CYTOKINE RECEPTOR CHIMAERAS

Description

FIELD OF THE INVENTION

[0001] The present invention relates to a cell which comprises a chimeric antigen receptor (CAR) and a constitutively active cytokine-signalling chimeric transmembrane protein.BACKGROUND TO THE INVENTIONChimeric antigen receptors (CARs)

[0002] A number of immunotherapeutic agents have been described for use in cancer treatment, including therapeutic monoclonal antibodies (mAbs), bi-specific T-cell engagers and chimeric antigen receptors (CARs).

[0003] Chimeric antigen receptors are proteins which graft the specificity of a monoclonal antibody (mAb) to the effector function of a T-cell. Their usual form is that of a type I transmembrane domain protein with an antigen recognizing amino terminus, a spacer, a transmembrane domain all connected to a compound endodomain which transmits T-cell survival and activation signals.

[0004] The most common form of these molecules are fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies which recognize a target antigen, fused via a spacer and a trans-membrane domain to a signaling endodomain. Such molecules result in activation of the T-cell in response to recognition by the scFv of its target. When T cells express such a CAR, they recognize and kill target cells that express the target antigen. Several CARs have been developed against tumour associated antigens, and adoptive transfer approaches using such CAR-expressing T cells are currently in clinical trial for the treatment of various cancers.CAR-based approaches to treat Prostate Cancer

[0005] Prostate cancer is the second most common cancer in men worldwide, and the sixth leading cause of cancer-related death. Globally, there are approximately 1,100,000 new cases and 300,000 mortalities every year, comprising 4 percent of all cancer deaths. It is estimated that 1 in every 6 men will be diagnosed with the disease during his lifetime.

[0006] Initial treatment for prostate cancer may consist of surgery, radiation, or hormone therapy, or any combination of each. Hormone therapy consists of lowering the levels of testosterone, the male hormone that fuels out-of-control cell growth. Chemotherapy is typically reserved for advanced-stage cancers.

[0007] When prostate cancers grow despite the lowering of testosterone levels by hormone therapy, treatment options are limited. Typically, the cancer vaccine sipuleucel-T (Provenge ®< ) a dendritic cell-based therapeutic cancer vaccine designed to induce an immune response targeted against the prostatic acid phosphatase ((PAP) antigen), a radiopharmaceutical agent (such as radium-223 chloride), secondary hormone therapies (such as abiraterone or enzalutamide), and / or chemotherapies (docetaxel and cabazitaxel) are added to the hormonal therapy in sequence. While each of these treatments can delay growth of the cancer for several months and palliate symptoms produced by the disease, the disease ultimately becomes resistant to them.

[0008] Preclinically, two antigens associated with prostate cancer have been targeted with CAR T-cell based therapies: prostate-specific membrane antigen (PSMA) and prostate stem cell antigen (PSCA).

[0009] Mice treated with PSCA CAR-engineered T cells showed delayed tumour growth (Hillerdal et al (2014) BMC Cancer 14:30; and Abate-Daga et al (2014) 25:1003-1012). Although the cells showed high in vitro cytotoxicity, in vivo, tumour growth was delayed but tumour-bearing mice were not cured.

[0010] This may be because, in vivo, CAR T-cells struggle to overcome the hostile microenvironment of a carcinoma. In particular CAR T-cells may fail to engraft and expand within a prostate cancer tumour bed.

[0011] CAR T-cell persistence and activity can be enhanced by administration of cytokines, or by the CAR T-cells producing cytokines constitutively. However, these approaches have limitations: systemic administration of cytokines can be toxic; constitutive production of cytokines may lead to uncontrolled proliferation and transformation (Nagarkatti et al (1994) PNAS 91:7638-7642; Hassuneh et al (1997) Blood 89:610-620).

[0012] There is therefore a need for alternative CAR T-cell approaches, which facilitate engraftment and expansion of T cells to counteract the effects of the hostile tumour microenvironment.On-target off-tumour toxicity

[0013] It is relatively rare for the presence of a single antigen effectively to describe a cancer, which can lead to a lack of specificity.

[0014] Most cancers cannot be differentiated from normal tissues on the basis of a single antigen. Hence, considerable "on-target off-tumour" toxicity occurs whereby normal tissues are damaged by the therapy. For instance, whilst targeting CD20 to treat B-cell lymphomas with Rituximab, the entire normal B-cell compartment is depleted, whilst targeting CD52 to treat chronic lymphocytic leukaemia, the entire lymphoid compartment is depleted, whilst targeting CD33 to treat acute myeloid leukaemia, the entire myeloid compartment is damaged etc.

[0015] The predicted problem of "on-target off-tumour" toxicity has been borne out by clinical trials. For example, an approach targeting ERBB2 caused death to a patient with colon cancer metastatic to the lungs and liver. ERBB2 is over-expressed in colon cancer in some patients, but it is also expressed on several normal tissues, including heart and normal vasculature.

[0016] There is therefore a need for improved approaches to cancer therapy in which such "on-target off-tumour" toxicity is reduced or eliminated.

[0017] Sogo et al., 2008, J Immunol Methods., 337(1):16-23 describes the selective expansion of genetically modified T cells using an antibody / interleukin-2 receptor chimera.

[0018] WO 2007 / 115230 describes a chimeric polypeptide comprising a first portion comprising a receptor domain, wherein the receptor domain comprises an intracellular region and a transmembrane region; and a second portion comprising a dimerization domain.

[0019] Wilkie et al., 2010, The Journal Of Biological Chemistry, 285(33):25538-25544 describes the selective expansion of chimeric antigen receptor-targeted T-cells with potent effector function using Interleukin-4.DESCRIPTION OF THE FIGURES

