Chimeric antigen receptor with spacers

Abstract

The present invention provides a chimeric antigen receptor (CAR) comprising: a. an antigen-binding domain; b. a spacer; c. a transmembrane domain; d. an intracellular signalling domain; wherein the spacer comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 or SEQ ID NO: 50, or a variant thereof having 1-5 amino acid modifications.

Description

[0001] CHIMERIC ANTIGEN RECEPTOR

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to novel spacer and transmembrane domains for use in chimeric antigen receptors.

[0004] BACKGROUND TO THE INVENTION

[0005] Adoptive cell therapy (ACT) is a personalised therapy that involves administration to the subject of immune cells with activity directed against a specific disease related antigen. ACT using naturally occurring tumour-reactive lymphocytes or tumourinfiltrating lymphocytes (TILs) has mediated durable, complete regressions in patients with melanoma. However, melanoma appears to be the only cancer which reproducibly gives rise to TIL cultures capable of specific antitumor recognition and reactivity.

[0006] Subsequent approaches have sought to more widely apply ACT to treat other diseases and cancers by genetically engineering cells to express anti-tumour receptors. For example, TCRs are composed of one a and one chain. These receptors recognise antigens that have been processed and presented by an MHC molecule. Normal circulating lymphocytes transduced with a retrovirus encoding a TCR that recognized the MART-1 melanoma-melanocyte antigen have been shown to mediate tumour regression.

[0007] Another approach is the administration of lymphocytes genetically expressed to express a CAR. CARs are artificial receptors that can be constructed by linking an antigen-binding domain, such as the variable regions of the antibody heavy and light chains, to intracellular signalling chains (alone or in combination with other signalling moieties) via extracellular spacer domains and membrane-localising transmembrane domains. CARs against the B cell antigen CD19 have been shown to mediate regression of an advanced B cell lymphoma.

[0008] Although CAR-T cell-mediated treatment have shown success towards compact target antigens such as CD19 or GD2, chimeric antigen receptors may fail to signal in response to other antigens. An optimum synaptic distance is required for efficient triggering of downstream signalling after antigen encounter. Upon T cell encounter with an antigen presenting cell (via TCR interaction with peptide MHC), proteins at the interface segregate passively based on size. Phosphatases such as CD45 and CD148, which have large ectodomains, are excluded from regions of close contact between the T cell and APC. The synapse formed through interaction of peptide MHC and TCR is optimal for occlusion of CD45. The size of the target antigen and the location of the target epitope may therefore determine the success of a particular CAR construct, due to the resulting size of the immune synapse. The size and structure of any given CAR construct plays an important role in determining the size of the immune synapse.

[0009] There is therefore a need for alternative CAR T-cell approaches, capable of killing target cells expressing different target antigens.

[0010] DESCRIPTION OF THE FIGURES

[0011] Figure 1 - Schematic of first, second, and third generations CARs, showing the different components found in these constructs.

[0012] Figure 2 - Schematic comparison on type I and type II transmembrane domains.

[0013] Figure 3 - Quantification of HA-tagged spacer expression. Cell surface expression of the HA-tagged spacer domains was achieved by calculating the median fluorescence intensity of 293T cells stained with an APC conjugated anti-HA antibody. CD8a stalk is labelled, to which all the novel spacers are compared. Numbered points represent novel spacers that have a higher cell surface expression than CD8a.

[0014] Figure 4 - Quantification of anti-CD19 CAR cell surface expression from the novel spacers with their endogenous transmembrane domains. Cell surface expression of the anti-CD19 CARs was quantified by calculating the median fluorescence intensity (MedFI) of anti-anti-CD19 idiotype staining in FlowJo. The anti- CD19 (aCD19) control CAR had the highest level of cell surface expression, as determined by its MedFI.

[0015] Figure 5 - Summary of the percentage of viable target cells after the addition of transduced PBMCs with anti-CD19 CARs with novel spacers at the 48-hour time point (set with endogenous transmembrane domains). (A) 1 :4 ratio, (B) 1 :8 ratio, (C) 1 :16 ratio of effector cells to target cells. Spacer 5 was not functional at all as the percentage of viable targets was around 100. Spacers 7 and 11 performed the best and facilitated more killing than the anti-CD19 CAR control. Figure 6 - Quantification of anti-CD19 CAR cell surface expression from the spacer set with a constant CD8a transmembrane domain. The median fluorescence intensity of the anti-CD19 CARs was determined by staining the transduced PBMCs with an anti-anti-CD19_CAT scFv idiotype antibody and quantifying the median fluorescence intensity using FlowJo software. The anti-CD19 (aCD19) control CAR had the highest level of cell surface expression, as determined by its MedFI once again.

[0016] Figure 7 - Summary of the percentage of viable target cells after the addition of transduced PBMCs with anti-CD19 CARs with novel spacers at the 48-hour time point (spacer set with a constant transmembrane domain). (A) 1 :4 ratio, (B) 1:8 ratio, (C) 1 :16 ratio of effector cells to target cells.

[0017] Figure 8 - Quantification of anti-CD22 CAR cell surface expression from the spacer set with endogenous transmembrane domains. The medium fluorescence intensity of the anti-CD22 CARs was determined by staining the transduced PBMCs with biotinylated soluble CD22, plus streptavidin-APC, and quantifying the median fluorescence intensity using FlowJo software. The anti-CD22 (aCD22) control CARs had the highest level of cell surface expression, as determined by their MedFI.

[0018] Figure 9 - Summary of the percentage of viable target cells after the addition of transduced PBMCs with anti-CD22 CARs with novel spacers at the 72-hour time point (spacer set with endogenous transmembrane domains). (A) 1 :1 ratio, (B) 1 :2 ratio, (C) 1 :4 ratio of effector cells to target cells.

[0019] Figure 10 - Normalised aCD19 CAR median fluorescent intensity in transduced primary T cells. Primary T cells were transduced with the CARs containing different spacers, stained with aaCD19 scFv idiotype antibody after 72 hours in culture, analysed by flow cytometry and the mean normalised median fluorescent intensity (MedFI) of the aCD19 CAR calculated (aaCD19 CAR MedFI / eGFP MedFI). The MedFI of positive control CAR, possessing a spacer derived from human CD8a, is also shown. The lead candidate spacers from the screen are labelled. Data represent mean measurements from 3 independent donors.

[0020] Figure 11 - Cytotoxicity data. The percentage of viable target cells was calculated by normalising to the non-transduced sample to account for non-specific lysis due to alloreactivity. The data show that the CAR bearing the CD166 spacer (spacer 12) was as effective as the CD8a control CAR at lysing target cells (evident at 1 :16 ratio). NT, non-transduced; and eGFP, enhanced green fluorescent protein.

[0021] Figure 12 - Interferon y secretion. The concentration of secreted IFNy in the target cell and CAR T cell co-cultures was determined by ELISA. CAR T cells bearing the CD166 spacer (spacer 12) secreted more IFNy than the 46 aa CD8a spacer positive control CAR T cells.

[0022] Figure 13 - Interleukin 2 secretion. The concentration of secreted IL-2 in the target cell and CAR T cell co-cultures was determined by ELISA. CAR T cells bearing the CD166 spacer (spacer 12) secreted more IL-2 than the 46 aa CD8a spacer positive control CAR T cells.

[0023] SUMMARY OF ASPECTS OF THE INVENTION

[0024] The present inventors provide herein improved chimeric antigen receptors (CARs). More specifically, the present inventors have shown that a variety of domains from different proteins may be used as spacers or transmembrane domains in a CAR format. These domains provide options for a variety of CAR constructs, allowing them to be tailored to different antigens.

[0025] Accordingly, in a first aspect the present invention provides a chimeric antigen receptor (CAR) comprising: a. an antigen-binding domain; b. a spacer; c. a transmembrane domain; d. an intracellular signalling domain; wherein the spacer comprises SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11 , SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 or SEQ ID NO: 50, or a variant thereof having 1-5 amino acid modifications.

[0026] In one aspect, the spacer comprises SEQ ID NO: 1. In another aspect, the spacer comprises SEQ ID NO: 2. In another aspect, the spacer comprises SEQ ID NO: 3. In another aspect, the spacer comprises SEQ ID NO: 4. In another aspect, the spacer comprises SEQ ID NO: 5. In another aspect, the spacer comprises SEQ ID NO: 6. In another aspect, the spacer comprises SEQ ID NO: 7. In another aspect, the spacer comprises SEQ ID NO: 8. In another aspect, the spacer comprises SEQ ID NO: 9. In another aspect, the spacer comprises SEQ ID NO: 10. In another aspect, the spacer comprises SEQ ID NO: 11. The transmembrane domain may comprise SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22, or a variant thereof having 1-5 amino acid modifications. Alternatively, wherein the transmembrane domain may comprise SEQ ID NO: 34.

