLFL GDYVDRGYFSIECVLYLWALKILYPKTLFLLRGNHECRHLTEYFTFKQECKIKYSERV YDACMDAFDCLPLAALMNQQFLCVHGGLSPEINTLDDIRKLDRFKEPPAYGPMCDIL WSDPLEDFGNEKTQEHFTHNTVRGCSYFYSYPAVCEFLQHNNLLSILRAHEAQDA GYRMYRKSQTTGFPSLITIFSAPNYLDVYNNKAAVLKYENNVMNIRQFNCSPHPYW LPNFMDVFEWSAPFVGEKVTEMLVNVLNICSDDELGSEEDGFDGATAAARKEVIRN KIRAIGKMARVFSVLREESESVLTLKGLTPTGMLPSGVLSGGKQTLQSATVEAIEAD EAIKGFSPQHKITSFEEAKGLDRINERMPPRRDAMPSDANLNSINKALTSETNGTDS NGSNSSNIQ SEQ ID NO: 95 – CNa22 GQ463595 MSEPKAIDPKLSTTDRVVKAVPFPPSHRLTAKEVFDNDGKPRVDILKAHLMKEGRLE ESVALRIITEGASILRQEKNLLDIDAPVTVCGDIHGQFFDLMKLFEVGGSPANTRYLFL GDYVDRGYFSIECVLYLWALKILYPKTLFLLRGNHECRHLTEYFTFKQECKIKYSERV YDACMDAFDCLPLAALMNQQFLCVHGGLSPEINTLDDIRKLDRFKEPPAYGPMCDIL WSDPLEDFGNEKTQEHFTHNTVRGCSYFYSYPAVCEFLQHNNLLSILRAHEAQDA GYRMYRKSQTTGFPSLITIFSAPNYLDRYNNKAAVLKYENNVMNIRQFNCSPHPFW LPNFMDVFTWSLPFVGEKVTEMLVNVLNICSDDELGSEEDGFDGATAAARKEVIRN KIRAIGKMARVFSVLREESESVLTLKGLTPTGMLPSGVLSGGKQTLQSATVEAIEAD EAIKGFSPQHKITSFEEAKGLDRINERMPPRRDAMPSDANLNSINKALTSETN GTDSNGSNSSNIQ SEQ ID NO: 96 – CNb30 GQ463597 MGNEASYPLEMCSHFDADEIKRLGKRFKKLDLDNSGSLSVEEFMSLPELQQNPLVQ RVIDIFDTDGNGEVDFKEFIEGVSQFSVKGDKEQKLRFAFRIYDMDKDGYISNGELF QVLKMMVGNNTKLADTQLQQIVDKTIINADKDGDGRISFEEFCAVVGGLDIHKKMVV DV SEQ ID NO: 97 MSEPKAIDPKLSTTDRVVKAVPFPPSHRLTAKEVFDNDGKPRVDILKAHLMKEGRLE ESVALRIITEGASILRQEKNLLDIDAPVTVCGDIHGQFFDLMKLFEVGGSPANTRYLFL GDYVDRGYFSI ECVL YLWALKI L YPKTLFLLRGN H ECRH L TEYFTFKQECKI KYSERVYDACMDAFDCLPLAALMNQQFLCVHGGLSPEINTLDDIRKLDRFKEPPAY GPMCDILWSDPLEDFGNEKTQEHFTHNTVRGCSYFYSYPAVCEFLQHNNLLSILRA HEAQDAGYRMYRKSQTTGFPSLITIFSAPNYLDVYNNKAAVLKYENNVMNIRQFNC SPHPYWLPNFMDVFTWSLPFVGEKVTEMLVNVLNICSDDELGSEEDGFDGATAAA RKEVIRNKIRAIGKMARVFSVLREESESVLTLKGLTPTGMLPSGVLSGGKQTLQSAT VEAIEADEAI K GFSPQH KITSFEEAKGLDRI N ERM PPR RDAM PS DAN LNSI N KALTSETNGTDSNGSN SSNIQ SEQ ID NO: 98 MG N EASYPLEMCSH FDADEI KRLG KRFKKLDLDNSGSLSVEEFMSLPELQQN PLVQRVIDIFDTDGNGEVDFKEFIEGVSQFSVKGDKEQKLRFAFRIYDMDKDGYISN GELFQVLKMMVGNNLKDTQLQQIVDKTIINADKDGDGRISFEEFCAVVGGLDIHKKM VVDV SEQ ID NO: 99 – c-Jun leucine zipper RIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMN SEQ ID NO: 100 – Fos,leucine zipper LTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEFILAAY SEQ ID NO: 101 – BZip (RR) leucine zipper domain MDPDLEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGK SEQ ID NO: 102 – Azip (EE) leucine zipper domain MDPDLEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK SEQ ID NO: 103 – pQTX-0340 MILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQA YGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKSGGGSGVQVETISP GDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGV AQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGGSGGGGSG NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAP EISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLVTRATNFSLLKQAGDVEEN PGPMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL PVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKT RAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFK IRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF VTAAGITLGMDELYK SEQ ID NO: 104 – pQTX-0370 MILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQA YGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKSGGGSGVQVETISP GDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGV AQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGGSGGGGSG NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAP EISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLVTREGRGSLLTCGDVEENP GPMALPVTALLLPLALLLHAAAPQVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYG MHWVRQAPGKGLEWVAFIRNDGSDKYYADSVKGRFTISRDNSEKTVSLQMSSLRA EDTAVYYCAKNGESGPLDYWYLDLWGRGTLVTVSSGGGGSGGGGSGGGGSTDV VMTQSPSSLSASVGDRVTITCQSSLDISHYLNWYQQKPGKAPKLLIYDASNLETGVP SRFSGSGSGTHFTFTISSLQPEDFATYYCQQYDNLPLTFGGGTKLEIKRTRKFVPVF LPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPL AGTCGVLLLSLVITLYCNHRPPAWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY APPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPRSQLASATNFSLLKQAGDVEENPGPMVSKGEELFTGVVPILV ELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFS RYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGI DFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQN TPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLG SEQ ID NO: 105 – STAT1 association motif QLLLQQDKVPEPASLSSNHSLTSCFTNQGYF SEQ ID NO: 106 – pQTX-0583 MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSF NQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKLEYSGGGSNCR NTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISP LEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVY FTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGG PSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPEL VLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQD PTHLVTRRRKRGSGATNFSLLKQAGDVEENPGPMVSKGEELFTGVVPILVELDGDV NGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHM KQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDG NILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDG PVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 107 – FKBP 109aa LEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQ EVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE SEQ ID NO: 108 – pQTX-0584 MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGK QEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESG GGSNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPG GLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDAL EIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLL FSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLV DFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLS LQELQGQDPTHLVTRRRKRGSGATNFSLLKQAGDVEENPGPMVSKGEELFTGVVP ILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQC FSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELK GIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQ QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 109 – pQTX-0585 MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGK QEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESG GGSERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIP PKGGALGEGPGASPCNQHSPYWAPPCYTLKPETTRRRKRGSGATNFSLLKQAGD VEENPGPMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQT AKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPE DGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIV EQYERAEGRHSTGGMDELYK SEQ ID NO: 110 – pQTX-0586 MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSF NQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKLEYSGGGSLEG VQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGG SGGGGSGGGGSGNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSS PFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQG YFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYC TFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGP PTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARL PLNTDAYLSLQELQGQDPTHLVTRRRKRGSGATNFSLLKQAGDVEENPGPMVSKG EELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLV TTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEG DTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDG SVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITL GMDELYK SEQ ID NO: 111 – pQTX-0587 MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGK QEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESG GGSASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKET SFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKLEYSGGGGS GGGGSGGGGSGERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYS ERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPETTRRRKRGSGATN FSLLKQAGDVEENPGPMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGE GRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEG FKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWE ASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDI TSHNEDYTIVEQYERAEGRHSTGGMDELYK SEQ ID NO: 112 – FRB99aa ASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQ AYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKLEY SEQ ID NO: 113 – FRB94aa MILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQA YGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK SEQ ID NO: 114 – STAT3 association motif YLRQ The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention. Example 1 – preparation, expression and testing of chimeric proteins Materials and Methods Cloning: A representative cytoplasmic construct was designed in-house and whole sequences were codon optimized for expression in human cells. This construct was cloned into a Lentiviral backbone (pcLV) which included the GFP reporter gene, and also into a Lentiviral backbone (pcLV) which contains a GFP reporter gene as well as an A2CAR. Two