[0020] Figure 1: Schematic diagram summarising the structure of various cytokine receptors, the cell types which produce the cytokines and the cell types which express the cytokine receptors. Figure 2: Schematic diagram showing proposed chimeric cytokine receptor (for reference only) (a) Cytokine IL2 and IL7 cytokine receptors signal through a common gamma chain and a cytokine specific alpha / beta chain. (b) One implementation of a chimeric cytokine receptor is to replace the ectodomain of the cytokine alpha / beta and gamma chain with different scFvs (or any other suitable binder) which recognize different epitopes of PSA. (c) An alternative approach is to replace the ectodomains of alpha / beta and gamma with the VH / VL of a PSA specific antibody, where both VH and VL are involved in binding so that binding brings them together. Figure 3: Aggregation-based cytokine signalling enhancer (for reference only) Schematic diagram showing a chimeric cytokine receptor and CAR combination system. The cell comprises two chimeric cytokine receptors which bind different epitopes on the same soluble ligand. In the absence of soluble ligand (e.g. PSA) but the presence of the cell-membrane antigen (e.g. PSMA) signalling occurs thought the CAR. In the presence of the soluble ligand, aggregation of the two chimeric cytokine receptors occurs, leading to cytokine-based signal enhancement. Figure 4: Theoretical construct map for the chimeric cytokine receptor / CAR combination system illustrated in Figure 3. Figure 5: Schematic diagram illustrating an example of a structure for the chimeric transmembrane protein of the present invention. The chimeric transmembrane protein comprises a dimerization domain and a cytokine receptor endodomain. The embodiment shown has a "Fab" type architecture, as the dimerization domain comprises antibody-type heavy and light chain constant regions. Constant dimerization between these domains brings together the IL2 receptor common γ chain with either the IL-2 receptor β chain or the IL-7 receptor α chain, leading to constitutive cytokine signalling. Figure 6: IL-2 signalling by the chimeric transmembrane protein. Two chimeric transmembrane proteins having the general structure shown in Figure 5 were tested for their ability to induce IL-2 signalling. One chimeric transmembrane protein comprised an IL2 receptor endodomain and the other comprised an IL-7 receptor endodomain. IL-2 signalling was tested using the murine cell line CTLL2 which is dependent on IL-2 signalling for growth. As a positive control, CTLL2 cells were cultured with 100 u / mL murine IL2. Cells expressing the chimeric transmembrane protein comprising the IL2 receptor endodomain (Fab_IL2endo) supported CTLL2 cell survival and growth, whereas cells expressing the chimeric transmembrane protein comprising the IL-7 receptor (Fab_IL7endo) did not. Figure 7: Schematic diagram illustrating panel of PSA chimeric cytokine receptors (for reference only) A panel of chimeric cytokine receptors (CCRs) targeting PSA was developed using scFvs derived from two antibodies which bind to different PSA epitopes: 5D5A5 and 5D3D11. Top-left panel: A CCR with an IL-2R endodomain having A5 on the chain with IL2R β chain and D11 on the chain with common γ chain; Top-right panel: A CCR with an IL7R endodomain having A5 on the chain with IL7R α chain and D11 on the chain with common γ chain; Bottom-left panel: A CCR with an IL-2R endodomain having D11 on the chain with IL2R β chain and A5 on the chain with common γ chain; and Bottom-right hand panel: A CCR with an IL-7R endodomain having D11 on the chain with IL7R α chain and A5 on the chain with common γ chain. A negative control was also created for each CCR, in which the IL2Rγ chain was replaced by a rigid linker. Figure 8: IL2 signalling from cells expressing a PSA chimeric cytokine receptor in the presence of PSA - CTLL2 proliferation (for reference only) CTLL2 cells were transduced with constructs expressing some of the PSA chimeric cytokine receptors illustrated in Figure 7. Cells were cultured in the presence of absence of IL2 (positive control) and the presence of absence of 5ng / mL or 5µg / mL PSA. CTLL2 proliferation was assessed after 3 and 7 days. The anti-PSA chimeric cytokine receptor with an IL2R endodomain supported CTLL2 cell proliferation in the absence of IL2 and the presence of PSA, but not the receptor having an IL7R endodomain or any of the CCRs comprising a rigid linker in the place of the common γ chain. Figure 9: IL2 signalling from cells expressing a PSA chimeric cytokine receptor in the presence of PSA - CTLL2 STAT5 phosphorylation (for reference only) CTLL2 cells were either left untransduced (WT); or transduced with a vector expressing a CCR against PSA (D11-CD8STK-IL2Rg_A5-Hinge-IL2Rb) or an equivalent construct having a rigid linker in the place of the common γ chain (D11-CD8STK-RL_A5-Hinge-IL2Rb). Cells were incubated with either 500µM Pervanadate or 500ng / mL PSA for 1 or 4 hours. Phosphorylation of Y694 of STAT5 was then investigated using phosphoflow. SUMMARY OF ASPECTS OF THE INVENTION

[0021] The present invention provides a cell which comprises: a chimeric antigen receptor (CAR); and a constitutively active cytokine-signalling chimeric transmembrane protein which comprises two polypeptides: (i) a first polypeptide which comprises: (a) the dimerization domain of a heavy chain constant domain (CH); and (b) a first chain of a type I cytokine receptor endodomain; and (ii) a second polypeptide which comprises: (a) the dimerization domain of a light chain constant domain (CL) which spontaneously heterodimerizes with the dimerization domain of the heavy chain constant domain (CH); and (b) a second chain of the type I cytokine-receptor endodomain.

[0022] The first polypeptide of the chimeric transmembrane protein may comprise a heavy chain constant domain (CH); and the second polypeptide of the chimeric transmembrane protein may comprise a light chain constant domain (CL).

[0023] The first and second chains for the cytokine receptor endodomains may be different and may be selected from type I cytokine receptor endodomain α-, β-, and γ-chains.

[0024] Alternatively the first and second chains for the cytokine receptor endodomains may be the same and may be selected from type I cytokine receptor endodomain α-, β-, and γ-chains.

[0025] For example, the cytokine receptor endodomain may comprise: (i) IL-2 receptor β-chain endodomain (ii) IL-7 receptor α-chain endodomain; (iii) IL-15 receptor α-chain endodomain; or (iv) common γ-chain receptor endodomain.

[0026] The first or second chain of the cytokine receptor endodomain may comprise (i), (ii) or (iii); and (iv).

[0027] The first polypeptide of the chimeric transmembrane protein may comprise a heavy chain variable domain (VH) and a heavy chain constant domain (CH); and the second polypeptide of the chimeric transmembrane protein may comprise a light chain variable domain (VL) and a light chain constant domain (CL).

[0028] The chimeric antigen receptor may bind a cell surface antigen associated with prostate cancer, such as prostate stem-cell antigen (PSCA) or prostate-specific membrane antigen (PSMA).

[0029] The present invention further provides a nucleic acid construct encoding a chimeric transmembrane protein as defined in the cell of the invention, the nucleic acid construct comprising a first nucleic acid sequence encoding the first polypeptide and a second nucleic acid sequence encoding the second polypeptide, the nucleic acid construct having the structure:         Dim1 -TM1-endo1-coexpr- Dim2 -TM2-endo2 in which Dim1 is a nucleic acid sequence encoding the dimerization domain of the first polypeptide; TM1 is a nucleic acid sequence encoding the transmembrane domain of the first polypeptide; endo 1 is a nucleic acid sequence encoding the endodomain of the first polypeptide; coexpr is a nucleic acid sequence encoding a cleavage or self-cleaving site enabling co-expression of first and second polypeptides Dim2 is a nucleic acid sequence encoding the dimerization domain of the second polypeptide; TM2 is a nucleic acid sequence encoding the transmembrane domain of the second polypeptide; endo 2 is a nucleic acid sequence encoding the endodomain of the second polypeptide, wherein the nucleic acid construct also comprises a nucleic acid sequence which encodes a chimeric antigen receptor (CAR).

[0030] The present invention provides a vector comprising a nucleic acid construct according to the invention.

[0031] The vector may be, for example, a retroviral vector or a lentiviral vector or a transposon.

[0032] The present invention provides a kit which comprises: i) a vector comprising a nucleic acid sequence encoding a first polypeptide as defined in the cell of the invention; and ii) a vector comprising a nucleic acid sequence encoding a second polypeptide as defined in the cell of the invention; and iii) a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor.

[0033] The kit may comprise: i) a vector comprising a nucleic acid sequence encoding a chimeric transmembrane protein as defined in the cell of the invention; and ii) a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor.

[0034] The present invention provides a method for making a cell according to the invention, which comprises the step of introducing: a nucleic acid construct according to the invention; a vector according to the invention; or a kit of vectors according to the invention, into a cell ex vivo.

