Chimeric antigen receptor

Abstract

The present invention relates to CARs that are specific for CD45, and cells comprising said CARs and their use in the treatment of disease, for example for myeloablation or for treating haematological cancer.

Description

[0001] CHIMERIC ANTIGEN RECEPTOR

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to CARs that are specific for CD45, cells comprising said CARs and their use in the treatment of disease, for example for myeloablation or for treating haematological cancer.

[0004] BACKGROUND TO THE INVENTION

[0005] Haemopoietic stem cell transplantation (SCT) is curative for patients with a wide range of malignant (including acute myeloid and acute lymphoblastic leukaemia, Non-Hodgkin’s lymphoma, myeloma) and non-malignant (e.g. haemoglobinopathies and bone marrow failure) haematological disorders as well as genetic diseases (e.g. primary immunodeficiency [PID] and metabolic diseases). For patients with genetic disorders of the haemopoietic system in whom no H LA-matched donor is available, gene therapy with autologous haemopoietic stem cells (HSCs) virally transduced with a corrected transgene are increasingly used (Kohn, D.B. Hematol Oncol Clin North Am. 2017 31(5):721-735; Frangoul H., Ho T.W. and Corbacioglu S. N Engl J Med. 2021 384(23):e91 ; Locatelli F., et al. N Engl J Med. 2022 386(5):415-427; Kohn D.B. et al. N Engl J Med. 384(21):2002-2013; Kohn D.B. et al. Nature medicine 2020 26(2):200-206).

[0006] However, for both SCT and gene therapy, intensive conditioning with chemo / radiotherapy has previously been necessary to achieve the myeloablation required to eradicate the patient’s own HSCs and create a niche for the incoming graft. In addition, in SCT immunosuppression is also used to prevent rejection and enable engraftment of donor HSCs. These conditioning regimens are often associated with severe organ toxicities such as veno-occlusive disease of the liver, gut mucositis and pneumonitis resulting in significant morbidity and mortality. Likewise, late adverse effects attributable to chemo / radiotherapy such as growth retardation, infertility, cardiotoxicity and secondary malignancy are common. These toxicities arise because conventional chemo / radiotherapy targets any dividing cell. There is a clear unmet need for conditioning that specifically targets HSCs without damaging other tissues.

[0007] Accordingly, there is a need for an efficacious, non-toxic conditioning approach, to enable transplant or gene therapy that would otherwise not have been carried out using existing chemo / radiotherapy-based conditioning methods, and also in blood cancers where chemotherapy is currently a primary treatment approach. Traditional conditioning regimes and chemotherapies are non-selective; they preferentially target dividing cells over non-dividing cells, but still affect many other cells in the body resulting in severe systemic toxicity. CAR-based technologies may be useful in selectively targeting and eliminating existing HSCs, with fewer side effects. Since CAR-Ts can be engineered to very selectively target antigens (proteins or other molecules), which are expressed only the surface of bone marrow stem cells and not on other cell types, they may be an effective means of stem cell ablation. CAR-Ts can perform targeted cell killing, for example, through cell- mediated cytotoxicity.

[0008] CD45 is a pan-haematopoietic specific antigen that is expressed on the surface of most normal haematopoietic cells, including HSCs as well as most malignant haematopoietic cells. CD45CAR T cells have potential for use as non-genotoxic conditioning agents for SCT and gene therapy as well as anti-tumour immunotherapy for haematological cancers. Since the CD45CAR T cells themselves naturally express CD45 on their cell surface, expression of the CD45-specific CAR results in fratricide of the CAR-T cells during manufacture.

[0009] There remains an unmet need to develop CAR-T cells specific for CD45 that are capable of being manufactured without inducing fratricide, that retain potency against CD45-expressing cells whilst being non-toxic and having minimal off-target activity.

[0010] SUMMARY OF THE INVENTION

[0011] The present inventors have engineered the antigen recognition domains of anti-CD45 antibodies to generate chimeric antigen receptors (CARs) which redirect engineered T cells to kill HSCs, progenitors and mature immune cells which express CD45, leading to myeloablation and immunosuppression. The CD45CAR T cells can be used as a targeted conditioning agent for SCT and gene therapy, enabling engraftment of allogeneic / gene- corrected HSCs in both malignant and non-malignant conditions without off-target toxicity to non-haemopoietic tissues. Moreover, since CD45 is also expressed on the surface of almost all haematological malignancies, the CD45CAR T cells could also have potent anti-tumour effects in diseases such as Acute Myeloid Leukaemia (AML), T-lineage acute lymphoblastic leukaemia / lymphoma and myeloma.