[0027] Each spacer may be combined with each transmembrane domain listed above.

[0028] The antigen binding domain may bind to any desired antigen. In particular, the antigen binding domain may bind to an antigen selected from the group comprising CD19, CD20, CD22, TRBC1, TRBC2, GD2, PSMA, CD33, CD123, CLL1 , FLT1, and Claudin.

[0029] The antigen binding domain may comprise any suitable antigen binding structure, such as those derived from antibodies. In particular, the antigen binding domain may comprise a single chain variable fragment (scFv), Fab fragment, domain antibody (dAb), or variable new antigen receptor (VNAR) domain.

[0030] The intracellular signalling domain may comprise CD3 endodomain. The intracellular signalling domain may further comprise a co-stimulatory domain selected from the list comprising CD28 endodomain, 4-1 BB endodomain, and 0X40 endodomain.

[0031] In a second aspect, the present invention provides a nucleic acid construct encoding a CAR according to the first aspect.

[0032] In a third aspect, the present invention provides a vector comprising a nucleic acid construct according to the second aspect.

[0033] In a fourth aspect, the present invention provides a cell comprising a CAR according to the first aspect. The cell may be a T cell or NK cell.

[0034] The cell in accordance with the present invention may be autologous. The cell in accordance with the present invention may be allogeneic.

[0035] The cell in accordance with the present invention may be isolated from a subject.

[0036] The cells may be expanded prior to incorporation into a pharmaceutical composition. The cells may be activated prior to the introduction of a nucleic acid which encodes a CAR.

[0037] In a fifth aspect, the present invention provides a method for making a cell according to the fourth aspect, the method comprising the step of transducing or transfecting a cell with a nucleic acid construct according to the second aspect or a vector according to the third aspect.

[0038] The CAR may be introduced into the cell by transduction, for example using a retroviral or lentiviral vector.

[0039] In a sixth aspect, the present invention provides a pharmaceutical composition which comprises a plurality of cells according to the fourth aspect, together with a pharmaceutically acceptable carrier, diluent or excipient.

[0040] In a seventh aspect, the present invention provides a method for treating cancer which comprises the step of administering a pharmaceutical composition according to the sixth aspect to a subject.

[0041] In an eight aspect, the present invention provides pharmaceutical composition according to the sixth aspect for use in treating cancer.

[0042] In one embodiment, the disease may be cancer. In one embodiment the cancer may be a solid tumour cancer.

[0043] In one embodiment the disease may be a haematological malignancy. Suitably, the disease may be a leukaemia. Suitably, the disease may be a lymphoma.

[0044] In a ninth aspect, the present invention provides the use of a cell according to the fourth aspect in the manufacture of a pharmaceutical composition for treating cancer. DETAILED DESCRIPTION

[0045] CHIMERIC ANTIGEN RECEPTOR (CAR)

[0046] A classical chimeric antigen receptor (CAR) is a chimeric type I trans-membrane protein which connects 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 antigen binding site, as described in more detail below. A spacer domain is usually necessary to isolate the binder from the membrane and to allow it a suitable orientation. A transmembrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.

[0047] The present inventors have identified several domains from naturally occurring proteins that may be used as spacer domains in a CAR format. Furthermore, several useful transmembrane domains have been identified. Useful combinations of spacer and transmembrane domain are also provided herein.

[0048] Early CAR designs had endodomains derived from the intracellular parts of either the y chain of the FCER1 or CD3 (Figure 1). 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 CD3 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 0X40 and 41 BB which transmit survival signals. Third generation CARs have also been described which have endodomains capable of transmitting activation, proliferation and survival signals.

[0049] CAR-encoding nucleic acids may be transferred to T cells using, for example, retroviral vectors. Lentiviral vectors may be employed. In this way, a large number of antigen-specific cells can be generated for adoptive cell transfer. When a 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 tumour cells expressing the targeted antigen.

[0050] CARs typically therefore comprise: (i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iii) an intracellular domain which comprises or associates with a signalling domain.

[0051] ANTIGEN BINDING DOMAIN

[0052] The antigen binding domain is the portion of the CAR which recognizes antigen.

[0053] 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 antibody; an artificial single binder such as a Darpin (designed ankyrin repeat protein); or a single-chain derived from a T-cell receptor.

[0054] The antigen binding domain may comprise a domain which is not based on the antigen binding site of an antibody. For example the antigen binding domain may comprise a domain based on a protein / peptide which is a soluble ligand for a tumour cell surface receptor (e.g. a soluble peptide such as a cytokine or a chemokine); or an extracellular domain of a membrane anchored ligand or a receptor for which the binding pair counterpart is expressed on the tumour cell.

[0055] The antigen binding domain may be based on a natural ligand of the antigen.

[0056] The antigen binding domain may comprise an affinity peptide from a combinatorial library or a de novo designed affinity protein / peptide.

[0057] One example of an anti-CD19 antigen binding domain that may be used in the CAR of the present invention has the following VH and VL sequences: >CAT19 VH ( SEQ ID NO : 39 ) QVQLQQSGPELVKPGASVKI SCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKAT LTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSS

[0058] >CAT19 VL ( SEQ ID NO : 40 ) QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTS YFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKR

[0059] An example of an anti-CD22 antigen binding domain that may be used in the CAR of the present invention has the following VH and VL sequences:

[0060] >9A8 VL ( SEQ ID NO : 41 )

[0061] DIQMTQSPSSLSASLGDRVTITCRSSQDIGNYLTWFQQKVGRSPRRMIYGAIKLEDGVPSRFS GSRSGSDYSLTISSLESEDVADYQCLQSIQYPFTFGSGTKLEIK

[0062] >9A8 VH ( SEQ ID NO : 42 )

[0063] EVQLVESGGGLVQPGRSLKLSCAASGFTFSNFAMAWVRQPPTKGLEWVASISTGGGNTYYRDS VKGRFTISRDDAKNTQYLQMDSLRSEDTATYYCARQRNYYDGSYDYEGYTMDAWGQGTSVTVS S

[0064] SPACER DOMAIN

[0065] CARs typically comprise a spacer sequence to connect the antigen-binding domain with the transmembrane domain and spatially separate the antigen-binding domain from the endodomain. A flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding.

[0066] The spacer domain of CARs has not received as much attention compared to the other domains and therefore, typically only a handful of spacers have been used in CARs. The present inventors therefore identified a need for alternative spacer options to allow for alternative CAR designs.

[0067] The spacer domains traditionally used in CARs are derived from the CD8a stalk, CD28 co-stimulatory receptor and the immunoglobulin (IgG) Fc region. The CD8a stalk is a well-characterised spacer and has been used in CARs taken to clinical trials and beyond to commercialisation. Alternative spacers include a spacer derived from the cartilage oligomeric matrix protein (COMP) that has been used by the present inventors in an anti-CD22 CAR, where its efficacy was tested in a clinical trial for the treatment of paediatric and young adult B cell acute lymphoblastic leukaemia. Patel et al. (1999, Gene Ther. 6(3):412-9) have shown that the spacer domain can influence the expression of CARs, especially in relation to signal transduction. In their work, the group modified the spacer and transmembrane domains of primary CARs to produce different configurations. They saw that changing these domains impacted the stability of the receptor as well as the affinity of the antigen-binding domain to its cognate antigen. Hudecek et al. (2015, Cancer Immunology Research 3, 125-135) also modified the spacer domain in CARs and found that exchanging traditional spacers used in CARs for novel spacers can increase T cell persistence in vivo. Recent work by Muller et al. (2021 , Frontiers in Immunology 12, 500) has shown that the choice of spacer domain has a significant impact on CAR function. CARs bearing a CD28 transmembrane domain can heterodimerise with endogenous CD28 through a core region, containing polar and cysteine residues present in the transmembrane domain, which increases the sensitivity and proliferative capacity of CAR T cells. The interaction between the CAR CD28 transmembrane domain and endogenous CD28 co-receptor is dependent on the spacer domain utilised, with lgG4 spacers blocking the interaction.

[0068] Schafer et al. (2020, Frontiers in Immunology 11, 1704) recently developed CARs with a spacer region derived from sialic acid-binding immunoglobulin-type lectins (Siglecs). The Siglec-derived spacer that promoted the activity of their anti-CD20 CAR the most was Siglec-4, which performed better than the lgG4 spacer domain and worked as well as a spacer derived from CD8a. Furthermore, they showed that the release of inflammatory cytokines was much lower when using a CAR with a spacer region derived from Siglec-4 than a CAR with a spacer derived from CD8a. Inflammatory cytokines are responsible for causing cytokine release syndrome (CRS) and neurotoxicity in patients undergoing CAR T cell therapy. They both can cause complications and even death in patients undergoing the treatment.