[0035] The cell may be from a sample isolated from a subject.

[0036] The invention provides a pharmaceutical composition comprising a plurality of cells according to the invention.

[0037] Described herein is a method for treating and / or preventing a disease, which comprises the step of administering a pharmaceutical composition according to the invention to a subject.

[0038] The method may comprise the following steps: (i) isolation of a cell-containing sample from a subject; (ii) transduction or transfection of the cells with: a nucleic acid construct according to the invention; a vector according to the invention; or a kit of vectors according to the invention; and (iii) administering the cells from (ii) to a the subject.

[0039] The sample may be a T-cell containing sample.

[0040] The disease may be a cancer.

[0041] The invention provides a pharmaceutical composition according to the invention for use in treating and / or preventing a disease.

[0042] Described herein is the use of a cell according to the invention in the manufacture of a medicament for treating and / or preventing a disease.DETAILED DESCRIPTIONCYTOKINE RECEPTORS AND SIGNALLING

[0043] Many cell functions are regulated by members of the cytokine receptor superfamily. Signalling by these receptors depends upon their association with Janus kinases (JAKs), which couple ligand binding to tyrosine phosphorylation of signalling proteins recruited to the receptor complex. Among these are the signal transducers and activators of transcription (STATs), a family of transcription factors that contribute to the diversity of cytokine responses.

[0044] The JAK-STAT system consists of three main components: (1) a receptor (2) Janus kinase (JAK) and (3) Signal Transducer and Activator of Transcription (STAT).

[0045] JAKs, which have tyrosine kinase activity, bind to cell surface cytokine receptors. The binding of the ligand to the receptor triggers activation of JAKs. With increased kinase activity, they phosphorylate tyrosine residues on the receptor and create sites for interaction with proteins that contain phosphotyrosine-binding SH2 domains. STATs possessing SH2 domains capable of binding these phosphotyrosine residues are recruited to the receptors, and are themselves tyrosine-phosphorylated by JAKs. These phosphotyrosines then act as binding sites for SH2 domains of other STATs, mediating their dimerization. Different STATs form hetero- or homodimers. Activated STAT dimers accumulate in the cell nucleus and activate transcription of their target genes.CYTOKINE RECEPTOR ENDODOMAIN

[0046] The constitutively active cytokine-signalling chimeric transmembrane protein comprises an endodomain which causes type I "cytokine-type" cell signalling .

[0047] The endodomain may be derived from a type I cytokine receptor. Type I cytokine receptors share a common amino acid motif (WSXWS) in the extracellular portion adjacent to the cell membrane.

[0048] Type I cytokine receptors include: (i) Interleukin receptors, such as the receptors for IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL13, IL-15, IL-21, IL-23 and IL-27; (ii) Colony stimulating factor receptors, such as the receptors for erythropoietin, GM-CSF, and G-CSF; and (iii) Hormone receptor / neuropeptide receptor, such as hormone receptor and prolactin receptor

[0049] Members of the type I cytokine receptor family comprise different chains, some of which are involved in ligand / cytokine interaction and others that are involved in signal transduction. For example the IL-2 receptor comprises an α-chain, a β-chain and a γ-chain.

[0050] The IL-2 receptor common gamma chain (also known as CD132) is shared between the IL-2 receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, IL-13 receptor and IL-15 receptor.IL-2

[0051] IL-2 binds to the IL-2 receptor, which has three forms, generated by different combinations of three different proteins, often referred to as "chains": α, β and γ; these subunits are also parts of receptors for other cytokines. The β and γ chains of the IL-2R are members of the type I cytokine receptor family.

[0052] The three receptor chains are expressed separately and differently on various cell types and can assemble in different combinations and orders to generate low, intermediate, and high affinity IL-2 receptors.

[0053] The α chain binds IL-2 with low affinity, the combination of β and γ together form a complex that binds IL-2 with intermediate affinity, primarily on memory T cells and NK cells; and all three receptor chains form a complex that binds IL-2 with high affinity (Kd ~ 10-11 M) on activated T cells and regulatory T cells.

[0054] The three IL-2 receptor chains span the cell membrane and extend into the cell, thereby delivering biochemical signals to the cell interior. The alpha chain does not participate in signalling, but the beta chain is complexed with the tyrosine phosphatase JAK1. Similarly the gamma chain complexes with another tyrosine kinase called JAK3. These enzymes are activated by IL-2 binding to the external domains of the IL-2R.

[0055] IL-2 signalling promotes the differentiation of T cells into effector T cells and into memory T cells when the initial T cells are also stimulated by an antigen. Through their role in the development of T cell immunologic memory, which depends upon the expansion of the number and function of antigen-selected T cell clones, they also have a key role in long-term cell-mediated immunity.

[0056] The constitutively active cytokine-signalling chimeric transmembrane protein may comprise the IL-2 receptor β-chain and / or the IL-2 receptor (i.e. common) γ-chain

[0057] The amino acid sequences for the endodomains of the IL-2 β-chain and common γ-chain are shown as SEQ ID No. 1 and 2 SEQ ID No. 1: Endodomain derived from human common gamma chain: SEQ ID No. 2: Endodomain derived from human IL-2Rβ:

[0058] The term "derived from" means that the endodomain of the constitutively active cytokine-signalling chimeric transmembrane protein has the same sequence as the wild-type sequence of the endogenous molecule, or a variant thereof which retains the ability to form a complex with JAK-1 or JAK-3 and activate one of the signalling pathways mentioned above.

[0059] A "variant" sequence having at least 80, 85, 90, 95, 98 or 99% sequence identity to the wild-type sequence (e.g. SEQ ID Nos. 1 or 2), providing that the variant sequence retains the function of the wild-type sequence i.e. the ability to form a complex with JAK-1 or JAK-3 and activate, for example, the JAK-STAT signalling pathway.

[0060] The percentage identity between two polypeptide sequences may be readily determined by programs such as BLAST which is freely available at http: / / blast.ncbi.nlm.nih.gov.IL-7

[0061] The interleukin-7 receptor is made up of two chains: the interleukin-7 receptor-α chain (CD127) and common-γ chain receptor (CD132). The common-γ chain receptors is shared with various cytokines, including interleukin-2, -4, -9, and -15. Interleukin-7 receptor is expressed on various cell types, including naive and memory T cells.

[0062] The interleukin-7 receptor plays a critical role in the development of lymphocytes, especially in V(D)J recombination. IL-7R also controls the accessibility of a region of the genome that contains the T-cell receptor gamma gene, by STAT5 and histone acetylation. Knockout studies in mice suggest that blocking apoptosis is an essential function of this protein during differentiation and activation of T lymphocytes.

[0063] The constitutively active cytokine-signalling chimeric transmembrane protein may comprise the IL-7 receptor α-chain and / or the IL-7 receptor (i.e. common) γ-chain, or a variant thereof.

[0064] The amino acid sequence for the endodomain of the IL-7 α-chain is shown as SEQ ID No. 3. IL-15

[0065] Interleukin 15 (IL-15) is a cytokine with structural similarity to IL-2. Like IL-2, IL-15 binds to and signals through a complex composed of IL-2 / IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 is secreted by mononuclear phagocytes (and some other cells) following viral infection. IL-15 induces cell proliferation of natural killer cells.