[0012] The present inventors have surprisingly found that a CAR comprising an antigen binding domain based on the variable region of the YTH24.5 antibody showed enhanced activity against CD45+target cells when compared to anti-CD45 CARs based on other CD45 binders known in the art. Accordingly, in a first aspect of the invention, there is provided a chimeric antigen receptor (CAR) comprising an antigen binding domain comprising:

[0013] (a) a heavy chain variable region that comprises:

[0014] (i) a heavy chain CDR1 comprising an amino acid sequence of DYYIH (SEQ ID NO: 1)

[0015] (ii) a heavy chain CDR2 comprising an amino acid sequence of YINPKSGFTNYNEKFRR (SEQ ID NO: 2). and

[0016] (iii) a heavy chain CDR3 comprising an amino acid sequence of RTGVIPMDA (SEQ ID NO: 3), and

[0017] (b) a light chain variable region that comprises:

[0018] (i) a light chain CDR1 comprising an amino acid sequence of RSSQSFVSSDGNTYLN (SEQ ID NO: 4),

[0019] (ii) a light chain CDR2 comprising an amino acid sequence of KVSNRLS (SEQ ID NO: 5), and

[0020] (iii) a light chain CDR3 comprising an amino acid sequence of GQASKIPLT (SEQ ID NO: 6).

[0021] In a further aspect of the invention, the CAR comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 8.

[0022] In one aspect of the invention, the antigen binding domain of the CAR is an scFv, wherein the heavy chain variable region and the light chain variable region are joined by a linker.

[0023] Suitably, the heavy chain variable region may be positioned to the N-terminal side of the linker, which is positioned to the N-terminal side of the light chain variable region.

[0024] In this regard, the present inventors surprisingly further found an advantageous arrangement of the variable heavy and variable light domains in an antigen binding domain based on the variable regions of the YTH24.5 binder. For example, a CAR comprising an antigen binding domain based on the variable region of YTH24.5 in the VH / VL orientation was highly expressed and demonstrated preferential in vitro activity.

[0025] In one aspect of the invention, the antigen binding domain of the CAR comprises an amino acid sequence of SEQ ID NO: 9.

[0026] In one aspect of the invention, the CAR comprises an intracellular costimulatory domain, wherein the intracellular costimulatory domain comprises a CD28 domain.

[0027] In such embodiments, the present inventors surprisingly found that use of certain costimulatory domains in the CAR of the present invention provided advantageous technical effects. The inventors found that use of a CD28 co-stimulatory domain limited in vivo persistence of a CAR cell, which may be beneficial in limiting CAR toxicity against an incoming graft following CAR administration.

[0028] In a further aspect of the invention, the intracellular signalling domain comprises or consists of an amino acid sequence of SEQ ID NO: 11.

[0029] In a further aspect of the invention, the intracellular signalling domain does not comprise a 4- 1 BB costimulatory domain.

[0030] In one aspect of the invention, the CAR comprises an endodomain that comprises or consists of: (i) an intracellular costimulatory domain comprising an CD28 domain, and (ii) an intracellular signalling domain comprising a CD3 domain, optionally wherein the total endodomain comprises or consists of an amino acid sequence with at least 95% identity to SEQ ID NO: 16.

[0031] In one aspect of the invention, the CAR comprises an amino acid sequence of SEQ ID NO: 12.

[0032] In a further aspect of the invention, there is provided a nucleic acid encoding a CAR of the present invention.

[0033] In a further aspect of the invention, the nucleic acid comprises a polynucleotide sequence of SEQ ID NO: 22.

[0034] In one aspect of the invention, there is provided a vector comprising a nucleic acid encoding a CAR of the present invention.

[0035] In a further aspect of the invention, the vector further comprises a nucleic acid encoding a transgene other than the CAR.

[0036] In certain aspects, the vector further comprises a nucleic acid encoding an inducible suicide gene, optionally this inducible suicide gene encodes iCaspase 9.