[0069] By screening a series of type I transmembrane domains, the present inventors have identified a set of 17 spacers that may be used in a CAR format as described herein. These spacers are shown in Table 1. Table 1. Spacer domains for use in a CAR format.

[0070] The present invention therefore provides CARs that make use of the spacers described in Table 1. These spacers may be combined with different transmembrane domains, such as the native domain from the protein from which they were derived or an alternative transmembrane domain such as the domain from CD8a or CD28. TRANSMEMBRANE DOMAIN

[0071] The transmembrane domain is the sequence of the CAR that spans the membrane.

[0072] A transmembrane domain may be any protein structure which is thermodynamically stable in a membrane. This is typically an alpha helix comprising of several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to supply the transmembrane portion of the invention.

[0073] The presence and span of a transmembrane domain of a protein can be predicted by those skilled in the art using bioinformatics tools such as the TMHMM algorithm (http: / / www.cbs.dtu.dk / services / TM HMM-2.0 / ). Further, given that the transmembrane domain of a protein is a relatively simple structure, i.e., a polypeptide sequence predicted to form a hydrophobic alpha helix of sufficient length to span the membrane, an artificially designed TM domain may also be used (for example as described in US 7052906 B1 which is incorporated herein by reference).

[0074] The transmembrane domain may be derived from CD28, which gives good receptor stability. The transmembrane domain from CD8a may also be used.

[0075] The present inventors have also found that the transmembrane domains of several type I membrane proteins can function as transmembrane domains in a CAR format. These transmembrane domains are shown in Table 2.

[0076] Table 2. Transmembrane domains corresponding to spacer domains of the present invention.

[0077] ENDODOMAIN

[0078] The endodomain is the signal-transmission portion of the chimeric receptor. It may be part of or associate with the intracellular domain of the chimeric receptor. After antigen recognition, receptors cluster, native CD45 and CD148 are excluded from the synapse and a signal is transmitted to the cell. The most commonly used endodomain component is that of CD3-zeta 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. Costimulatory signals promote T-cell proliferation and survival. There are two main types of co-stimulatory signals: those that belong the Ig family (CD28, ICOS) and the TNF family (0X40, 41 BB, CD27, GITR etc). For example, chimeric CD28 and 0X40 can be used with CD3-Zeta to transmit a proliferative I survival signal, or all three can be used together.

[0079] The endodomain may comprise:

[0080] (i) an ITAM-containing endodomain, such as the endodomain from CD3 zeta; and / or

[0081] (ii) a co-stimulatory domain, such as the endodomain from CD28 or ICOS; and / or

[0082] (iii) a domain which transmits a survival signal, for example a TNF receptor family endodomain such as OX-40, 4-1 BB, CD27 or GITR.

[0083] The sequence of the CD3zeta endodomain is provided below:

[0084] >CD3_zeta ( SEQ ID NO : 35 ) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ

[0085] KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0086] The sequence of the 4-1 BB sequence is also provided below:

[0087] >4 -lBB ( SEQ ID NO : 36 ) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0088] The CARs of the invention may also include part of the intracellular domain of the original type I transmembrane domains from which the novel spacer and / or transmembrane domains have been isolated. These sequences are listed in table 3.

[0089] Table 3. Intracellular domains corresponding to spacer domains of the present invention.

[0090] A number of systems have been described in which the antigen recognition portion is on a separate molecule from the signal transmission portion, such as those described in W0015 / 150771 ; WO2016 / 124930 and WO2016 / 030691. The chimeric receptor of the present invention may therefore comprise an antigen-binding component comprising an antigen-binding domain and a transmembrane domain; which is capable of interacting with a separate intracellular signalling component comprising a signalling domain. The vector of the invention may express a chimeric receptor signalling system comprising such an antigen-binding component and intracellular signalling component.

[0091] The chimeric receptor may comprise a signal peptide so that when it is expressed inside a cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed. The signal peptide may be at the amino terminus of the molecule. TARGET ANTIGEN

[0092] A “target antigen” as used herein refers to the antigen which the CAR has specificity for, i.e. the antigen which the antigen binding domain of the CAR has been engineered to have specificity for.

[0093] A target antigen may be a disease associated antigen.

[0094] Suitably the target antigen may be associated with chronic infection.

[0095] Suitably the target antigen may be associated with autoimmunity.

[0096] A target antigen may be a tumour associated antigen e.g., a cancer related antigen.

[0097] Various target antigens are known, as shown table 4. The antigen-binding domain used in the present invention may be a domain which is capable of binding an antigen as indicated therein.

[0098] Table 4: Example target antigens

[0099] CELL

[0100] The present invention also relates to a genetically modified cell, comprising a CAR of the invention.

[0101] A “starting population of cells” as used herein refers to a sample of cells which will be used to produce genetically modified cells which comprise a CAR. The starting population of cells may be obtained from any source of blood cells or peripheral blood mononuclear cells (PBMCs). The source cells may be provided fresh or may be cryopreserved prior to use. The starting population of cells may be used without any further manipulation or may be used after an isolation or enrichment step. Methods for isolating or enriching white blood cells are known in the art. For example, white blood cells or PBMCs may be obtained from whole blood by various methods e.g., density gradient separation, such as using Ficoll-Paque density gradient media; by magnetic bead separation, such as MACS Milteyni Biotec CD3, CD4 or CD8 beads; by elutriation or any other method. Separation or isolation of cells may be automated or may be performed manually.

[0102] The starting population of cells may be from blood e.g., from a peripheral blood sample or from a biopsy. The starting population of cells may be peripheral blood mononuclear cells. The starting population of cells may be a leukapheresate.

[0103] Suitably, the starting population of cells may be obtained from the subject (1stparty).

[0104] Suitably, the starting population of cells may be obtained from a donor (2ndparty).

[0105] Suitably, the starting population of cells may be obtained from a donor who is an unconnected donor (3rdparty).

[0106] Alternatively, the cells may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to, for example, T cells. Alternatively, an immortalized cell line which retains its lytic function and could act as a therapeutic may be used. Suitably, the starting population may be whole blood obtained from the subject. Suitably, the starting population may be PBMCs obtained from the subject. Suitably, the starting population may be a leukapheresate obtained from the subject.

[0107] Suitably, the starting population may be whole blood obtained from a donor. Suitably, the starting population may be PBMCs obtained from a donor. Suitably, the starting population may be a leukapheresate obtained from a donor.

[0108] “Transduced or transfected cells” as used herein refers to the cell population which has undergone the transduction or transfection process. This population of cells may contain a mixture of cells which have been successfully genetically modified and those which have not.

[0109] An “genetically modified cell” as used herein means a cell which has been modified to comprise or express a CAR. 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. Any suitable method may be used to introduce a nucleic acid sequence into a cell which encodes a CAR.

[0110] Suitably, a genetically modified cell is a cell whose genome has been modified e.g., by transduction or by transfection. Suitably, a genetically modified cell is a cell whose genome has been modified by retroviral transduction. Suitably, a genetically modified cell is a cell whose genome has been modified by lentiviral transduction.

[0111] As used herein, the term “introduced” refers to methods for inserting foreign 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.

[0112] Genetically modified cells according to the invention may be generated by introducing DNA or RNA coding for the CAR 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 the introduction of a nucleic acid sequence encoding a CAR , for example by treatment with an anti-CD3 monoclonal antibody or both anti-CD3 and anti-CD28 monoclonal antibodies.

[0113] Suitably, the genetically modified cell may be autologous.

[0114] Suitably, the cell may be allogeneic.

[0115] In one embodiment, the genetically modified cell may be a PBMC.

[0116] Suitably, the genetically modified cell may be a B cell. Suitably the genetically modified cell may be an NK cell. Suitably the genetically modified cell may be a T cell.

[0117] The genetically modified cell may be a cytolytic immune cell.

[0118] “Cytolytic immune cell” as used herein is a cell which directly kills other cells. Cytolytic cells may kill cancerous cells; virally infected cells or other damaged cells. Cytolytic immune cells include T cells and Natural killer (NK) cells.

[0119] Cytolytic immune cells can be T cells or T lymphocytes which are a type of lymphocyte that play a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes, such as B cells and NK cells, by the presence of a TCR on their cell surface. There are various types of T cell, as summarised below.

[0120] Helper T 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, TH 17, Th9, or TFH, which secrete different cytokines to facilitate different types of immune responses.

[0121] Cytolytic T cells (TC cells, or CTLs) destroy virally infected cells and tumour cells and are also implicated in transplant rejection. CTLs express the CD8 at their surface. CTLs may be known as CD8+ T cells. 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.

[0122] 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.

[0123] 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.

[0124] Two major classes of CD4+ Treg cells have been described — naturally occurring Treg cells and adaptive or induced Treg cells.

[0125] 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.

[0126] 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 increased expression of PD-1 (programmed cell death-1 , pdcdl), 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.