[0066] Interleukin-15 receptor consists of an interleukin 15 receptor alpha subunit and shares common beta and gamma subunits with the IL-2 receptor.SPACER

[0067] The constitutively active cytokine-signalling chimeric transmembrane protein may comprise a spacer.

[0068] Where a cell comprises two or more constitutively active cytokine-signalling chimeric transmembrane protein, the spacers may be the same or different. Where a cell comprises a constitutively active cytokine-signalling chimeric transmembrane protein and a chimeric antigen receptor (CAR), the spacer of the constitutively active cytokine-signalling chimeric transmembrane protein and the CAR may be different, for example, having a different length. The spacer of the CAR may be longer than the spacer of the or each constitutively active cytokine-signalling chimeric transmembrane protein.

[0069] The spacer sequence may, for example, comprise an IgG1 Fc region, an IgG1 hinge or a CD8 stalk. The linker may alternatively comprise an alternative linker sequence which has similar length and / or domain spacing properties as an IgG1 Fc region, an IgG1 hinge or a CD8 stalk.

[0070] A human IgG1 spacer may be altered to remove Fc binding motifs.

[0071] Examples of amino acid sequences for these spacers are given below: SEQ ID No. 4 (hinge-CH2CH3 of human IgG1) SEQ ID No. 5 (human CD8 stalk): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI SEQ ID No. 6 (human IgG1 hinge): AEPKSPDKTHTCPPCPKDPK TRANSMEMBRANE DOMAIN

[0072] The transmembrane domain is the sequence of a constitutively active cytokine-signalling chimeric transmembrane protein that spans the membrane. It may comprise a hydrophobic alpha helix. The transmembrane domain may be derived from CD28, which gives good receptor stability.

[0073] Alternatively the transmembrane domain may be derived from a cytokine receptor, for example the same cytokine from which the endodomain is derived.

[0074] The transmembrane domain may, for example be derived from IL-2R, IL-7R or IL-15R. SEQ ID No. 7 - Transmembrane derived from human common gamma chain: VVISVGSMGLIISLLCVYFWL SEQ ID No. 8 - Transmembrane derived from human IL-2Rβ: IPWLGHLLVGLSGAFGFIILVYLLI SEQ ID No. 9 - Transmembrane derived from human IL-7Rα: PILLTISILSFFSVALLVILACVLW SEQ ID No. 10 - Transmembrane derived from human IL-15Rα: AISTSTVLLCGLSAVSLLACYL CHIMERIC ANTIGEN RECEPTORS (CAR)

[0075] The cell of the present invention comprises one or more chimeric antigen receptor(s). The CAR(s) may be specific for a tumour-associated antigen.

[0076] Classical CARs are chimeric type I trans-membrane proteins which connect an extracellular antigen-recognizing domain (binder) to an intracellular signalling domain (endodomain). The binder is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats which comprise an antibody-like or ligand-based antigen binding site. A trans-membrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.

[0077] Early CAR designs had endodomains derived from the intracellular parts of either the γ chain of the FcεR1 or CD3ζ. Consequently, these first generation receptors transmitted immunological signal 1, which was sufficient to trigger T-cell killing of cognate target cells but failed to fully activate the T-cell to proliferate and survive. To overcome this limitation, compound endodomains have been constructed: fusion of the intracellular part of a T-cell co-stimulatory molecule to that of CD3ζ results in second generation receptors which can transmit an activating and co-stimulatory signal simultaneously after antigen recognition. The co-stimulatory domain most commonly used is that of CD28. This supplies the most potent co-stimulatory signal - namely immunological signal 2, which triggers T-cell proliferation. Some receptors have also been described which include TNF receptor family endodomains, such as the closely related OX40 and 41BB which transmit survival signals. Even more potent third generation CARs have now been described which have endodomains capable of transmitting activation, proliferation and survival signals.

[0078] CAR-encoding nucleic acids may be transferred to T cells using, for example, retroviral vectors. In this way, a large number of antigen-specific T cells can be generated for adoptive cell transfer. When the CAR binds the target-antigen, this results in the transmission of an activating signal to the T-cell it is expressed on. Thus the CAR directs the specificity and cytotoxicity of the T cell towards cells expressing the targeted antigen.

[0079] The cell of the present invention comprises one or more CAR(s).

[0080] The CAR(s) may comprise an antigen-binding domain, a spacer domain, a transmembrane domain and an endodomain. The endodomain may comprise or associate with a domain which transmit T-cell activation signals.CAR ANTIGEN BINDING DOMAIN

[0081] The antigen-binding domain is the portion of a CAR which recognizes antigen.

[0082] Numerous antigen-binding domains are known in the art, including those based on the antigen binding site of an antibody, antibody mimetics, and T-cell receptors. For example, the antigen-binding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a natural ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain binder such as a camelid; an artificial binder single as a Darpin; or a single-chain derived from a T-cell receptor.

[0083] The term "ligand" is used synonymously with "antigen" to mean an entity which is specifically recognised and bound by the antigen-binding domain of a CAR.CELL SURFACE ANTIGEN

[0084] The CAR may recognise a cell-surface antigen, i.e. an entity, such as a transmembrane protein which is expressed on the surface of a target cell, such as a tumour cell.

[0085] The CAR may specifically bind a tumour-associated cell-surface antigen.

[0086] Various tumour associated antigens (TAA) are known, some of which are shown in Table 1. The antigen-binding domain used in the present invention may be a domain which is capable of binding a TAA as indicated therein. Table 1Cancer type TAA Diffuse Large B-cell LymphomaCD19, CD20, CD22Breast cancerErbB2, MUC1AMLCD13, CD33NeuroblastomaGD2, NCAM, ALK, GD2B-CLLCD19, CD52, CD160Colorectal cancerFolate binding protein, CA-125Chronic Lymphocytic LeukaemiaCD5, CD19GliomaEGFR, VimentinMultiple myelomaBCMA, CD138Renal Cell CarcinomaCarbonic anhydrase IX, G250Prostate cancerPSMABowel cancerA33 PROSTATE-CANCER ASSOCIATED ANTIGENS

[0087] The CAR may specifically bind a cell-surface antigen associated with prostate cancer, such as prostate stem cell antigen (PSCA) or prostate-specific membrane antigen (PSMA).

[0088] PSCA is a glycosylphosphatidylinositol-anchored cell membrane glycoprotein. It is is up-regulated in a large proportion of prostate cancers and is also detected in cancers of the bladder and pancreas.

[0089] Various anti-PSCA antibodies are known, such as 7F5 (Morgenroth et al (Prostate (2007) 67:1121-1131); 1G8 (Hillerdal et al (2014) BMC Cancer 14:30); and Ha1-4.117 (Abate-Daga et al (2014) 25:1003-1012).

[0090] The cell of the invention may also express an anti-PSCA CAR which may comprise an antigen binding domain based on one of these antibodies.