[0037] For example, to overcome the issue of the CD45CAR-T cells potentially showing disadvantageous activity against an incoming graft, the present inventors further incorporated an inducible suicide gene to enable deletion of the CD45CAR-T cells prior to infusion of the graft cells. This has the advantageous effect that the incoming graft cells do not need to be edited to avoid CD45CAR-T-mediated toxicity. In a further aspect of the invention, the vector further comprises one or more gene editing systems, preferably wherein the vector comprises one or more nucleic acid encoding said one or more gene editing systems.

[0038] In one aspect of the invention, there is provided a cell comprising a CAR of the invention, a nucleic acid encoding the CAR of the invention, and / or a vector comprising a nucleic acid encoding the CAR of the invention.

[0039] In a further aspect, the cell further:

[0040] (i) comprises an inducible suicide gene,

[0041] (ii) has reduced or abolished expression of CD45, optionally wherein the cell is a PTPRC knock-down or knock-out cell, and / or

[0042] (iii) has reduced or abolished expression of TCR, optionally wherein the cell is a TRAC knock-down or knock-out cell.

[0043] In some embodiments, in particular to overcome the issue of fratricide, the present inventors have engineered CD45 knockout CD45CAR-T cells using a CRISPR-Cas9 gene-editing approach. The inventors surprisingly found that a CRISPR-Cas9 knockout of CD45 did not prevent T-cell functionality in vivo, in contrast to previous studies. The inventors surprisingly found that CAR-T cells of the present invention show improved functionality over prior art CAR- T cells in the context of CD45 deficiency. In addition, the present inventors have simultaneously knocked out the T-cell receptor a constant (TRAC) gene, enabling generation of a universal CD45CAR-T product generated from healthy 3rd party T cells which can be used to treat multiple patients without causing graft-versus-host disease (GVHD), further broadening the utility of these CAR T cells.

[0044] In one aspect, there is provided a population of cells or a pharmaceutical composition comprising a cell or population of cells of the invention. The population of cells may also be referred to as a plurality of cells.

[0045] In one aspect, there is provided a CAR, a nucleic acid, a vector, a cell, a population of cells or a pharmaceutical composition of the invention for use as a medicament.

[0046] In one aspect, there is provided a CAR, a nucleic acid, a vector, a cell, a population of cells or a pharmaceutical composition of the invention for use in a method of myeloablation, optionally wherein the myeloablation is carried out to eradicate a subject’s own haematopoietic stem cells to allow engraftment of a stem cell or gene edited stem cell graft. In one aspect, there is provided a CAR, a nucleic acid, a vector, a cell, a population of cells or a pharmaceutical composition of the invention for use in a method of treating cancer, wherein the cancer is a haematological malignancy.

[0047] In one aspect, there is provided a CAR, a nucleic acid, a vector, a cell, a population of cells or a pharmaceutical composition of the invention for use in a method of treating a non-malignant disease or condition of haematopoietic cells, wherein the CAR, a nucleic acid, a vector, a cell, a population of cells or a pharmaceutical composition of the invention results in lymphodepletion of the affected haematopoietic cells, optionally wherein the method comprises a subsequent administration of a stem cell graft derived from a healthy donor and / or a gene edited stem cell graft.

[0048] DESCRIPITON OF THE FIGURES

[0049] Figure 1 - Human CD45 expression is limited to hematopoietic tissue and is expressed at highly levels on human CD34+haemopoietic stem and progenitor cells.

[0050] (A) Northern blot analysis of CD45 RNA expression in various human tissues. Low levels of CD45 RNA in heart and liver is due to the presence of contaminating hematopoietic cells. PBMN, peripheral blood mononuclear cells. (B) Western blot using YTH24.5 MAb showing restricted expression of human CD45 to hematopoietic tissues. Of the 19 tissues analyzed using the YTH24.5 MAb, the strongest CD45 expression was detected in the spleen with a weaker signal in the thymus and kidney. No CD45 was detected in the remaining non- hematopoietic tissues analyzed. (C) Mean CD45 antigen density as assessed on different fractions of hematopoietic cells from healthy donors (n=3) and PBMCs (n=1) used as a control. HSCs were defined as CD34+ / CD387CD45RA7CD90+, multi-lymphoid progenitors (MLPs) as CD34+ / CD387CD45RA+ / CD90' and multi-potent progenitors (MPPs) as CD34+ / CD38_ / CD45RA7CD90-.