[0127] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originate during a normal immune response. Peripherally generated Tregs may be referred to as induced Treg (iTreg) cells.

[0128] As used herein, the term “induced regulatory T cell” (iTreg) means a CD4+ CD25+ FOXP3+ Helios- Neuropilin 1- T cell which develops from mature CD4+ conventional T cells outside of the thymus. For example, iTregs can be induced in vitro from CD4+ CD25-FOXP3- cells in the presence of IL-2 and TGF-p.

[0129] Suitably the cell may be a T cell. Suitably the cell may be a helper T cell. Suitably the cell may be a cytolytic T cell. Suitably the cell may be a memory T cell. Suitably the cell may be a regulatory T cell (Treg). Suitably the cell may be a naturally occurring Treg or an adaptive Treg.

[0130] Natural Killer Cells (or NK cells) are a type of cytolytic cell which form part of the innate immune system. NK cells provide rapid responses to innate signals from virally infected cells in an MHC independent manner.

[0131] 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.

[0132] Suitably the cell may be a natural killer cell.

[0133] Suitably, the cell may be a stem cell.

[0134] In one embodiment, the cell may be a progenitor cell.

[0135] As used herein, the term “stem cell” means an undifferentiated cell which is capable of indefinitely giving rise to more stem cells of the same type, and from which other, specialised cells may arise by differentiation. Stem cells are multipotent. Stem cells may be for example, embryonic stem cells or adult stem cells.

[0136] As used herein, the term “progenitor cell” means a cell which is able to differentiate to form one or more types of cells but has limited self-renewal in vitro. Suitably, the cell may be any cell capable of differentiating into a cytolytic immune cell.

[0137] Suitably, the cell may be capable of being differentiated into a T cell or NK cell.

[0138] Suitably, the cell may be an embryonic stem cell (ESC). Suitably, the cell may be a haematopoietic stem cell or haematopoietic progenitor cell. Suitably, the cell may be an induced pluripotent stem cell (iPSC). Suitably, the cell may be obtained from umbilical cord blood. Suitably, the cell may be obtained from adult peripheral blood.

[0139] In some aspects, hematopoietic stem and progenitor cell (HSPCs) may be obtained from umbilical cord blood. Cord blood can be harvested according to techniques known in the art (e.g., U.S. Pat. Nos. 7,147,626 and 7,131 ,958 which are incorporated herein by reference).

[0140] In one aspect, HSPCs may be obtained from pluripotent stem cell sources, e.g., induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs).

[0141] As used herein, the term “hematopoietic stem and progenitor cell” or “HSPC” refers to a cell which expresses the antigenic marker CD34 (CD34+) and populations of such cells. In particular embodiments, the term “HSPC” refers to a cell identified by the presence of the antigenic marker CD34 (CD34+) and the absence of lineage (lin) markers. The population of cells comprising CD34+ and / or Lin(-) cells includes haematopoietic stem cells and hematopoietic progenitor cells.

[0142] HSPCs can be obtained or isolated from bone marrow of adults, which includes femurs, hip, ribs, sternum, and other bones. Bone marrow aspirates containing HSPCs can be obtained or isolated directly from the hip using a needle and syringe. Other sources of HSPCs include umbilical cord blood, placental blood, mobilized peripheral blood, Wharton's jelly, placenta, fetal blood, fetal liver, or fetai spleen. In particular embodiments, harvesting a sufficient quantity of HSPCs for use in therapeutic applications may require mobilizing the stem and progenitor cells in the subject.

[0143] As used herein, the term “induced pluripotent stem cell” or “iPSC” refers to a non- pluripotent cell that has been reprogrammed to a pluripotent state. Once the cells of a subject have been reprogrammed to a pluripotent state, the cells can then be programmed to a desired cell type, such as a hematopoietic stem or progenitor cell (HSC and HPC respectively).

[0144] As used herein, the term “reprogramming” refers to a method of increasing the potency of a cell to a less differentiated state.

[0145] As used herein, the term “programming” refers to a method of decreasing the potency of a cell or differentiating the cell to a more differentiated state.

[0146] The cells of the invention may be any of the cell types mentioned above.

[0147] A “population of genetically modified cells” as used herein means one or more genetically modified cells according to the present invention.

[0148] Suitably a population of genetically modified cells as used herein may mean two or more (or a plurality of) genetically modified cells according to the present invention.

[0149] In one embodiment, the cell or population of cells may be activated before the CAR is introduced into the cell (iv).

[0150] In another embodiment, the cell or population of cells may not be activated before the CAR is introduced into the cell (iv).

[0151] Optionally, the method may additionally comprise isolating a cell containing sample from a subject. This cell containing sample may be used as the staring population of cells.

[0152] Optionally, the cells for use in the present invention may be activated and / or expanded prior to the introduction of a nucleic acid sequence which encodes a CAR.

[0153] Any method known in the art for activating and / or expanding cells may be used in the method of the invention. For example cells for use in the present invention e.g. T cells may be activated and / or expanded by treatment with an anti-CD3 monoclonal antibody or both anti-CD3 and anti-CD28 monoclonal antibodies. Suitably, interleukin 7 (IL- 7) and / or interleukin 15 (IL-15) may be used to expand cells e.g. T cells in vitro. Suitably, interleukin 2 (IL-2) may be used for the expansion of cells in vitro.

[0154] NK cells for use in the present invention may be activated and / or expanded by treatment with cytokines such as interleukin 2 (IL-2) and / or interleukin 15 (IL-15). Incubation with accessory cells such as monocytes, B-lymphoblastoid cells or cell lines which express stimulatory molecules may be used to provide additional signals for expansion of NK cells.

[0155] As used herein “activated” means that a cell has been stimulated, causing the cell to proliferate, differentiate or initiate an effector function.

[0156] Methods for measuring cell activation are known in the art and include, for example, measuring the expression of activation markers by flow cytometry, such as the expression of CD69, CD25, CD38 or HLA-DR or measuring intracellular cytokines.

[0157] As used herein “expanded” means that a cell or population of cells has been induced to proliferate.

[0158] 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.

[0159] In one aspect, the method according to the present invention produces a population of engineered cells (e.g., genetically modified cells) which comprise a chimeric antigen receptor.

[0160] Suitably a genetically modified cell or population of genetically modified cells according to the present invention may be made by the method according to the invention.

[0161] In one aspect, the population of genetically modified cells according to the present invention or obtainable (e.g. obtained) by a method according to the present invention are less differentiated. As used herein “differentiated” refers to the stage of development of a particular cell within the linear progression of differentiation of that cell type. For example, CD4+ and CD8+ T cells can be categorized into distinct memory subsets based on their differentiation states. CD4+ and CD8+ T cells follow a progressive pathway of differentiation from naive T cells into central memory and effector memory cell populations. The differentiation state of CD8+ T cells is inversely related to their capacity to proliferate and persist.

[0162] Preclinical studies suggest that improved antitumor responses are achieved when genetically modified T cells are in the early stages of differentiation (such as naive or central memory cells). Central memory cells have improved in vivo persistence compared with effector memory cells.

[0163] In one aspect, the population of genetically modified cells according to the invention or obtainable by a method according to the invention are more naive.

[0164] As used herein, “naive” means a cell which is not fully differentiated. A naive T cell may not have encountered antigen.

[0165] Naive T cells may be characterised by the surface expression of L selection (CD62L), the absence of activation markers CD25, CD44 or CD69 and the absence of memory CD45RO isoform e.g., naive T cells may be CD62LHiCD25LoCD44LoCD69Lo. Naive T cells also express functional IL-7 receptors, consisting of subunits IL-7 receptor-a, CD127, and common-y chain, CD132.

[0166] In one aspect, a naive cell subset may be defined as CCR7+ / CD45RA+ cells. Suitably, a naive cell subset may be further defined as CCR7+ / CD45RA+ / CD62L+ / CD27+ cells.

[0167] Suitably, the genetically modified cells according to the present invention or obtainable (e.g. obtained) by a method according to the present invention may have increased expression of CD27 and / or CD62L.

[0168] Without wishing to be bound by theory, a more naive or immature genetically modified cell population is advantageous for use in therapy because naive cells exhibit enhanced persistence in vivo and enhanced cytolytic activity when compared to cells with a more differentiated phenotype. As used herein “exhaustion” or “exhausted” means that the cell exhibits decreased effector functions and / or altered phenotype. Immune cell exhaustion describes the status of dysfunction of immune cells, usually under the setting of tumours or chronic infection. Exhaustion may be accompanied by phenotypic changes, epigenetic modifications and alterations in transcriptional profiles.

[0169] Effector functions may include the production of effector cytokines and direct cytotoxic activity.

[0170] Suitably the population of genetically modified cells according to the present invention or obtainable (or obtained) by a method according to the present invention may have decreased expression of one or more exhaustion markers.