[0091] PSMA is a zinc metalloenzyme that resides in membranes. PSMA is strongly expressed in the human prostate, being a hundredfold greater than the expression in most other tissues. In cancer, it is upregulated in expression and has been called the second-most-upregulated gene in prostate cancer, with increase of 8- to 12-fold over the noncancerous prostate. In addition to the expression in the human prostate and prostate cancer, PSMA is also found to be highly expressed in tumor neovasculature but not normal vasculature of all types of solid tumors, such as kidney, breast, colon, etc.

[0092] Various anti-PSMA antibodies are known, such as 7E11, J591, J415, and Hybritech PEQ226.5 and PM2J004.5 each of which binds a distinct epitope of PSMA (Chang et al (1999) Cancer Res 15:3192-8).

[0093] The cell of the invention may also express an anti-PSMA CAR which may comprise an antigen binding domain based on one of these antibodies.

[0094] For example, the CAR may comprise an scFv based on J591, having the sequence shown as SEQ ID No. 20. SEQ ID No. 20 (J591 scFv) CAR TRANSMEMBRANE DOMAIN

[0095] The transmembrane domain is the sequence of a CAR that spans the membrane. It may comprise a hydrophobic alpha helix. The CAR transmembrane domain may be derived from CD28, which gives good receptor stability.CAR SIGNAL PEPTIDE

[0096] The CAR and the constitutively active cytokine-signalling chimeric transmembrane protein may comprise a signal peptide so that when it / they is expressed in a cell, such as a T-cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed.

[0097] The core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix. The signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation. At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase. Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. The free signal peptides are then digested by specific proteases.

[0098] The signal peptide may be at the amino terminus of the molecule.

[0099] The signal peptide may comprise the sequence shown as SEQ ID No. 21, 22 or 23 or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions) provided that the signal peptide still functions to cause cell surface expression of the CAR. SEQ ID No. 21: MGTSLLCWMALCLLGADHADG

[0100] The signal peptide of SEQ ID No. 21 is compact and highly efficient and is derived from TCR beta chain. It is predicted to give about 95% cleavage after the terminal glycine, giving efficient removal by signal peptidase. SEQ ID No. 22: MSLPVTALLLPLALLLHAARP

[0101] The signal peptide of SEQ ID No. 22 is derived from IgG1. SEQ ID No. 23: MAVPTQVLGLLLLWLTDARC

[0102] The signal peptide of SEQ ID No. 23 is derived from CD8a.CAR ENDODOMAIN

[0103] The endodomain is the portion of a classical CAR which is located on the intracellular side of the membrane.

[0104] The endodomain is the signal-transmission portion of a classical CAR. After antigen recognition by the antigen binding domain, individual CAR molecules cluster, native CD45 and CD148 are excluded from the synapse and a signal is transmitted to the cell.

[0105] The CAR endodomain may be or comprise an intracellular signalling domain. In an alternative embodiment, the endodomain of the present CAR may be capable of interacting with an intracellular signalling molecule which is present in the cytoplasm, leading to signalling.

[0106] The intracellular signalling domain or separate intracellular signalling molecule may be or comprise a T cell signalling domain.

[0107] The most commonly used signalling domain component is that of CD3-zeta endodomain, which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signalling may be needed. For example, chimeric CD28 and OX40 can be used with CD3-Zeta to transmit a proliferative / survival signal, or all three can be used together.

[0108] The CAR may comprise the CD3-Zeta endodomain alone, the CD3-Zeta endodomain with that of either CD28 or OX40 or the CD28 endodomain and OX40 and CD3-Zeta endodomain.

[0109] The CAR endodomain may comprise one or more of the following: an ICOS endodomain, a CD27 endodomain, a BTLA endodomain, a CD30 endodomain, a GITR endodomain and an HVEM endodomain.

[0110] The endomain may comprise the sequence shown as SEQ ID No. 24 to 32 or a variant thereof having at least 80% sequence identity. SEQ ID No. 24 - CD3 Z endodomain SEQ ID No. 25 - CD28 and CD3 Zeta endodomains SEQ ID No. 26 - CD28, OX40 and CD3 Zeta endodomains SEQ ID No. 27 - ICOS endodomain CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL SEQ ID No. 28 - CD27 endodomain QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP SEQ ID No. 29 - BTLA endodomain SEQ ID No. 30 - CD30 endodomain SEQ ID No. 31 - GITR endodomain SEQ ID No. 32 - HVEM endodomain

[0111] A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID No. 24 to 32, provided that the sequence provides an effective intracellular signalling domain.NUCLEIC ACID CONSTRUCT

[0112] The present invention provides a nucleic acid construct encoding a constitutively active cytokine-signalling chimeric transmembrane protein as defined in the cell of the invention, the nucleic acid construct comprising a first nucleic acid sequence encoding the first polypeptide and a second nucleic acid sequence encoding the second polypeptide, the nucleic acid construct having the structure:         Dim1 -TM1-endo1-coexpr- Dim2 -TM2-endo2 in which Dim1 is a nucleic acid sequence encoding the dimerization domain of the first polypeptide; TM1 is a nucleic acid sequence encoding the transmembrane domain of the first polypeptide; endo 1 is a nucleic acid sequence encoding the endodomain of the first polypeptide; coexpr is a nucleic acid sequence encoding a cleavage or self-cleaving site enabling co-expression of first and second polypeptides Dim2 is a nucleic acid sequence encoding the dimerization domain of the second polypeptide; TM2 is a nucleic acid sequence encoding the transmembrane domain of the second polypeptide; endo 2 is a nucleic acid sequence encoding the endodomain of the second polypeptide, wherein the nucleic acid construct also comprises a nucleic acid sequence which encodes a chimeric antigen receptor (CAR).

[0113] As used herein, the terms "polynucleotide", "nucleotide", and "nucleic acid" are intended to be synonymous with each other.

[0114] 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.

[0115] Nucleic acids according to the invention 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 to oligonucleotides 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.

[0116] 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.

[0117] In the structure above, "coexpr" is a nucleic acid sequence enabling co-expression of both first and second CARs. It may be a sequence encoding a cleavage site, such that the nucleic acid construct produces a constitutively active cytokine-signalling chimeric transmembrane protein and a CAR, joined by a cleavage site(s). The cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into individual peptides without the need for any external cleavage activity.

[0118] The cleavage site may be any sequence which enables the constitutively active cytokine-signalling chimeric transmembrane protein and CAR to become separated.

[0119] The term "cleavage" is used herein for convenience, but the cleavage site may cause the peptides to separate into individual entities by a mechanism other than classical cleavage. For example, for the Foot-and-Mouth disease virus (FMDV) 2A self-cleaving peptide (see below), various models have been proposed for to account for the "cleavage" activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect (Donnelly et al (2001) J. Gen. Virol. 82:1027-1041). The exact mechanism of such "cleavage" is not important for the purposes of the present invention, as long as the cleavage site, when positioned between nucleic acid sequences which encode proteins, causes the proteins to be expressed as separate entities.

[0120] The cleavage site may be a furin cleavage site.