[0051] Figure 2 - CD45 is expressed on leukaemic blasts and LSCs from almost all patients with poor risk AML whereas expression of other CAR T cell targets is variable and CD45 is highly expressed on leukaemia stem cells in a high risk paediatric AML sample

[0052] Comparison of CD45, CD33, CD123 and CLL-01 expression on leukaemic blasts and LSCs from a cohort of poor risk adult AML diagnostic blood / BM samples using CyTOF analysis. Live nucleated singlets were gated for bulk blasts (CD34+ / CD117+ / CD45int, left panel), LSC-like blasts (CD34+CD38-, right panel) and then analysed for CD45, CD33, CD123 and CLL1 expression. Comparison of CD45, CD33, CD123 and CLL-01 expression on leukaemic stem cells (LSCs) from 15 high risk paediatric patients determined using single cell CITEseq.

[0053] Figure 3 - Generation of CD45CAR T cells with knockout of CD45 with / without knockout of TRAC.

[0054] General manufacture protocol for the generation of gene-edited CD45 CAR T cells used in functional assays. Peripheral blood mononuclear cells were isolated from health donor PBMCs by Ficoll density gradient centrifugation and T cells were activated with CD3 / CD28 Beads or TransAct™. CRISPR-mediated knockout of CD45 ± TRAC was performed on day 2 and transduction of the CD45 CAR carried out on the following day. Cells were cultured with IL-2 from day 2 (between 40 U / rnL to 100 U / rnL) until functional assays were performed or gradually weaned off of IL-2 until before functional assays were performed. Flow cytometry and functional assays were performed after day 6 or until the percentage CD45+cells remaining in the cultures was <1 %.

[0055] Figure 4 - CD45 CAR1 is optimal for transduction and expression.

[0056] (A) Four CD45 CARs were generated which consisted of scFvs from anti-CD45 YTH24.5 and YTH54.12, CD8 transmembrane (TM) and CD8 stalk and costimulatory domains from CD28 and CD3zeta. The CARs were cloned into a lentiviral vector. (B) Primary human T cells that were unedited or gene-edited to knock out CD45 were transduced with the four CAR constructs. Expression of the ~60 KDa CAR was determined by Western blotting using an anti-CD3zeta antibody at 5 days post-transduction. Endogenous CD3z is 15 KDa (not shown). Loss of CD45 expression was also confirmed Western blotting with an anti-CD45 antibody. (C) Cell surface expression of the CD45 CARs was confirmed by flow cytometry using protein L to detect the YTH24.5 and YTH54.12 scFvs at 4 days post-transduction. A control CD19 CAR was also generated and detected using and anti-idiotype antibody.

[0057] Figure 5 - Enrichment of CD45~ CAR T cells through fratricide in bulk transduced CD45KO CAR T cells.

[0058] Expression of CD45 by flow cytometry at the indicated number of days post activation. Primary T cells were activated with CD3 / CD28 beads (day 0), CRISPR edited to KO CD45 and TRAC (day 2) and then transduced with lentivirus for either CD45 CAR1 (MOI 3) or CD19 CAR (MOI 2) on day 3. Unedited cells with or with CAR were also generated and were used to determine the CD45+gate in flow analysis. Figure 6 - Enrichment of CD45 / TRAC DKO cells after CRISPR-mediated knockout of CD45 and TRAC.

[0059] (A and B). Inference of CRISPR Edits (ICE) analysis. Indel plot displays distribution of INDELs with most in a window -30 to +5 from the cutting site. (C) Flow cytometry was performed at day 10 of manufacture to assess CAR expression using protein L for CD45 CAR1 , anti-CD45 (H130) for CD45 and an anti-TCR a / palpha antibody for TCR expression.

[0060] Figure 7 - Antigen density on human leukaemia cell lines and non-haemopoietic cell lines.

[0061] AML cell lines (MOLM14, MOLM14 (luc / GFP) OCIM1 , HL-60 and MV4-11), T-ALL cell lines (Jurkat, J45.01 , SupT1 , SupT1 CD19 and SupT1 CD45 KO), B-ALL (Nalm6) and non- haemopoietic HEK293T cells were stained with anti-CD45 (HI30)-PE and the antigen densities were determined using BD Quantibrite PE following manufacturer’s instructions.

[0062] Figure 8 - CD45 / TRAC DKO-CD45CAR T cells show potent cytotoxicity against haemopoietic but not non-haemopoietic targets.