[0171] Suitably, one or more exhaustion markers may be two exhaustion markers. Suitably, one or more exhaustion markers may be three exhaustion markers. Suitably, one or more exhaustion markers may be four exhaustion markers. Suitably, one or more exhaustion markers may be five exhaustion markers. Suitably, one or more exhaustion markers may be six exhaustion markers. Suitably, one or more exhaustion markers may be seven exhaustion markers.

[0172] For example, in the context of NK cells, effector functions may include production of interferon gamma (IFN-y). Other effector functions of NK cells include direct cytotoxic activity, such as activity dependent on perforin and granzyme, or induction of target cell apoptosis by tumour necrosis factor alpha (TNF-a), Fas ligand (FasL) and TNF- related apoptosis-inducing ligand (TRAIL).

[0173] Suitably, exhausted NK cells may produce decreased amounts of effector cytokines e.g., IFN-y, compared with non-exhausted NK cells. Suitably, exhausted NK cells may have decreased cytolytic activity and may, for example, produce decreased amounts of CD107a and / or granzyme B and / or perforin compared with non-exhausted NK cells.

[0174] Suitably the one or more exhaustion markers may be selected from the group consisting of: IFN-y, TNF-a, FasL, TRAIL, CD107a, granzyme B and perforin. Suitably the one or more exhaustion markers may be selected from the group consisting of: IFN-y, TNF-a, FasL, TRAIL, CD107a, granzyme B and perforin wherein the genetically modified cell is an NK cell.

[0175] Suitably, the one or more exhaustion markers may comprise decreased IFN-y production. Suitably, the one or more exhaustion markers may comprise decreased TNF-a production. Suitably, the one or more exhaustion markers may comprise decreased expression of FASL. Suitably, the one or more exhaustion markers may comprise decreased expression of TRAIL. Suitably, the one or more exhaustion markers may comprise decreased expression of CD107a. Suitably, the one or more exhaustion markers may comprise decreased production of granzyme B. Suitably, the one or more exhaustion markers may comprise decreased production of perforin.

[0176] For example, in the context of T cells, exhaustion may be defined by poor effector function, sustained expression of inhibitory receptors and / or a transcriptional state distinct from that of functional effector or memory T cells. For example, exhausted T cells may express high levels of PD1 , Tim3, Lag3, CD43 (1 B11), CD69 and inhibitory receptors but low levels of CD62L and CD127 and decreased interleukin-2 (IL-2), TNF-a and IFN-y production.

[0177] Suitably, the one or more exhaustion markers may comprise increased (e.g. high) expression of PD1. Suitably, the one or more exhaustion markers may comprise increased (e.g. high) expression of Tim3. Suitably, the one or more exhaustion markers may comprise increased (e.g., high) expression of Lag3. Suitably, the one or more exhaustion markers may comprise increased (e.g. high) expression of CD43 (1 B11). Suitably, the one or more exhaustion markers may comprise increased (e.g. high) expression of CD69. Suitably, the one or more exhaustion markers may comprise increased (e.g. high) expression of inhibitory receptors. Suitably, the one or more exhaustion markers may comprise decreased (e.g. low) expression of CD62L. Suitably, the one or more exhaustion markers may comprise decreased (e.g. low) expression of CD127. Suitably, the one or more exhaustion markers may comprise decreased (e.g., low) IL-2 production upon target encounter. Suitably, the one or more exhaustion markers may comprise decreased (e.g. low) TNF-a production upon target encounter. Suitably, the one or more exhaustion markers may comprise decreased (e.g. low) IFN-y production upon target encounter.

[0178] Suitably, the one or more exhaustion markers may be selected from the group consisting of: PD1 , Lag3 and Tim3. Suitably, the one or more exhaustion markers may comprise PD1. Suitably, the one or more exhaustion markers may comprise Lag3. Suitably, the one or more exhaustion markers may comprise Tim3. Suitably, the one or more exhaustion markers may be selected from the group consisting of: PD1, Lag3 and Tim3 wherein the genetically modified cell is a T cell.

[0179] In one aspect, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, or more than 35% of the population of genetically modified cells according to the present invention or obtainable (or obtained) by a method according to the present invention may be naive (CCCR7+ / CD45RA+) and CD62L+ / CD27+. Suitably more than 10% of the genetically modified cells according to the invention may be naive (CCCR7+ / CD45RA+) and CD62L+ / CD27+. Suitably more than 15% of the genetically modified cells according to the invention may be naive (CCCR7+ / CD45RA+) and CD62L+ / CD27+. Suitably more than 20% of the genetically modified cells according to the invention may be naive (CCCR7+ / CD45RA+) and CD62L+ / CD27+. Suitably more than 30% of the genetically modified cells according to the invention may be naive (CCCR7+ / CD45RA+) and CD62L+ / CD27+. Suitably more than 40% of the genetically modified cells according to the invention may be naive (CCCR7+ / CD45RA+) and CD62L+ / CD27+.

[0180] Suitably, the proportion of naive cells may be measured in the CD8+ T cell subset.

[0181] In one aspect, fewer than 30%, fewer than 25%, fewer than 20%, fewer than 15%, fewer than 10%, fewer than 5% of the population of genetically modified cells according to the present invention or obtainable (or obtained) by a method according to the present invention may express multiple exhaustion markers. Suitably fewer than 30% of the genetically modified cells according to the invention may exhibit multiple exhaustion markers. Suitably fewer than 25% of the genetically modified cells according to the invention may exhibit multiple exhaustion markers. Suitably fewer than 20% of the genetically modified cells according to the invention may exhibit multiple exhaustion markers. Suitably fewer than 15% of the genetically modified cells according to the invention may exhibit multiple exhaustion markers.

[0182] Suitably the multiple exhaustion markers may be selected from increased expression (e.g. high levels) of PD1, Tim3, Lag3, CD43 (1B11), CD69 and inhibitory receptors and decreased expression of (e.g. low levels) of CD62L and CD127 and decreased (e.g. low) interleukin-2 (IL-2), TNF-a and IFN-y production. Suitably the multiple exhaustion markers may be selected from increased expression of (e.g. high levels) of Lag3, PD1 and Tim3.

[0183] In one aspect, the population of genetically modified cells according to the present invention or obtainable (or obtained) by a method according to the present invention have increased levels of CAR transduction efficiency.

[0184] METHOD

[0185] There is provided a method of preparing a population of genetically modified cells which comprise a chimeric antigen receptor (CAR).

[0186] Methods of preparing a population of genetically modified cells for cellular therapy are known generally in the art. Methods of preparing genetically modified cells for cellular therapy may include some or all of the following steps:

[0187] The starting material may initially be frozen e.g. the starting population of cells may be frozen once it is obtained from the donor e.g. source of cells. If frozen material is used then thawing and an optional rest period may occur before proceeding to the next step. Alternatively, fresh starting material may be used. The starting material may undergo initial purification / enrichment for white cells (e.g., Ficoll gradient) of for T cells.

[0188] The starting material may then be activated e.g., the T cells may be activated. This may be performed by any methods known in the art e.g. using soluble CD3 / CD28 antibodies, or CD3 / CD28 beads (e.g. Dynabeads), or CD3 / 28 nanomatrix (e.g TransAct). As is understood in the art, the length of the activation step may be varied e.g. from under an hour to beyond 72 hours before proceeding to the next step.

[0189] The activated cells may then be transduced with the viral vector (e.g. retroviral or lentiviral). This may be done in the presence of a transduction enhancer (e.g. retronectin, or polybrene), or by spinoculation or by simple incubation. Non-viral vectors may also be used for the genetic modification step (e.g. using RNA electroporation or transposition using DNA).

[0190] The cells may then undergo an expansion step that may last from hours to several days, depending on the final dose of cells required. Generally the more cells required, the longer the expansion step.

[0191] The cells at the end of the manufacturing process may be used fresh, or preferably may be frozen before use. The overall process may therefore take from 2 to 18 days. Typically, the overall process takes 6-10 days.

[0192] During the process the cells may be cultured in cell growth medium that may contain additional supplements. Such supplements may be human serum, fetal bovine serum, human serum albumin and / or cytokines (such as IL2, IL7 and / or IL15, IL21).

[0193] “MOI7”multiplicity of infection” as used herein indicates the number of infectious vector particles per cell used in transduction. For example, a MOI of 1 means the addition of 104infectious vector particles to 104cells. The number of infectious particles is obtained by titration of the viral vector on a permissive cell line.

[0194] Suitably, the transduced cell type may be the same cell type that has been used for the titration. In this case, a MOI of 1 should result in a mean number of vector integrations per cell of 1 as estimated by quantitative PCR or other suitable method.

[0195] Cells which express the target antigen may be "not transduced" in that such cells are undetectable, or transduced at minimal or very low levels, such as 1% or less.