constructs were therefore generated for testing (plus and minus A2CAR), as shown in Figure 5. These constructs were designated pQTX-0340 (without CAR) and pQTX-0370 (with CAR). These constructs were transformed into Stble3 bacteria with the selection agent Kanamycin. Colonies were picked and DNA was extracted using a Mini-prep kit (Promega). DNA was checked and confirmed using Sanger sequencing and correct clones were amplified and purified using a Maxi-prep kit (Qiagen). Transfection and lentiviral particle production: HEK293T/17 cells were seeded and cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) + 10% FBS (Fetal Bovine Serum) for 24 hours. Fugene HD (Promega) transfection reagent was brought to room temperature and mixed with the construct DNA, packaging plasmid (pD8.91) and envelope plasmid (pVSV-G). This mixture was incubated for 10 minutes and then added dropwise to the HEK293T/17 cells. Supernatant was harvested 48 hours post-transfection, sterile filtered and concentrated via ultra-centrifugation. Isolation, Transduction and Expansion of Human Treg cells: CD45RA+ human Tregs were isolated from healthy donor blood. Tregs were transduced with various constructs using the required multiplicity of infection (MOI). Non-tissue culture treated well plates were coated with RetroNectin® (Takara Bio, Shiga, Japan) prior to addition of viral particles and Tregs followed by spinoculation. Tregs were expanded using Dynabeads™ Human T-Activator CD3/CD28 for T Cell Expansion and Activation (ThermoFisher Scientific, Massachusetts, USA), X-VIVO™ 15 media (Lonza, Basel, Switzerland) supplemented with 5% heat-inactivated human serum (Merck Life Science UK Limited, Dorset, UK) and interleukin-2 (IL-2; Proleukin®, Clinigen, Burton upon Trent, UK). IL-2 was supplemented at 300 international units (IU) per 1 ml of culture volume every 2 days. The protocol is illustrated in Figure 6. Flow cytometry staining to determine transduction efficiency: Once Tregs had sufficiently expanded, cells were taken for staining to check phenotype and transduction efficiency. Cells were washed with FACS buffer (phosphate buffer saline (PBS) with 2% fetal bovine serum (FBS) and 2mM EDTA). Cells were washed in PBS before LIVE/DEAD™ Fixable Near-IR (ThermoFisher Scientific) was used to stain the dead cells for 15 mins at RT. Cells were washed in FACS buffer before Fc blocking with Human TruStain FcX™ (Biolegend) and stained with cell surface antibodies: Brilliant Violet 510™ anti-human CD4 (A161A1; Biolegend, California, USA), PE/Cyanine7 anti-human CD25 (BC96; Biolegend). Washed cells were then fixed and permeabilized using the eBioscience™ FoxP3/Transcription Factor Staining Buffer Set before addition of Alexa Fluor® 488 anti-GFP (FM264G; Biolegend) and PE anti-human FoxP3 antibodies (206D; Biolegend). Cells were acquired by flow cytometry and data was analysed on FlowJo_V10 software. pSTAT5 assay set up for human Tregs Setup: The protocol is shown in Figure 6. Tregs were rested for 24-36 hours prior to setting up the assay. This assay was setup in U bottom 96 well plates with 150,000-200,000 cell per cells per well. Cells were treated with serial dilutions of Rapamycin (1, 10, 100,1000, and 10000nMol), with 0nMol Rapamycin acting as a negative control. Cells treated with 100IU/ml and 1000IU/ml IL-2 for 30min period to staining were used as a positive control for the pSTAT5 detection. All cultures were performed in X- VIVO™ 15 media (Lonza) supplemented with 5% heat-inactivated human serum (Merck Life Science UK Limited) and incubated at 37°C and 5% CO2. Readouts were taken at 4hr and 24hr post Rapamycin treatment. Subsequently, the cells were stained as detailed below. pSTAT5 Staining: Cells were washed in PBS and the supernatant was discarded. Cells were then incubated in 50ul Live/Dead- Near IF (ThermoFisher) and Human TruStain FcX: Fc receptor blocking solution (Biolegend), in PBS respectively. Cells were washed in FACS Buffer and the supernatant was discarded.50μl of surface staining panel containing 0.5ul of both CD4- BV510 (Biolegend) and CD25- PE-cy7 (Biolegend) in FACS buffer (PBS + 2% FBS) was then added and samples were incubated in the fridge.100ul of FACS buffer was added per well to wash cells and the supernatant was discarded.125ul of Buffer 1 from the PerFix EXPOSE: Phospho-Epitopes Exposure kit (Beckman Coulter) diluted in FBS at a 1:4 ratio (25ul Buffer 1: 100ul FBS) was added and samples were incubated before centrifuging and discarding supernatents.100ul of Buffer 2 from the PerFix EXPOSE: Phospho-Epitopes Exposure kit (Beckman Coulter) was diluted in FBS at a 1:1 ratio (50ul Buffer 1: 50ul FBS)and added to the wells. Samples were again incubated, before centrifuging and discarding the supernatant. 