[0063] (A) Unedited / edited CD45CAR or CD19CAR T cells were co-cultured for 6 hours with51Cr labelled SLIPT1 target cells which expressed CD45 alone, CD45 and CD19 or neither. Unedited / DKO T-cells which expressed no CAR were used as controls. (B) Unedited / edited CD45CAR or CD19CAR T-cells were co-cultured for 72 hours with MOLM14, Raji (B-ALL) or HEK293T cells and % viable targets determined flow cytometrically.

[0064] Figure 9 - DKO CD451 CAR T cells proliferate specifically in response to CD45+ cell lines only.

[0065] (A) DKO UT (B) CDKO CD45 CAR1 and (C) DKO CD19 CAR T cells were co-cultured with target cells at E:T of 1 :1 for 72h hours. Cells were pulsed with3H-thymidine for the last 18 hours before harvesting. PHA, phytohaemagglutinin. (D) to (F) Supernatants from the wells in panels A-C were harvested at 48h and the IFN-gamma levels were determined by ELISA. Data is mean of triplicate wells ±s.d.

[0066] Figure 10 - CD45CAR1 shows enhanced functionality compared to BC8-based CAR T cells.

[0067] (A) DKO T cells were transduced with lentiviral vectors encoding CD45CAR1 , TanCARI , Binder A-based CARs, BC8-based CD45CARs, CAR1 T2A iCasp9 or a CD19 CAR. CD45 and CAR expression on CAR T cells were determined by flow at day 10 of manufacture. (B) CAR T cells were co-cultured with cell lines expressing high level (Molm14 and Jurkat) or low levels (J45.01) of CD45 in a flow based cytotoxicity assay. Flow cytometry was performed at 72 hours and the absolute number of target cells was determined using Precision Count Beads™. CD19 CAR T cells were used as a control. Data is mean ± s.d. for triplicate wells. The number of effector cells were adjusted with DKO / LIT (MACS sorted to remove residual CD45+cells) cells to achieve equivalent transduction efficiencies of -25%. (C)3H-thymidine incorporation assays were carried out with CAR T cells co-cultured with irradiated target cells at 1 :1 E / T ratio for 72 hours. P / I=phorbol 12-myristate 13-acetate / ionomycin. Data is mean ± s.d. for triplicate wells. (D) Cytometric bead assays were performed using supernatant taken at 48h co-culture from the same wells as for panel C to determine levels of IFN-y and IL-2. Data is mean ± s.d. for triplicate wells.

[0068] Figure 11 - DKO CD45CAR T-cells prevent engraftment in NSG mice.

[0069] (A) Clonogenic assays were performed after CD34+ cells from PBSCs were co-cultured with effector cells for 72h and scored after 14 days. Data are mean colony numbers ± s.d. from triplicate wells using two health donors. (B) Schematic indicating experimental set up. 0.5x106human CD34+cells from healthy donor mobilised peripheral blood were injected into sublethally irradiated (2.5Gy) NSG mice and CAR T-cells injected on the following day. Mice were culled at 5 weeks post-transplant of CD34+cells. (C) Engraftment of human CD45+cells in the bone marrow (BM). (D) Absolute numbers of human HSCs in the BM. Statistical significance was determined using unpaired Students t-test. * = p <0.05, ** = p <0.01 , **** = p <0.0001.

[0070] Figure 12 - DKO CD45CAR T cells prevent development of AML and improve survival in MOLM14 AML xenograft mice.

[0071] (A) Schematic indicating experimental setup. (B) Bioluminescence images of the indicated experimental groups on day 14 after LUC GFP+ MOLM14 injection. (C) Tumour burden of indicated groups measured with bioluminescence imaging. (D) LUC GFP+ MOLM14 AML cells as a percentage of total mononuclear cells in mouse bone marrow at point of cull. Determined by flow cytometry. (E) Survival curves for experimental mice. DKO CD45CAR T cells significantly prolonged survival vs DKO untransduced T cell treated mice (**p<0.01 , Log-rank test).

[0072] Figure 13 - DKO CD45CAR T cells induce tumour regression of established AML in a MOLM14 AML xenograft mice.