[0196] As is known in the art, the use of transduction enhancers may alter the MOI required for transduction e.g. may lower the MOI required for transduction. Transduction enhancers are known in the art, such as VectoFusin or RetroNectin.

[0197] Suitably, the MOI may be chosen to achieve about 10-50% transduction. Suitably, the MOI may be chosen to achieve about 15-40% transduction. Suitably, the MOI may be chosen to achieve about 20-30% transduction.

[0198] PHARMACEUTICAL COMPOSITION

[0199] The present invention also relates to a pharmaceutical composition comprising a genetically modified cell of the invention or a population of genetically modified cells according to the invention.

[0200] In one aspect, there is provided a pharmaceutical composition which comprises a population of genetically modified cells according to the present invention or obtainable by a method according to the present invention. Suitably, the pharmaceutical composition may comprise cryopreserved genetically modified cells according to the present invention or obtainable by a method according to the present invention.

[0201] 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.

[0202] Suitably, the cells may be expanded prior to incorporation into a pharmaceutical composition. For example, the cells may be expanded after the introduction of the nucleic acid which encodes the CAR against the target antigen.

[0203] Suitably, the cells may be activated prior to the introduction of a nucleic acid which encodes a CAR. For example, the cells may be activated before the introduction of the nucleic acid which encodes the CAR against the target antigen.

[0204] The cells may be activated and / or expanded by any method known in the art, for example by treatment with an anti-CD3 monoclonal antibody or both anti-CD3 and anti-CD28 monoclonal antibodies.

[0205] Suitably, the starting population of cells may previously have been frozen. If frozen cells are used, then the cells may be thawed and optionally, may be allowed to recover in culture before being processed.

[0206] The method may additionally comprise a step of enriching the starting population for white blood cells. Any methods for isolating or enriching white blood cells known in the art may be used. For example, white blood cells or PBMCs may be obtained from whole blood by various methods e.g. density gradient separation, such as using Ficoll-Paque density gradient media; by magnetic bead separation, such as MACS Milteyni Biotec CD3, CD4 or CD8 beads; by elutriation or any other method. Separation or isolation of cells may be automated or may be performed manually.

[0207] Successful transduction or transfection of a cell with a nucleic acid encoding a CAR may be identified by methods known in the art, for example by flow cytometry. In one aspect, the population of genetically modified cells according to the present invention is the active ingredient of the pharmaceutical composition.

[0208] METHOD OF TREATMENT

[0209] The genetically modified cells of the present invention may be capable of killing target cells, such as cancer cells, virally infected cells or other damaged cells.

[0210] The genetically modified cells of the present invention may be used in therapy. The genetically modified cells of the present invention may be used for the treatment and / or prevention of disease. Suitably, a pharmaceutical composition comprising genetically modified cells according to the present invention may be used in therapy. Suitably, a pharmaceutical composition comprising genetically modified cells according to the present invention may be used for the treatment and / or prevention of disease.

[0211] It will be understood that the target antigen of the CAR will be chosen based on the required therapy. For example, if the CAR is for treating cancer, the target antigen of the CAR may be an antigen associated with cancer.

[0212] The genetically modified cells of the present invention may be used for the treatment of an infection, such as a viral infection.

[0213] The genetically modified cells of the invention may also be used for the control of pathogenic immune responses, for example in autoimmune diseases, allergies and graft-vs-host rejection.

[0214] The present invention provides a method for treating and / or preventing a disease which comprises the step of administering an genetically modified cell of the present invention to a subject.

[0215] The present invention provides a method for treating and / or preventing a disease which comprises the step of administering a pharmaceutical composition of the present invention to a subject.

[0216] The present invention also provides a genetically modified cell of the present invention for use in treating and / or preventing a disease. The present invention also provides a pharmaceutical composition of the present invention for use in treating and / or preventing a disease.

[0217] The invention also relates to the use of an genetically modified cell according to the present invention in the manufacture of a medicament for treating and / or preventing a disease.

[0218] Suitably, the present methods of treatment may relate to the administration of a pharmaceutical composition of the present invention to a subject.

[0219] Suitably, the method may additionally comprise a cell expansion step before administration to the patient e.g. the cells may be cultured before administration to the patient.

[0220] The genetically modified cells or pharmaceutical composition of the present invention may be used for the treatment and / or prevention of a cancerous disease, such as a haematological malignancy, bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), lung cancer, melanoma, pancreatic cancer, prostate cancer and thyroid cancer, cancers of the oral cavity and pharynx which includes cancer of the tongue, mouth and pharynx; cancers of the digestive system which includes oesophageal, gastric and colorectal cancers; cancers of the liver and biliary tree which includes hepatocellular carcinomas and cholangiocarcinomas; cancers of the respiratory system which includes bronchogenic cancers and cancers of the larynx; cancers of bone and joints which includes osteosarcoma; cancers of the skin which includes melanoma; breast cancer; cancers of the genital tract which include uterine, ovarian and cervical cancer in women, prostate and testicular cancer in men; cancers of the renal tract which include renal cell carcinoma and transitional cell carcinomas of the utterers or bladder; brain cancers including gliomas, glioblastoma multiforme and medullobastomas; cancers of the endocrine system including thyroid cancer, adrenal carcinoma and cancers associated with multiple endocrine neoplasm syndromes; and cancers of other and unspecified sites including neuroblastoma.

[0221] Suitably, the genetically modified cells or pharmaceutical composition of the present invention may be used for the treatment and / or prevention of a haematological malignancy. As used herein, “haematological malignancy” refers to a cancer which affects the blood and lymph system and includes leukaemia, lymphoma, myeloma and related blood disorders.

[0222] Suitably, the genetically modified cells or pharmaceutical composition of the present invention may be used for the treatment and / or prevention of a haematological malignancy.

[0223] Suitably, the genetically modified cells or pharmaceutical composition of the present invention may be used in the treatment and / or prevention of leukaemias both acute and chronic, myeloid or lymphoid including: acute lymphoblastic leukaemia (ALL), acute myeloid leukaemia (AML), acute premyelocytic leukaemia (APL), and B- or T- cell acute lympoblastic leukaemia (B-ALL or T-ALL respectively), chronic lymphocytic leukaemia (CLL), chronic myeloid leukaemia (CML), chronic myelomonocytic leukaemia (CMML), hairy cell leukaemia (HCL) and large granular lymphocytic leukaemia (LGLL); lymphomas including Hodgkin's lymphoma and non-Hodgkin lymphoma (NHL), both Low-grade NHL and High-grade NHL; myeloma (Multiple Myeloma (MM)), including: smouldering or asymptomatic myeloma and symptomatic myeloma and other conditions related to blood cancer such as monoclonal gammopathy of undetermined significance (MGUS), myelodysplastic sydromes (MDS), solitary plasmacytoma, and myeloproliferative neoplasms (MPN), including essential thrombocythaemia (ET), myelofibrosis (MF), and polycythaemia vera (PV).

[0224] Treatment with the genetically modified cells of the present invention or pharmaceutical composition according to the present invention may help prevent the escape or release of tumour cells which often occurs with standard approaches.

[0225] The term “treat / treatment / treating” refers to administering an genetically modified cell, population of genetically modified cells, or pharmaceutical composition according to the present invention 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.

[0226] Reference to “prevention” / ”preventing” (or prophylaxis) as used herein refers to delaying or preventing the onset of the symptoms of the disease. Prevention may be absolute (such that no disease occurs) or may be effective only in some individuals or for a limited amount of time.

[0227] In a preferred embodiment of the present invention, the subject of any of the methods described herein is a mammal, preferably a cat, dog, horse, donkey, sheep, pig, goat, cow, mouse, rat, rabbit or guinea pig. Preferably the subject is a human.

[0228] ADMINISTRATION

[0229] The administration of the pharmaceutical composition can be accomplished using any of a variety of routes that make the genetically modified cells comprised in the pharmaceutical composition bioavailable to the subject. For example, the composition can be administered by oral and parenteral routes, intraperitoneally, intravenously, subcutaneously, transcutaneously, intramuscularly, via local delivery for example by catheter or stent.

[0230] Suitably, the genetically modified cell according to the invention or the pharmaceutical composition according to the invention is administered intravenously.

[0231] Those skilled in the art will appreciate, for example, that route of delivery (e.g., oral vs intravenous vs subcutaneous, etc.) may impact dose amount and / or required dose amount may impact route of delivery. For example, where particularly high concentrations of an agent within a particular site or location are of interest, focused delivery may be desired and / or useful. Other factors to be considered when optimizing routes and / or dosing schedule for a given therapeutic regimen may include, for example, the disease being treated (e.g., type or stage, etc.), the clinical condition of a subject (e.g., age, overall health, etc.), the presence or absence of combination therapy, and other factors known to medical practitioners.