50ul of intracellular staining panel containing 1ul of GFP- AF488 (Biolegend) and 2.5ul pSTAT5-PE (Biolegend) in Buffer 3 from the PerFix EXPOSE: Phospho- Epitopes Exposure kit (Beckman Coulter) was added and incubated, followed by 150ul of 1X Buffer 4 (diluted 1:20 in H2O) from the PerFix EXPOSE: Phospho- Epitopes Exposure kit (Beckman Coulter). Samples were centrifuged and supernatants discarded. All samples were resuspended in 200ul of 1X Buffer 4 (diluted 1:20 in H2O) from the PerFix EXPOSE: Phospho-Epitopes Exposure kit (Beckman Coulter), before analysing samples by flow cytometry. Set up of the Survival Assays This is depicted in Figure 8. Transduced CAR Tregs were rested prior to set up of the survival assays: Treg cultures were depleted of the Dynabeads™ Human T-Activator CD3/CD28 for T Cell Expansion and Activation (ThermoFisher Scientific) via magnetic technology. Cells were washed and resuspended in X-VIVO™ 15 media (Lonza) supplemented with 1% heat-inactivated human serum (Merck Life Science UK Limited); no IL-2 was supplemented. Cells were rested for 24 hours prior to use in assays. CAR Tregs were co-cultured in the presence of Dynabeads™ Human T-Activator CD3/CD28 for T Cell Expansion and Activation beads or media alone for 6-10 days with or without 10 IU/ml of IL2. CAR Tregs were treated with 100nM, 1000nM or 2740nM rapamycin (Sigma-Aldrich) or vehicle alone every day. The vehicle used was dimethyl sulfoxide (DMSO; Sigma-Aldrich). On day 6 or 10, cells were washed in FACS buffer before Fc blocking with Human TruStain FcX™ (Biolegend) and staining with cell surface antibodies: Brilliant Violet 510™ anti-human CD4 (Biolegend), PE/Cyanine7 anti-human CD25 (Biolegend). Washed cells were then stained with Annexin V/7AAD using the PE Annexin V Apoptosis Detection Kit with 7AAD (Biolegend) according to the manufacturer’s guidelines. Cells were acquired by flow cytometry. Data was analysed on FlowJo_V10 software. Graphs were produced using GraphPad Prism 8 software. Results A representative cytoplasmic construct was designed comprising an interaction domain comprising two dimerization domains and a signalling domain comprising a JAK1-binding domain and a STAT5 association motif to determine if constructs lacking a transmembrane domain could result in increased levels of pSTAT5 within cells. Tregs were transduced with construct pQTX 0340 and were treated with different levels of Rapamycin. As can be seen from Figure 7, pSTAT5 expression was shown to be increased in transduced Tregs as compared to non-transduced cells or mock cells, or as compared to transduced cells in the absence of Rapamycin (23.4% as compared to 5.82%). A survival assay further demonstrated that cells transduced with pQTX 0340 had an increase in survival after treatment with Rapamycin, both under bead stimulated conditions and media only. Two different gating strategies were employed as shown in Figures 9 and 10, and both demonstrated an increase in survival for pQTX 0340 transduced cells in the presence of Rapamycin. Figure 11 in particular shows an increase in survival in transduced cells after Rapamycin treatment from 4.34% to 18.4% (with beads) and from 37.9% to 59.8% (media alone). No increase in survival is seen for non-transduced cells. Experiments carried out using cells transduced with pQTX 0370 (pQTX 0340 plus HLA A2 CAR) further demonstrate the survival advantage shown for cells transduced with the construct (as can be seen from Figures 12-13). The data therefore indicate that cytoplasmic constructs capable of dimerization and having a JAK1-binding motif and a STAT5 association motif are capable of increasing pSTAT5 and survival of transduced cells, demonstrating that it is not necessary to use membrane bound constructs and removing the requirement to ensure membrane trafficking and expression.