[0073] A) Schematic indicating the experimental setup: Following establishment of AML tumours at D12 post inoculation with LUC GFP+ MOLM14 cells, mice were treated with either untransduced DKO T cells or 5 x 106CD45CAR1 T cells or 5 x 106CD19CAR T cells. B) Bioluminescence images of the indicated experimental groups up to day 26 after LUC GFP+ M0LM14 injection. C) Tumour burden measured with bioluminescence imaging. (D) Survival curves. Mice with tumour burden corresponding to a bioluminescence signal >2E9 p / s were culled.

[0074] Figure 14: Efficient killing of DKO T cells transduced with CD45CAR and iCaspase 9 after treatment with Rimiducid.

[0075] 2.5x105untransduced DKO T cells, DKO T cells expressing CAR1 alone or co-expressing CAR1 and iCaspase 9 were treated for 48 hours with either vehicle alone or 20 mM AP1903 (Rimiducid). The level of viable (Annexin V- / 7AAD-) CAR1+cells was determined by flow cytometry using recombinant CD45-biotin.

[0076] Figure 15: DKO CD45CAR1 T cells lead to complete regression of tumour burden in established AML xenograft mice.

[0077] (A) Xenograft experimental schema. NSG mice were injected i.v. with GFP+ / luciferase+expressing MOLM-14. Engraftment in all mice was confirmed by bioluminescence imaging and tumor burden was allowed to develop for 12 days prior to CAR T cell injection. (B) Bioluminescence images of effector treated MOLM-14 xenograft mice. (C) Average total bioluminescence for each cohort throughout the experiment. Two-way ANOVA was performed. (D) Survival curves for xenograft mice up to 40 days post MOLM-14 injection. Logrank test was performed. (E) Human CD2+T cells as a percentage of total mononuclear cells in mouse bone marrow at point of cull, as determined by flow cytometry. One-way ANOVA was performed. (F) LUC+ GFP+ MOLM-14 cells as a percentage of total mononuclear cells in mouse bone marrow at point of cull, as determined by flow cytometry. One-way ANOVA was performed. *P < 0.05, **P < 0.01.

[0078] Figure 16: DKO CD45CAR1 T cells have enhanced functionality compared to DKO BC8-based CAR T cells in xenograft and PDX models of AML in vivo.

[0079] (A) Xenograft experimental schema. NSG mice were injected i.v. with GFP+ / Luc+ expressing MOLM-14. Engraftment in all mice was confirmed by bioluminescence imaging and tumour burden was allowed to develop for 8 days prior to CAR T cell injection. (B) Bioluminescence images of effector treated MOLM-14 xenograft mice. (C) Average total bioluminescence for each cohort throughout the experiment. Two-way ANOVA was performed. (D) Survival curves for xenograft mice up to 60 days post MOLM-14 injection. Logrank test was performed. *P < 0.05, **P < 0.01 , ***P < 0.001 , ****P < 0.0001. Figure 17: DKO CD45CAR1 T cells showed significant specific cytotoxicity against adult de novo primary AML cells and CD34+ stem / progenitor cells in vitro.

[0080] (A) De novo primary AML samples from the bone marrow of patients taken at time of diagnosis were co-cultured with DKO Unt, DKO CD45CAR1 or DKO CD19CAR T cells for 48 hours at different EffectorTarget ratios. Numbers of viable AML cells were subseguently determined by flow cytometry and percentage specific killing was determined. Mean ± s.d. from pooled data from 3 patients is shown. MOLM-14 and Nalm6 cell lines were used for controls (data not shown). (B) Health donor CD34+cells are co-cultured with untransduced DKO or DKO CD45CAR1 T cells for 72 hours at an EffectorTarget ratio of 1 :1. Numbers of viable CD34+cells were determined by flow cytometry using counting beads.

[0081] Figure 18: DKO CD45CAR1 T cells showed significant anti-leukaemic efficacy in PDX model of AML in vivo.

[0082] (A) PDX experimental schematic. NSGs were injected with 1x105PDX AML cells i.v. After 7 days, mice were treated with 2x106DKO Unt, DKO CD45CAR1 , or DKO CD19CAR T cells. Mice were sacrificed after 5 weeks and flow cytometry was used to assess tumour burden.

[0083] (B) Absolute count of human CD45+hCD33+(AML) cells per leg (tibia and femur) at week 5. Mann-Whitney test was performed. *P < 0.05. Mean ± s.d. is plotted.