[0121] Furin is an enzyme which belongs to the subtilisin-like proprotein convertase family. The members of this family are proprotein convertases that process latent precursor proteins into their biologically active products. Furin is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at their paired basic amino acid processing sites. Examples of furin substrates include proparathyroid hormone, transforming growth factor beta 1 precursor, proalbumin, pro-beta-secretase, membrane type-1 matrix metalloproteinase, beta subunit of pro-nerve growth factor and von Willebrand factor. Furin cleaves proteins just downstream of a basic amino acid target sequence (canonically, Arg-X-(Arg / Lys)-Arg') and is enriched in the Golgi apparatus.

[0122] The cleavage site may be a Tobacco Etch Virus (TEV) cleavage site.

[0123] TEV protease is a highly sequence-specific cysteine protease which is chymotrypsin-like proteases. It is very specific for its target cleavage site and is therefore frequently used for the controlled cleavage of fusion proteins both in vitro and in vivo. The consensus TEV cleavage site is ENLYFQ\S (where '\' denotes the cleaved peptide bond). Mammalian cells, such as human cells, do not express TEV protease. Thus in embodiments in which the present nucleic acid construct comprises a TEV cleavage site and is expressed in a mammalian cell - exogenous TEV protease must also expressed in the mammalian cell.

[0124] The cleavage site may encode a self-cleaving peptide.

[0125] A 'self-cleaving peptide' refers to a peptide which functions such that when the polypeptide comprising the proteins and the self-cleaving peptide is produced, it is immediately "cleaved" or separated into distinct and discrete first and second polypeptides without the need for any external cleavage activity.

[0126] The self-cleaving peptide may be a 2A self-cleaving peptide from an aphtho- or a cardiovirus. The primary 2A / 2B cleavage of the aptho- and cardioviruses is mediated by 2A "cleaving" at its own C-terminus. In apthoviruses, such as foot-and-mouth disease viruses (FMDV) and equine rhinitis A virus, the 2A region is a short section of about 18 amino acids, which, together with the N-terminal residue of protein 2B (a conserved proline residue) represents an autonomous element capable of mediating "cleavage" at its own C-terminus (Donelly et al (2001) as above).

[0127] "2A-like" sequences have been found in picornaviruses other than aptho- or cardioviruses, 'picornavirus-like' insect viruses, type C rotaviruses and repeated sequences within Trypanosoma spp and a bacterial sequence (Donnelly et al (2001) as above). The cleavage site may comprise one of these 2A-like sequences, such as: YHADYYKQRLIHDVEMNPGP (SEQ ID No. 33) HYAGYFADLLIHDIETNPGP (SEQ ID No. 34) QCTNYALLKLAGDVESNPGP (SEQ ID No. 35) ATNFSLLKQAGDVEENPGP (SEQ ID No. 36) AARQMLLLLSGDVETNPGP (SEQ ID No. 37) RAEGRGSLLTCGDVEENPGP (SEQ ID No. 38) TRAEIEDELIRAGIESNPGP (SEQ ID No. 39) TRAEIEDELIRADIESNPGP (SEQ ID No. 40) AKFQIDKILISGDVELNPGP (SEQ ID No. 41) SSIIRTKMLVSGDVEENPGP (SEQ ID No. 42) CDAQRQKLLLSGDIEQNPGP (SEQ ID No. 43) YPIDFGGFLVKADSEFNPGP (SEQ ID No. 44)

[0128] The cleavage site may comprise the 2A-like sequence shown as SEQ ID No. 38 (RAEGRGSLLTCGDVEENPGP).VECTOR

[0129] Described herein is a vector, or kit of vectors, which comprises one or more nucleic acid sequence(s) encoding a constitutively active cytokine-signalling chimeric transmembrane protein as described herein and a CAR. Such a vector may be used to introduce the nucleic acid sequence(s) into a host cell so that it expresses a a constitutively active cytokine-signalling chimeric transmembrane protein as described herein.

[0130] The vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon based vector or synthetic mRNA.

[0131] The vector may be capable of transfecting or transducing a T cell or a NK cell.CELL

[0132] The present invention provides a cell which comprises: a chimeric antigen receptor (CAR); and a constitutively active cytokine-signalling chimeric transmembrane protein which comprises two polypeptides: (i) the dimerization domain of a first polypeptide which comprises: (a) a heavy chain constant domain (CH); and (b) a first chain of a type I cytokine receptor endodomain; and (ii) a second polypeptide which comprises: (a) the dimerization domain of a light chain constant domain (CL) which spontaneously heterodimerizes with the dimerization domain of the heavy chain constant domain (CH); and (b) a second chain of the type I cytokine-receptor endodomain.

[0133] The cell may comprise a nucleic acid or a vector of the present invention.

[0134] The cell may be a cytolytic immune cell such as a T cell or an NK cell.

[0135] T cells or T lymphocytes are a type of lymphocyte that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. There are various types of T cell, as summarised below.

[0136] Helper T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells become activated when they are presented with peptide antigens by MHC class II molecules on the surface of antigen presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines to facilitate different types of immune responses.

[0137] Cytolytic T cells (TC cells, or CTLs) destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. CTLs express the CD8 at their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of all nucleated cells. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevent autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0138] Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with "memory" against past infections. Memory T cells comprise three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells may be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0139] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus.

[0140] Two major classes of CD4+ Treg cells have been described - naturally occurring Treg cells and adaptive Treg cells.

[0141] Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and have been linked to interactions between developing T cells with both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells that have been activated with TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations of the FOXP3 gene can prevent regulatory T cell development, causing the fatal autoimmune disease IPEX.

[0142] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originate during a normal immune response.

[0143] The cell may be a Natural Killer cell (or NK cell). NK cells form part of the innate immune system. NK cells provide rapid responses to innate signals from virally infected cells in an MHC independent manner

[0144] NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph node, spleen, tonsils and thymus where they then enter into the circulation.

[0145] The cells of the invention may be any of the cell types mentioned above.

[0146] T or NK cells according to the invention may either be created 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).

[0147] Alternatively, T or NK cells according to the invention may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to T or NK cells. Alternatively, an immortalized T-cell line which retains its lytic function and could act as a therapeutic may be used.

[0148] In all these embodiments, cells are generated by introducing DNA or RNA coding for the chimeric transmembrane protein by one of many means including transduction with a viral vector, transfection with DNA or RNA.

[0149] The cell of the invention may be an ex vivo T or NK cell from a subject. The T or NK cell may be from a peripheral blood mononuclear cell (PBMC) sample. T or NK cells may be activated and / or expanded prior to being transduced with nucleic acid encoding the molecules providing the chimeric transmembrane protein as defined in the cell of the invention, for example by treatment with an anti-CD3 monoclonal antibody.

[0150] The T or NK cell of the invention may be made by: (i) isolation of a T or NK cell-containing sample from a subject or other sources listed above; and (ii) transduction or transfection of the T or NK cells with one or more a nucleic acid sequence(s) encoding the chimeric transmembrane protein as defined in the cell of the invention.

[0151] The T or NK cells may then by purified, for example, selected on the basis of expression of the antigen-binding domain of the antigen-binding polypeptide.PHARMACEUTICAL COMPOSITION

[0152] The present invention also relates to a pharmaceutical composition containing a plurality of cells according to the invention.