Claims

Claims 1. A chimeric protein, said chimeric protein being a non-membrane associated protein comprising, linked together in any order: (i) an interaction domain comprising at least one dimerization domain; (ii) a signalling domain comprising a tyrosine kinase activating domain comprising a JAK1-binding motif and a tyrosine effector domain comprising one or more tyrosine residues that can be phosphorylated by JAK1; wherein the dimerization domain of said chimeric protein is capable of dimerizing with a cognate dimerization domain of at least a second, non- membrane associated, chimeric protein providing a second component of the signalling protein, wherein the at least a second chimeric protein may be the same chimeric protein, or a different chimeric protein, and wherein dimerization of the chimeric protein with the at least a second chimeric protein provides a signal in a cell expressing the chimeric protein and the at least a second protein.
2. The chimeric protein of claim 1, wherein the tyrosine effector domain comprises at least one STAT association motif, preferably a STAT5 association motif.
3. The chimeric protein of claim 1 or claim 2, wherein the signalling domain of said chimeric protein further comprises a JAK3-binding domain.
4. The chimeric protein of any one of claims 1 to 3, wherein said second chimeric protein comprises a different signalling domain, optionally wherein the signalling domain of the second chimeric protein comprises a JAK3- binding motif.
5. The chimeric protein of claim 3, wherein the signalling domain of the second chimeric protein does not comprise a tyrosine effector domain.
6. A pair of dimerizable chimeric proteins, which together when dimerized are capable of providing a signal in a cell expressing the proteins, each protein of the pair being a non-membrane associated protein, wherein the first chimeric protein of the pair comprises, linked together in any order: (i) an interaction domain comprising at least one dimerization domain; (ii) a signalling domain A comprising a tyrosine kinase activating domain comprising a JAK1-binding motif and a tyrosine effector domain comprising one or more tyrosine residues that can be phosphorylated by JAK1; and the second chimeric protein of the pair comprises, linked together in any order: (i) an interaction domain comprising at least one dimerization domain; and (ii) a signalling domain B comprising a tyrosine kinase activating domain comprising a JAK3-binding motif; and wherein the dimerization domain of the first chimeric protein is capable of dimerizing with the dimerization domain of the second chimeric protein causing the signalling domains of pair of chimeric proteins to dimerize to enable signalling.
7. The chimeric protein of any one of claims 1 to 5 or the pair of chimeric proteins of claim 6, wherein the interaction domain (i) comprises a single dimerization domain; or (ii) comprises 2 dimerization domains D1 and D2, which may be the same or different.
8. The chimeric protein or pair of chimeric proteins of any one of claims 1 to 7, wherein the said dimerization domain(s) heterodimerize.
9. The chimeric protein or pair of chimeric proteins of any one of claims 1 to 7, wherein the said dimerization domain(s) homodimerize.
10. The chimeric protein or pair of chimeric proteins of any one of claims 1 to 9, wherein the chimeric protein has the formula: (i) D - [Endo]; (ii) [Endo] – D; (iii) [Endo] – D1- D2; (iv) D1-D2 –[Endo]; (v) D1- [Endo] - D2 wherein [Endo] is the signalling domain, D, D1 or D2 are dimerization domains and the linkage (-) between domains may be a direct or indirect, optionally wherein an additional domain is included at one or both ends, or in between [Endo] and a dimerization domain.
11. The chimeric protein or pair of chimeric proteins of claim 10, wherein the dimerization domains D1 and D2 are heterodimerization domains Ht1 and Ht2.
12. The chimeric protein or pair of chimeric proteins of any one of claims 1 to 11, wherein the dimerization is inducible, and the dimerization domain(s) is/are capable of dimerizing in the presence of a dimerization inducer.
13. The chimeric protein or pair of chimeric proteins of any one of claims 1 to 5, wherein the dimerization domain comprises a FK506-bindng protein (FKBP) or a FRB domain of mTOR (FRB).
14. The chimeric protein or pair of chimeric proteins of claim 6, wherein the interaction domain comprises 2 dimerization domains and D1 comprises FKBP and D2 comprises FRB.
15. The chimeric protein or pair of chimeric proteins of any one of claims 13 to 14, wherein the dimerization inducer is rapamycin or a rapamycin analogue.