[0084] DETAILED DESCRIPTION OF THE INVENTION

[0085] Chimeric Antigen Receptor (CAR)

[0086] The term “chimeric antigen receptor” or “CAR” or “chimeric T cell receptor” or “artificial T cell receptors” or “chimeric immunoreceptors”, as used herein, refer to a synthetic chimeric transmembrane protein which connects an extracellular antigen-binding domain (binder) to an intracellular section (endodomain). Chimeric antigen receptors can provide both antigenbinding and T cell activating functions when expressed in T cells. Chimeric antigen receptors can be designed to be directed against a broader range of antigens compared to conventional T cells, which activation reguires antigen peptides to be presented by MHC molecules. Chimeric antigen receptors contain costimulatory signalling domains within the intracellular moiety and as such provide improved activation of T cells than endogenous TCR.

[0087] The present CAR may have an antigen-binding domain derived from single chain variable fragments (scFvs) or fragment antigen-binding regions (Fabs) derived from immunoglobulins such as antibodies, or natural or synthetic ligands that engage a receptor / binding partner. The antigen-binding domain (binder) may be connected to endodomain via a spacer domain and a transmembrane domain. The spacer domain may function to separate the antigen-binding domain from the T cell membrane to facilitate its binding activity and allow the antigen-binding domain to adopt a suitable orientation. The transmembrane domain anchors the protein in the cell membrane and connects the spacer and antigen-binding domain to the endodomain.

[0088] CARs are commonly classified into ‘generations’, by virtue of the composition of their intracellular signalling domain(s). All CARs typically comprise an extracellular antigen-binding domain, historically an scFv, joined to a membrane-anchoring transmembrane domain by a linker or spacer sequence. Whilst first generation CARs comprise a single intracellular signalling domain that is typically a single CD3 zeta chain, second and third generation CARs additionally comprise one or two further co-stimulatory domains (respectively), typically CD28, 4-1 BB, and / or OX-40. Fourth generation CARs are structurally similar to second generation CARs, however, are typically provided alongside an expression cassette (e.g., in a CAR T cell) that encodes an additional transgene such as a cytokine.

[0089] In one embodiment the CAR of the invention is a first generation CAR.

[0090] In one embodiment the CAR of the invention is a second generation CAR.

[0091] A CAR according to the present invention thus comprises a CD45 binding domain, a transmembrane domain, optionally one or more co-stimulatory domains, and an intracellular signalling domain.

[0092] The target-antigen binding domain of a CAR is commonly fused via a spacer and transmembrane domain to a signalling endodomain, wherein said signalling endodomain is capable of directly transducing an activation signal into the T cell activation signalling cascade. 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 of the present invention is able to activate the T cell it is expressed on following binding of the CAR to CD45 expressed on the surface of target cells.

[0093] The present CD45CAR cells are able to target haematopoietic cells (including HSCs) whilst sparing non-haematopoietic tissue. This specific targeting leads to depletion of HSCs from the bone marrow niche as well as deletion of mature T-, B- and NK cells leading to both myeloablation and lymphodepletion and enabling engraftment allogeneic HSCs or autologous gene-corrected cells. In patients with CD45+ malignancies, CD45CAR T cells will kill CD45+ tumour cells resulting in tumour cytoreduction as well as normal HSCs allowing a subsequent allogeneic transplant to be given. CD45 binding domain

[0094] The antigen-binding domain of the present CAR is capable of binding to CD45.

[0095] CD45 is a receptor tyrosine phosphatase that is specifically expressed by haemopoietic tissue including HSCs and progenitors, but is absent from non-hematopoietic tissues (Straathof K.C. , et al., Lancet 2009: 374: 912-20). In humans, the CD45 protein is encoded by the PTPRC gene. CD45 is involved in regulation of B- and T- cell antigen receptor signalling, and regulates lymphocyte survival, cytokine response, and TCR signalling (Tchilian EZ, Beverley PCL. Trends Immunol 2006; 27:146-53). CD45 is expressed on most normal haematopoietic cells, including haematopoietic stem cells (HSCs). The present inventors have shown that CD45 is also expressed on most malignant haematopoietic cells.