[0153] The pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and / or compounds. Such a formulation may, for example, be in a form suitable for intravenous infusion.METHOD OF TREATMENT

[0154] Described is a method for treating and / or preventing a disease which comprises the step of administering the cells of the present invention (for example in a pharmaceutical composition as described above) to a subject.

[0155] A method for treating a disease relates to the therapeutic use of the cells of the present invention. 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 and / or to slow down, reduce or block the progression of the disease.

[0156] The method for preventing a disease relates to the prophylactic use of the cells of the present invention. Herein such cells may be administered to a subject who has not yet contracted the disease and / or who is not showing any symptoms of the disease to prevent or impair the cause of the disease or to reduce or prevent development of at least one symptom associated with the disease. The subject may have a predisposition for, or be thought to be at risk of developing, the disease.

[0157] The method may involve the steps of: (i) isolating a T or NK cell-containing sample; (ii) transducing or transfecting such cells with a nucleic acid sequence or vector provided by the present invention; (iii) administering the cells from (ii) to a subject.

[0158] The T or NK cell-containing sample may be isolated from a subject or from other sources, for example as described above. The T or NK cells may be isolated from a subject'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).

[0159] Described is a constitutively active cytokine-signalling chimeric transmembrane protein -expressing cell as described herein for use in treating and / or preventing a disease.

[0160] Described is the use of a constitutively active cytokine-signalling chimeric transmembrane protein-expressing cell as described herein in the manufacture of a medicament for the treatment and / or prevention of a disease.

[0161] The disease to be treated and / or prevented by the methods described herein may be a cancerous disease, such as bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), leukaemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer and thyroid cancer.

[0162] The cells of the present invention may be capable of killing target cells, such as cancer cells. The target cell may be characterised by the presence of a tumour secreted ligand or chemokine ligand in the vicinity of the target cell. The target cell may be characterised by the presence of a soluble ligand together with the expression of a tumour-associated antigen (TAA) at the target cell surface.

[0163] The cells and pharmaceutical compositions of present invention may be for use in the treatment and / or prevention of the diseases described above.

[0164] The cells and pharmaceutical compositions of present invention may be for use in any of the methods described above.CHIMERIC TRANSMEMBRANE PROTEIN

[0165] The present chimeric transmembrane protein comprises a dimerization domain; and a cytokine receptor endodomain.

[0166] Dimerisation of the chimeric transmembrane protein occurs spontaneously, such that the chimeric transmembrane protein will be constitutively active.

[0167] In the present invention, the dimerization domain spontaneously heterodimerizes based on the dimerization domain of an antibody. The constitutively active cytokine-signalling chimeric transmembrane protein comprises the dimerization portion of a heavy chain constant domain (CH) and a light chain constant domain (CL) which spontaneously heterodimerizes with the dimerization domain of the heavy chain constant domain (CH). The "dimerization portion" of a constant domain is the part of the sequence which forms the inter-chain disulphide bond.

[0168] The constitutively active cytokine-signalling chimeric transmembrane protein may comprise the Fab portion of an antibody as exodomain, for example as illustrated schematically in Figure 5.

[0169] 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.EXAMPLESExample 1 - In vitro testing (for reference only)

[0170] T-cells are transduced with either a PSMA-specific CAR, or transduced with a construct which co-expresses a PSMA-specific CAR with a PSA-specific CCR. T-cells are co-cultured with PSMA expressing target cells which secrete or do not secrete PSA. This co-culture is conducted in the presence or absence of exogenous IL2. This co-culture is conducted at different effector to target ratios. This co-culture is repeated serially with T-cells challenged with repeated target cells. Proliferation of T-cells and killing of target cells is determined. In this way, the contribution to proliferation and survival of T-cells the CCR makes can be measured. Further, the ability contribution to repeated rechallenge the ability of serialExample 2 - In vivo testing (for reference only)

[0171] NSG mice are engrafted with a human prostate cancer cell line which expresses PSMA and secretes PSA and which expresses firefly Luciferase. T-cells are transduced with either a PSMA-specific CAR, or transduced with a construct which co-expresses the PSMA-specific CAR with a PSA-specific CCR. T-cells are administered to the mice. The tumour burden can be serially measured using bioluminescent imaging and the response to CAR T-cells evaluated. Mice within each cohort can be sacrificed at different time-points and tumour burden directly measured by macroscopic measurements and by immunohistochemistry. Further, engraftment / expansion of T-cells at the tumour bed or within lymphoid tissues such as lymph nodes, spleen and bone-marrow measured by flow cytometry of said tissues.Example 3 - Creation of and testing a constitutively active cytokine-signalling molecule

[0172] A constitutively active cytokine-signalling chimeric transmembrane protein was produced by linking cytokine receptor endodomains to a "Fab" type exodomain (Figure 5). This structure uses the natural dimerization components of antibodies, namely the dimerization domain from the heavy and light chain constant regions. The chimeric transmembrane protein has two chains; a first polypeptide which comprises the antibody light κ chain and the IL2 receptor common γ chain as endodomain; and a second polypeptide which comprises the antibody heavy chain CH1 and an endodomain which comprises either: the IL2 receptor β chain (giving a constitutively active IL2-signalling molecule); or the IL7 receptor (giving a constitutively active IL7-signalling molecule). The constitutively active cytokine-signalling chimeric transmembrane proteins tested in this study included the scFv heavy and light chain variable regions. These domains are not needed for dimerization to occur. The signal is independent of antigen binding and the structure could equally be "headless" (as shown in Figure 5) or comprise another entity such as a protein tag.

[0173] Nucleic acid sequences encoding these two polypeptides were cloned in frame separated by a 2A-peptide encoding sequence.

[0174] CTLL-2 (ATCC ®< TIB-214 ™< ) are murine cytotoxic T lymphocyte cells which are dependent upon IL-2 for growth. In the absence of IL-2 the cells undergo apoptosis. CTLL-2 cells were transduced with a vector expressing the chimeric protein comprising an IL2-receptor endodomain (Fab_IL2endo) or a vector expressing the chimeric protein comprising an IL7 receptor endodomain (Fab_IL7endo) or left untransduced (WT). As a positive control, cells of all three types were co-cultured with 100 U / ml murine IL2. Cell proliferation was assessed after 3 and 7 days of culture and the results are shown in Figure 6.

[0175] Untransduced CTLL2 cells, together with CTLL2 cells transduced with either construct (Fab_IL2endo or Fab_IL7endo) proliferated in the presence of 100U / mL murine IL2 (Figure 6, left-hand panel). However, in the absence of exogenously added IL2, only cells transduced with the construct having an IL2R endodomain (Fab_IL2endo) survived and proliferated. This shows that the chimeric transmembrane receptor provides the CTLL2 cells with the necessary IL2 signal.Example 4 - Generation and testing of a chimeric cytokine receptor against PSA (for reference only)

[0176] A panel of chimeric cytokine receptors targeting PSA was developed using scFvs derived from two antibodies which bind to different PSA epitopes: 5D5A5 and 5D3D11. The crystal structure of PSA has been obtained in a sandwich complex with these two (Stura et al (2011) as above).

[0177] Schematic diagrams illustrating some of the panel of CCRs is illustrated in Figure 7.