16. The chimeric protein or pair of chimeric proteins of any one of claims 1 to 12, wherein: (i) a single dimerization domain or one of a pair of dimerization domains Ht1 and Ht2 comprises a FKBP, and the other comprises calcineurin or an FK506-binding fragment thereof, and the dimerization inducer is FK506; or (ii) a single dimerization domain or one of a pair of dimerization domains Ht1 and Ht2 comprises a cyclophilin (CyP) or a cyclosporine (CsA)- binding fragment thereof, and the other comprises calcineurin or a CsA-binding fragment thereof, and the dimerization inducer is CsA; or (iii) a single dimerization domain or one of a pair of dimerization domains Ht1 and Ht2 comprises a cyclophilin (CyP), or a cyclosporine (CsA)- binding fragment thereof and the other comprises a FKBP, and the dimerization inducer is FKCsA; or (iv) a single dimerization domain or one of a pair of dimerization domains Ht1 and Ht2 comprises a 14-3-3 protein or a fusicoccin-binding fragment thereof, and the other comprises a C-terminal peptide of a plant plasma membrane H+-ATPase (PMA), and the dimerization inducer is fusicoccin; or (v) a single dimerization domain or one of a pair of dimerization domains Ht1 and Ht2 comprises a SNAP-tag, and the other comprises a HALO- tag, and the dimerization inducer is a HaXS molecule; or (vi) the dimerization domain comprises a FKBP dimerization domain sequence as shown in any one of SEQ ID NOs: 36-40 and the dimerization inducer is AP1903 or AP20187; or (vii) the dimerization domain comprises a FKBP and the dimerization inducer is FK1012.
17. The chimeric protein or pair of chimeric proteins of any one of claims 1 to 11, wherein the dimerization is constitutive, optionally wherein the dimerization domain comprises a leucine zipper sequence.
18. The chimeric protein or pair of chimeric proteins of any one of claims 1 to 17, wherein in the signalling domain the tyrosine kinase activating domain is located N-terminal to the tyrosine effector domain.
19. The chimeric protein or pair of chimeric proteins of any one of claims 1 to 18, wherein the signalling domain comprises, N- to C-terminal: (i) JAK1 motif – STAT5 motif; or (ii) JAK1 motif – STAT5 motif – JAK3 motif; wherein the motifs are linked directly or indirectly to one another, particularly wherein the JAK3 motif is in reverse orientation.
20. The chimeric protein or pair of chimeric proteins of any one of claims 1 to 19, wherein any two domains are linked together by a linker, optionally wherein (i) the interaction domain is linked to the signalling domain by a linker; (ii) where the interaction domain comprises D1 and D2, D1 and D2 in a 1- part interaction domain are linked together by a linker; (iii) where the interaction domain comprises D1 and D2, D1 and D2 in a 2- part interaction domain are each separately linked to an end of the signalling domain by a linker.
21. The chimeric protein or pair of chimeric proteins of claim 20, wherein the linker: (i) is a flexible linker; and/or (ii) comprises at least one Gly-Ser domain composed solely of Ser and Gly residues; and/or (iii) comprises at least one Gly-Ser domain having the formula: (S)q-[(G)m-(S)m]n-(G)p, wherein q is 0 or 1; m is an integer from 1-8; n is an integer of at least 1 (e.g. from 1 to 8, or preferably 1 to 6); and p is 0 or an integer from 1 to 3.
22. The chimeric protein or pair of chimeric proteins of any one of claims 1 to 21, wherein the tyrosine effector domain comprises a STAT5 association motif which: (i) comprises an amino acid sequence as set out in any one of SEQ ID NOs: 9-15 or a variant which is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NOs: 9-15; and/or (ii) comprises the amino acid motif YXXF/L (SEQ ID NO: 16); wherein X is any amino acid; and/or (iii) comprises the amino acid motif YCTF (SEQ ID NO: 17), YFFF (SEQ ID NO: 18), YLSL (SEQ ID NO: 19), or YLSLQ (SEQ ID NO: 20).
23. The chimeric protein or pair of chimeric proteins of any one of claims 1 to 22, wherein: (i) the JAK1-binding motif comprises an amino acid motif as shown in any one of SEQ ID NOs: 21-27 or a variant thereof which is capable of binding JAK1; and/or (ii) the JAK2-binding motif comprises an amino acid motif as shown in any one of SEQ ID NOs: 28-30 or a variant thereof which is capable of binding JAK1; and/or (iii) the JAK3-binding motif comprises an amino acid motif as shown in any one of SEQ ID NOs: 33 or 34 or a variant thereof which is capable of binding JAK1.