[0232] The dosage is such that it is sufficient to stabilise or improve symptoms of the disease.

[0233] Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient. The dosage is such that it is sufficient to reduce or deplete the number of cells expressing the target antigen. USE

[0234] The present invention also provides a pharmaceutical composition or population of genetically modified cells according to the invention for use in treating disease. The pharmaceutical composition or population of genetically modified cells may be any as defined above.

[0235] The present invention also relates to the use of a population of genetically modified cells of the present invention as defined above in the manufacture of a medicament for the treatment of a disease.

[0236] 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.

[0237] EPITOPE TAG

[0238] The present invention also provides a polypeptide comprising: a. an epitope tag; b. a spacer; and c. a transmembrane domain; wherein the spacer comprises SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11 , or a variant thereof having 1-5 amino acid modifications.

[0239] The polypeptides provided in accordance with this aspect of the invention are useful for cell production, for example as purification or selection markers.

[0240] The transmembrane domain may comprise SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , or SEQ ID NO: 22, or a variant thereof having 1-5 amino acid modifications. Alternatively, the transmembrane domain may comprise SEQ ID NO: 34, or a variant thereof having 1-5 amino acid modifications. The epitope tag may be any suitable amino acid sequence that functions as an epitope. The epitope tag may be selected from the group comprising HA tag, V5 tag, and streptavidin.

[0241] The HA tag is derived from the Human influenza hemagglutinin molecule, corresponding to amino acids 98-106.

[0242] >HA tag ( SEQ ID NO : 37 ) YPYDVPDYA

[0243] The V5 tag is derived from the P and V protein of the simian virus 5 (SV5, a paramyxovirus), corresponding to amino acid residues 95 to 108 of RNA polymerase alpha subunit.

[0244] >V5 tag ( SEQ ID NO : 38 ) GKPIPNPLLGLDST

[0245] Epitope tags derived from streptavidin are described in WO2016030690, the contents of which are incorporated herein by reference.

[0246] EXAMPLES

[0247] Example 1 - Candidate Selection

[0248] To create novel spacer domains, 93 cluster of differentiation (CD) proteins - cell surface proteins used to determine cell type - with a type I transmembrane domain were investigated (Figure 2). The amino acid sequences of the CDs were considered when making the spacer domains and any signalling domains were avoided during the truncation process. Each cut was made between structural domains to increase the likelihood of obtaining a correctly folded subdomain that would not misfold and aggregate.

[0249] Once the sequences of the truncated proteins had been isolated, plasmid constructs were designed. The widely adopted haemagglutinin (HA) epitope derived from influenza virus was used for detection of the novel spacers at the cell surface. To confirm successful transfection, an enhanced green fluorescence protein marker (eGFP) was placed downstream of the spacer. This placement was chosen deliberately because cells that transcribe and translate the construct will fluoresce and be detected regardless of whether the HA-tagged spacer is expressed on the cell surface or not. A T2A self-cleaving polypeptide sequence facilitates translation of the two separate polypeptide sequences - eGFP and the HA-tagged novel spacer.

[0250] In each case, the endogenous transmembrane domain of the protein was included. This was done to maintain maximal stability of each protein during the initial screen, which was deemed a more efficient way to eliminate proteins that would not be successful spacers.

[0251] To test the spacers 293T cells were transiently transfected with the constructs and compared to a construct with the CD8a stalk inserted to act as a control. After 48 hours, the cells were stained with an APC conjugated anti-HA antibody, followed by Sytox cell viability dye to distinguish between live and dead cells. In this experiment, the eGFP produced by the cells acted as a control for successful transfection and therefore, it was clear that non-fluorescing cells had not taken up the plasmid. The median fluorescence intensity was calculated to compare the level of expression of the HA tag on the surface of cells: cells which had a higher median fluorescence intensity than the CD8a stalk were of interest.

[0252] As shown in Figure 3, 11 out of the 93 spacers had a higher median fluorescence intensity than the CD8a stalk.

[0253] Example 2 - Novel spacer screening in T cells

[0254] Next the novel spacers were tested in peripheral blood mononuclear cells (PBMCs) from four different donors. The PBMCs were stimulated with soluble anti-CD3 and anti-CD28 antibodies for 48 hours, and interleukin-2 (IL-2) for 24 hours prior to transduction. After transduction, the PBMCs were left for 72 hours to allow for proviral integration and expression of the transgenes. The cells were then stained with an anti-HA antibody, as well as anti-CD3 and anti-CD8 antibodies to enable identification of T cell sub-populations, in preparation for flow cytometry analysis. Gating was carried out on CD8+or CD8' single, viable, CD3+T cells.

[0255] Analysis of the transduced PBMCs showed similar trends in spacer domain cell surface expression to those observed in 293T cells, with the highest expressing spacers in 293T cells also being the highest expressing in PBMCs. Comparison of spacer expression in CD8+and CD8' populations also demonstrated that there was little difference between the two, indicating that there was no cell type specific expression of the spacer domains and that they could be used for the cell surface expression of molecules in both cell types. This is important because although CTLs are the main T cells that target cancer antigens in the body, CD4+T cells can also be activated in response to exposure to antigen-bearing tumour cells when they are modified to express a CAR.

[0256] While there was no spacer that had a higher level of cell surface expression of HA than the CD8a stalk, at least one spacer, spacer 3, had a similar level of expression. This was observed in CD8+CTLs as well as in the CD8' T cell population. It should be noted that this was a lead candidate in transfected 293T cells, showing that expression of this spacer is tolerated in different cell types (epithelial and T cell).

[0257] Example 3 - Functional testing of novel spacers in an anti-CD19 CAR

[0258] To test the function of the 11 leading candidate spacers, they were inserted into a well characterised anti-CD19 CAR. New constructs were designed where the CD8a spacer and transmembrane domains were extracted and replaced with the novel spacer domains and their endogenous transmembrane domains (Tables 1 & 2). The RQR8 marker, described in WO2013 / 153391 , was also included to identify cells that had been successfully transduced.

[0259] After isolating the plasmid constructs, 293T cells were transfected with the new constructs as well as lentiviral packaging plasmids to produce lentiviral supernatants. After 48 hours, the supernatants were harvested and a titration with Jurkat cells was carried out to calculate the optimal infectious titre. Jurkat cells were stained with an APC conjugated anti-CD34 antibody to be able to detect successfully transduced cells that express RQR8. Cells were also stained with a PE conjugated anti-anti- CD19 CAT antibody to detect cells expressing the CAR on their surface.

[0260] After determining the optimal infectious titre, PBMCs were transduced with the correct volume of supernatant to achieve a multiplicity of infection (MOI) of 2.5. Results show that 10 out of 11 of the spacers are functional since they express RQR8 and the CAR on their cell surface. Spacer 5 did not facilitate the presentation of the antigen-binding domain of the CAR, but cell RQR8 surface expression was detected to a comparable level to the other CARs. Many of the spacers facilitate expression of the anti-CD19 scFv on the cell surface. Spacer 8 had the highest surface expression of the CAR from the novel spacer set as shown in Figure 4. The rest of the spacers had a very similar level of expression of the CAR on the cell surface.

[0261] Cytotoxicity assays were conducted four days after PBMC transduction. Raji cells were used as target cells, using effector to target (E:T) ratios of: 1 :0, 1 :4, 1 :8 and 1 :16 at two time points: 24 hours and 48 hours. After each time point, the cells were stained with PE-Cy7 conjugated anti-CD3 and PE-conjugated anti-CD22 antibodies to detect T cells and target cells respectively. The Raji target cells are both CD19 and CD22 positive; however, staining was not carried out with an anti-CD19 antibody due to blocking of the antigen by the anti-CD19 CAR.

[0262] The 24-hour time point showed limited cytolytic activity; however, after 48 hours, cytolysis of target cells was observed. The 48-hour time point data revealed that many of the CARs with the novel spacers outperformed the CD8a control CAR in terms of targeting Raji cells for destruction, especially at a ratio of 1 :4. This is shown by Figure 5.

[0263] In conclusion, novel spacer CARs were capable of cytolysing target cells suggesting that the spacer domains adopted favourable conformations to allow the CARs to engage with the target antigen and deliver a strong activation signal to the T cell.

[0264] Example 4 - Functional testing of novel spacers in an anti-CD19 CAR with a CD8a transmembrane domain

[0265] To further investigate the novel spacers, additional constructs using a typical CD8a transmembrane domain that is most frequently used in CARs were created to enable a direct comparison between the control CD19 CAR and the CARs with novel spacers.

[0266] Plasmid constructs, viral particles and transductions were carried out as before. Figure 6 shows a comparison of the surface expression of these constructs.