[0178] The panel included the following constructs: A5-CD8stk-IL2Rg_D11-Hinge-IL2Rb: A CCR with an IL-2R endodomain having A5 on the chain with common γ chain and D11 on the chain with the IL2R β chain; D11-CD8stk- IL2Rg_A5-Hinge-IL2Rb: A CCR with an IL-2R endodomain having D11 on the chain with common γ chain and A5 on the chain with IL2R β chain; D11-CD8stk- RL _A5-Hinge -IL2Rb: A negative control construct which is equivalent D11-CD8stk- IL2Rg_A5-Hinge-IL2Rb, but in which the IL2Rγ chain is replaced by a rigid linker; D11-CD8stk- IL2Rg_A5-Hinge-IL7Ra: A CCR with an IL-7R endodomain having D11 on the chain with common γ chain and A5 on the chain with IL7R α chain; and D11-CD8stk- RL _A5-Hinge -IL7Ra: A negative control construct which is equivalent D11-CD8stk- IL2Rg_A5-Hinge-IL7Ra, but in which the IL2Rγ chain is replaced by a rigid linker; CTLL2 cells were transduced with vectors expressing these constructs. Cells were cultured in the presence or absence of IL2 (the presence of IL2 acting as a positive control) and the presence or absence of 5ng / mL or 5µg / mL PSA. CTLL2 cell proliferation was assessed after 3 and 7 days and the results are shown in Figure 8.

[0179] CTLL2 cells expressing a CCR with an IL7 endodomain did not support CTLL2 cell survival and proliferation (Figure 8, last two panels). The presence of murine IL-2 in these cells supported CTLL2 cell growth and proliferation at day 3, but by day 7 the majority of cells had undergone apoptosis.

[0180] The anti-PSA chimeric cytokine receptors with an IL2R endodomain supported CTLL2 cell proliferation in the absence of IL2 and the presence of PSA at both 5ng / ml and 5µg / ml (Figure 8, first panel), with 5µg / ml giving greater survival and proliferation, particularly at day 7.

[0181] Both the anti-PSA chimeric cytokine receptors with an IL2R endodomain, i.e. A5-CD8stk-IL2Rg_D11-Hinge-IL2Rb and D11-CD8stk- IL2Rg_A5-Hinge-IL2Rb, indicating that the relative positioning of the two PSA-binding domains: 5D5A5 and 5D3D11, is not important for function.

[0182] Substitution of the common γ chain with a rigid linker abolished the capacity of the CCR to support CTLL2 cell survival and proliferation (Figure 8, third panel).

[0183] As another read-out for IL2 signalling, the phosphorylation of Y694 of STATS was investigated using phosphoflow.

[0184] CTLL2 cells were either untransduced (WT); transduced with a PSA CCR constructs having an IL2R endodomain (D11-CD8STK-IL2Rg_A5-Hinge-IL2Rb); or transduced with an equivalent negative control construct in which the IL2Rγ chain is replaced with a rigid linker (D11-CD8STK-RL_A5-Hinge-IL2Rb). The cells were incubated overnight in the absence of exogenously added IL-2. The following day, the cells were incubated with either Pervanadate at 500µM (a positive control which inhibits phosphatase and will lead to STAT5 phoshorylation) or 500ng / mL PSA for 1 or 4 hours. After incubation the cells were fixed, permeabilised and analysed by flow cytometry.

[0185] The results are shown in Figure 9. In the cells expressing the PSA CCR, the presence of PSA lead to increasing STATS phosphorylation with time (Figure 9, central panel). No such increase in phosphorylation was seen with untransduced CTLL2 cells, or with CTLL2 cells transduced with an equivalent construct in which the IL2Rγ chain is replaced with a rigid linker (Figure 9, right hand panel).

[0186] These results are consistent with the CTLL2 survival / proliferation data shown in Figure 8 and demonstrate that a chimeric cytokine receptor against a soluble ligand (here, PSA) can be used to trigger cytokine signalling in a T-cell.

Claims

1. A cell which comprises: a chimeric antigen receptor (CAR); and a constitutively active cytokine-signalling chimeric transmembrane protein which comprises two polypeptides: (i) a first polypeptide which comprises: (a) the dimerization domain of a heavy chain constant domain (CH); and (b) a first chain of a type I cytokine receptor endodomain; and (ii) a second polypeptide which comprises: (a) the dimerization domain of a light chain constant domain (CL) which spontaneously heterodimerizes with the dimerization domain of the heavy chain constant domain (CH); and (b) a second chain of the type I cytokine-receptor endodomain.

2. A cell according to claim 1, wherein the first polypeptide of the chimeric transmembrane protein comprises a heavy chain constant domain (CH); and the second polypeptide of the chimeric transmembrane protein comprises a light chain constant domain (CL).

3. A cell according to claim 1 or 2, wherein the first or second chain of the cytokine receptor endodomain comprises: (i) IL-2 receptor β-chain endodomain (ii) IL-7 receptor α-chain endodomain; or (iii) IL-15 receptor α-chain endodomain; and / or (iv) common γ-chain receptor endodomain.

4. A cell according to any preceding claim, wherein the first polypeptide of the chimeric transmembrane protein comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH); and the second polypeptide of the chimeric transmembrane protein comprises a light chain variable domain (VL) and a light chain constant domain (CL).

5. A nucleic acid construct encoding a chimeric transmembrane protein as defined in claim 1, the nucleic acid construct comprising a first nucleic acid sequence encoding the first polypeptide and a second nucleic acid sequence encoding the second polypeptide, the nucleic acid construct having the structure:         Dim1 -TM1-endo1-coexpr-Dim2 -TM2-endo2 in which Dim1 is a nucleic acid sequence encoding the dimerization domain of the first polypeptide; TM1 is a a nucleic acid sequence encoding the transmembrane domain of the first polypeptide; endo 1 is a nucleic acid sequence encoding the endodomain of the first polypeptide; coexpr is a nucleic acid sequence encoding a cleavage or self-cleaving site enabling co-expression of first and second polypeptides Dim2 is a nucleic acid sequence encoding the dimerization domain of the second polypeptide; TM2 is a a nucleic acid sequence encoding the transmembrane domain of the second polypeptide; endo 2 is a nucleic acid sequence encoding the endodomain of the second polypeptide, wherein the nucleic acid construct also comprises a nucleic acid sequence which encodes a chimeric antigen receptor (CAR).

6. A vector comprising a nucleic acid construct according to claim 5.

7. A kit which comprises: i) a vector comprising a nucleic acid sequence encoding a first polypeptide as defined in claim 1 or 2; and ii) a vector comprising a nucleic acid sequence encoding a second polypeptide as defined in claim 1 or 2; and iii) a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor.

8. A kit which comprises: i) a vector comprising a nucleic acid sequence encoding a chimeric transmembrane protein as defined in any of claims 1 to 4; and ii) a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor.

9. A method for making a cell according to any of claims 1 to 4, which comprises the step of introducing: a nucleic acid construct according to claim 5; a vector according to claim 6; or a kit of vectors according to claim 7 or 8, into a cell ex vivo.

10. A pharmaceutical composition comprising a plurality of cells according to any of claims 1 to 4.

11. A pharmaceutical composition according to claim 10 for use in treating and / or preventing a disease.