24. The chimeric protein or pair of chimeric proteins of any one of claims 1 to 23, wherein the signalling domain comprises an IL2Rβ sequence selected from an amino acid sequence as set out in any one of SEQ ID NOs: 9, 31, or 32, or a sequence which has at least 80% sequence identity to any one of SEQ ID NOs: 9, 31, or 32.
25. A nucleic acid molecule comprising a nucleotide sequence encoding a chimeric protein as defined in any one of claims 1 to 24.
26. A construct comprising a nucleic acid molecule as defined in claim 25, and one or more further nucleotide sequences.
27. The construct of claim 26, wherein a further nucleotide sequence: (i) is a regulatory sequence; and/or (ii) encodes a protein of interest; and/or wherein the protein of interest is (iii) a therapeutic protein; or (iv) an antigen receptor; or (v) a CAR or TCR; or (vi) a safety switch polypeptide; (vii) a FOXP3 polypeptide.
28. The construct of claim 26 or 27, wherein the further nucleotide sequence encodes a mutant calcineurin which is resistant to at least one calcineurin inhibitor, optionally wherein said mutant calcineurin is sensitive to at least one calcineurin inhibitor.
29. The construct of claim 28, wherein said calcineurin mutant is resistant to FK506 and sensitive to CsA or wherein said calcineurin mutant is resistant to CsA and sensitive to FK506.
30. The construct of any one of claims 26 to 29, wherein the nucleotide sequence encoding the chimeric protein and one or more further nucleotide sequences encoding a protein of interest, or nucleotide sequences respectively encoding a pair of chimeric proteins, are separated from one another by a co- expression sequence.
31. A vector comprising a nucleic acid molecule or construct of any one of claims 25 to 29.
32. A cell which expresses a chimeric protein or a pair of chimeric proteins as defined in any one of claims 1 to 24, or which comprises a nucleic acid molecule, construct or vector as defined in any one of claims 25 to 31.
33. The cell of claim 33 which co-expresses the chimeric protein in the cytoplasm and a protein of interest, particularly wherein the protein of interest is a CAR or TCR which is expressed at its cell surface.
34. The cell of claim 32 or claim 33 which is a Treg or a precursor therefor.
35. A cell population comprising a cell as defined in any one of claims 32 to 34.
36. A method for making a cell according to any one of claims 32 to 34, which comprises the step of introducing into the cell a nucleic acid molecule, construct or vector according to any one of claims 25 to 31.
37. A method of promoting the survival or persistence of a cell, said method comprising introducing into the cell, a nucleic acid molecule, construct or vector as defined in any one of claims 25 to 31, and optionally exposing the cell to a dimerization inducer.
38. A method of selectively expanding a cell according to any one of claims 32 to 34, wherein a nucleic acid molecule comprising a mutant calcineurin which is resistant to at least one calcineurin inhibitor has been introduced into the cell, and wherein the cell is cultured in the presence of said calcineurin inhibitor.
39. A pharmaceutical composition comprising a cell or cell population as defined in any one of claims 32 to 35 or a vector as defined in claim 31.
40. A cell or cell population as defined in any one of claims 32 to 35 or a vector as defined in claim 31 or a pharmaceutical composition as defined in claim 39 for use in therapy.
41. A cell of any one of claims 32 to 34, a cell population of claim 35, a vector of claim 31 or a pharmaceutical composition of claim 39 for treating cancer, an infectious, neurodegenerative or inflammatory disease, or for inducing immunosuppression.
42. The cell, cell population, vector or pharmaceutical composition of claim 41, for use in induction of tolerance to a transplant; treating and/or preventing graft- versus-host disease (GvHD), an autoimmune or allergic disease; to promote tissue repair and/or tissue regeneration; or to ameliorate inflammation in a subject, preferably wherein the cell is a Treg cell.
43. A combination product comprising (a) a cell or cell population as defined in any one of claims 32 to 35 or a vector as defined in claim 31 or a pharmaceutical composition as defined in claim 39, and (b) a dimerization inducer, for use in therapy, preferably ACT or gene therapy.
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