[0267] Cytotoxicity assays were also carried out as before, using Raji cells and the same effector to target ratios as in the previous experiment: 1 :0, 1 ;4, 1 :8 and 1 :16. Two time points (24-hour and the 48-hour) were used. Cells were stained after each time point with PE-Cy7 conjugated anti-CD3 and PE-conjugated anti-CD22 antibodies to detect T cells and target cells respectively. The results from the cytotoxicity assay showed that most of the spacers were able to facilitate much higher cytolytic activity than the anti-CD19 control CAR as shown in Figure 7.

[0268] Example 5 - Functional testing of novel spacers in an anti-CD22 CAR

[0269] A second round of testing was carried out in which the novel spacers were inserted into a well characterised anti-CD22 CAR. The CD22 molecule is bulkier than CD19 and therefore represents a very different target.

[0270] In these experiments eGFP marker was used instead of RQR8.

[0271] Constructs using the novel spacers and their endogenous transmembrane domains were produced as before. Virus production and transduction into 293T cells was carried out as before.

[0272] All the novel spacers were able to facilitate the expression of the CAR on the cell surface as shown in Figure 8.

[0273] Interestingly, spacer 3 and spacer 11 are 26 and 23 amino acids in length, respectively, suggesting that longer spacers favour higher levels of expression of the anti-CD22 scFv at the cell surface. The rest of the spacers had very similar levels of CAR expression, which was very different from the results seen with the anti-CD19 CAR.

[0274] Cytotoxicity assays were conducted using Raji cells as target cells. Effector to target ratios used were: 1 :0, 1 : 1 , 1 :2 and 1 :4. Cells were stained after 48 and 72 hours with PE-Cy7 conjugated anti-CD3 and PE-conjugated anti-CD19 antibodies to detect T cells and target cells, respectively. An anti-CD22 antibody was not used for staining to avoid steric hinderance with the CD22 CAR.

[0275] As shown in Figure 9, all the spacers displayed some cytolytic activity when in an anti-CD22 CAR format. Example 6 - Functional screening of 96 spacers derived from type-1 transmembrane proteins

[0276] The functionality of a further 96 spacer candidates was initially tested by transducing peripheral blood mononuclear cells (PBMCs) from 3 independent donors with the aCD19_CAT CAR lentiviral vectors and staining the cells with aCD3 and aaCD19_CAT idiotype antibodies to determine if the scFv was expressed on the surface of T cells. The lentiviral vectors also contained enhanced green fluorescent protein (eGFP) as a transduction marker, which enabled normalisation of aCD19_CAT CAR expression. Transduced cells were analysed by flow cytometry and the median fluorescent intensity (MedFI) of eGFP and the aCD19_CAT CAR determined to enable normalisation of the CAR MedFI (Error! Reference source not found.0). As a positive control, an aCD19_CAT CAR with a 46 amino acid spacer derived from human CD8a (residues 137 to 182) was included (91799; SEQ ID NO: 34).

[0277] >CD8 spacer ( SEQ ID NO : 34 ) PTTTPAPRPP TPAPTIASQP LSLRPEACRP AAGGAVHTRG LDFACD

[0278] Cytotoxicity assays

[0279] Peripheral blood mononuclear cells from two independent donors were transduced with lentiviral vectors encoding the candidate spacers and three control spacers derived from CD8a, CD28 or the hinge region of human lgG1 (Table 5).

[0280] Table 5. Control spacer domains

[0281] After transduction, cells were stained with aCD3 and aaCD19_CAT idiotype antibodies and analysed by flow cytometry. Transduction efficiencies ranged from 25- 58% for T cells transduced with CAR-containing lentiviral vectors, depending on the donor. The transduction efficiency of the eGFP alone control was considerably higher and ranged from 77-81%. The CAR-T cells were normalised to a achieve a transduction efficiency of 30%, where possible, with non-transduced PBMCs and cytotoxicity assays set up using effector to target (E:T) ratios of 1 :1, 1:4, 1:8 and 1:16. Cell culture supernatant was removed from the cytotoxicity assays after 48 hours and the cells harvested for staining with aCD3 and aCD20 antibodies and analysis by flow cytometry.

[0282] Raji cells within the target cell gate were enumerated and the percentage of viable target cells calculated by normalising to the number of target cells in the nontransduced T cell sample. This accounted for non-specific lysis due to al loreactivity. The data showed that the CAR containing the spacer derived from CD166 was as effective as the control CAR, bearing the 46 aa CD8a spacer, in mediating the lysis of the Raji target cells (Figure 11).

[0283] Cytokine secretion

[0284] To determine how the spacer domains influenced cytokine secretion, cell culture supernatant was collected from the co-cultures prior to harvesting the cells and ELISA carried out to detect secreted IFNy (Figure 12) and IL-2 (Figure 13).

[0285] The data showed that T cells expressing the CAR with the CD166-derived spacer secreted more IFNy and IL-2 than the 46 aa CD8s bearing CAR T cells. These data illustrate that the spacer domain can influence cytokine secretion by T cells in response to antigenic stimulation.

[0286] The lead candidate from the screening of 96 spacer domains derived from human type-l transmembrane proteins was a 40 amino acid spacer derived from CD166. This spacer facilitated expression of the aCD19_CAT scFv at the cell surface, endowed T cells with the ability to recognise and lyse target cells and promoted a high level of inflammatory cytokine secretion (IFNy and IL-2).

[0287] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS1. A chimeric antigen receptor (CAR) comprising: a. an antigen-binding domain; b. a spacer; c. a transmembrane domain; d. an intracellular signalling domain; wherein the spacer comprises SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 or SEQ ID NO: 50, or a variant thereof having 1-5 amino acid modifications.

2. The CAR according to claim 1 , wherein the spacer comprises SEQ ID NO: 1.

3. The CAR according to claim 1, wherein the spacer comprises SEQ ID NO: 2.

4. The CAR according to claim 1, wherein the spacer comprises SEQ ID NO: 3.

5. The CAR according to claim 1, wherein the spacer comprises SEQ ID NO: 4.

6. The CAR according to claim 1, wherein the spacer comprises SEQ ID NO: 5.

7. The CAR according to claim 1, wherein the spacer comprises SEQ ID NO: 6.

8. The CAR according to claim 1, wherein the spacer comprises SEQ ID NO: 7.

9. The CAR according to claim 1, wherein the spacer comprises SEQ ID NO: 8.

10. The CAR according to claim 1, wherein the spacer comprises SEQ ID NO: 9.

11. The CAR according to claim 1 , wherein the spacer comprises SEQ I D NO: 10.

12. The CAR according to claim 1, wherein the spacer comprises SEQ ID NO: 11.

13. The CAR according to any one of claims 1 to 12, wherein the transmembrane domain comprises SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, or SEQ ID NO: 34, or a variant thereof having 1-5 amino acid modifications.

14. The CAR according to any one of claims 1 to 13, wherein the antigen binding domain binds to an antigen selected from the group comprising CD19, CD20, CD22, TRBC1, TRBC2, GD2, PSMA, CD33, CD123, CLL1 , FLT1, and Claudin.

15. The CAR according to any one of claims 1 to 14, wherein the antigen binding domain comprises a single chain variable fragment (scFv), Fab fragment, domain antibody (dAb), or variable new antigen receptor (VNAR) domain.

16. The CAR according to any one of claims 1 to 15, wherein the intracellular signalling domain comprises CD3 endodomain.

17. The CAR according to claim 16, wherein the intracellular signalling domain further comprises a co-stimulatory domain selected from the list comprising CD28 endodomain, 4-1 BB endodomain, and 0X40 endodomain.

18. A nucleic acid construct encoding a CAR according to any one of claims 1 to 17.

19. A vector comprising a nucleic acid construct according to claim 18.

20. A cell comprising a CAR according to any one of claims 1 to 17.

21. The cell of claim 20, wherein the cell is a T cell or NK cell.

22. A method for making a cell according to claim 20 which comprises the step of transducing or transfecting a cell with a nucleic acid construct according to claim 18.

23. A pharmaceutical composition which comprises a plurality of cells according to claim 20, together with a pharmaceutically acceptable carrier, diluent or excipient.

24. A method for treating cancer which comprises the step of administering a pharmaceutical composition according to claim 23 to a subject.

25. A pharmaceutical composition according to claim 23 for use in treating cancer.

26. The use of a cell according to claim 20 in the manufacture of a pharmaceutical composition for treating cancer.

27. A polypeptide comprising: a. an epitope tag; b. a spacer; and c. a transmembrane domain; wherein the spacer comprises SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , or SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 or SEQ ID NO: 50, or a variant thereof having 1-5 amino acid modifications.

28. The polypeptide according to claim 27, wherein the transmembrane domain comprises SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, or SEQ ID NO: 34, or a variant thereof having 1-5 amino acid modifications.

29. The polypeptide according to any one of claims 27 to 28, wherein the epitope tag is selected from the group comprising HA tag, V5 tag, and streptavidin.

Images