[0096] Human CD45 is a relatively large gene with 33 total exons. Human CD45 has at least 8 isoforms, which are generated by differential inclusion of exons 4, 5 and 6. Isoform CD45RO, for example, lacks exons 4-6, and is associated with Memory T cells, whereas CD45RA and CD45RB have exons 4 and 5 respectively, and are associated with Naive T cells. CD45RABC has all three exons 4-6, and is associated with B cells.

[0097] Suitably, the antigen-binding domain of the present CAR is capable of binding to any of the isoforms of CD45, for example the antigen-binding domain is capable of binding CD45RABC, CD45RAB, CD45RBC, CD45RA, CD45RB, CD45RC, CD45RAC, and / or CD45RO.

[0098] An illustrative CD45 amino acid sequence is the human CD45RABC sequence, designated by UniProt accession number P08575-3. An illustrative amino acid sequence for CD45 is shown as SEQ ID NO: 17:

[0099] CD45 (SEQ ID NO: 17)

[0100] MTMYLWLKLLAFGFAFLDTEVFVTGQSPTPSPTGLTTAKMPSVPLSSDPLPTHTTAFSPASTFEREND FSETTTSLSPDNTSTQVSPDSLDNASAFNTTGVSSVQTPHLPTHADSQTPSAGTDTQTFSGSAANAKL NPTPGSNAISDVPGERSTASTFPTDPVSPLTTTLSLAHHSSAALPARTSNTTITANTSDAYLNASETT TLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYLYNKETKLFTAKLNVNENVECGNNTCTNNEVHN LTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHDCTQVEKADTTICLKWKNIETFTCDTQNITY RFQCGNMI FDNKEIKLENLEPEHEYKCDSEILYNNHKFTNASKI IKTDFGSPGEPQII FCRSEAAHQG VITWNPPQRSFHNFTLCYIKETEKDCLNLDKNLIKYDLQNLKPYTKYVLSLHAYIIAKVQRNGSAAMC HFTTKSAPPSQVWNMTVSMTSDNSMHVKCRPPRDRNGPHERYHLEVEAGNTLVRNESHKNCDFRVKDL QYSTDYTFKAYFHNGDYPGEPFILHHSTSYNSKALIAFLAFLIIVTSIALLVVLYKIYDLHKKRSCNL DEQQELVERDDEKQLMNVEPIHADILLETYKRKIADEGRLFLAEFQSIPRVFSKFPIKEARKPFNQNK NRYVDILPYDYNRVELSEINGDAGSNYINASYIDGFKEPRKYIAAQGPRDETVDDFWRMIWEQKATVI VMVTRCEEGNRNKCAEYWPSMEEGTRAFGDVVVKINQHKRCPDYIIQKLNIVNKKEKATGREVTHIQF TSWPDHGVPEDPHLLLKLRRRVNAFSNFFSGPIVVHCSAGVGRTGTYIGIDAMLEGLEAENKVDVYGY VVKLRRQRCLMVQVEAQYILIHQALVEYNQFGETEVNLSELHPYLHNMKKRDPPSEPSPLEAEFQRLP SYRSWRTQHIGNQEENKSKNRNSNVIPYDYNRVPLKHELEMSKESEHDSDESSDDDSDSEEPSKYINA SFIMSYWKPEVMIAAQGPLKETIGDFWQMI FQRKVKVIVMLTELKHGDQEICAQYWGEGKQTYGDIEV DLKDTDKSSTYTLRVFELRHSKRKDSRTVYQYQYTNWSVEQLPAEPKELISMIQVVKQKLPQKNSSEG NKHHKSTPLLIHCRDGSQQTGI FCALLNLLESAETEEVVDIFQVVKALRKARPGMVSTFEQYQFLYDV IASTYPAQNGQVKKNNHQEDKIEFDNEVDKVKQDANCVNPLGAPEKLPEAKEQAEGSEPTSGTEGPEH SVNGPASPALNQGS

[0101] Suitably, the CD45 amino acid sequence may comprise the sequence shown as SEQ ID NO: 17 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.

[0102] Suitably, the CD45 amino acid sequence may be of a CD45 isoform. Suitably, the CD45 amino acid sequence may be an amino acid sequence of a CD45RABC, CD45RAB, CD45RBC, CD45RA, CD45RB, CD45RC, CD45RAC, or CD45RO isoform, or variants thereof having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.

[0103] The antigen-binding domains described herein are able to specifically bind to CD45.

[0104] Thus, the present invention provides a CAR comprising a CD45 binding domain that is capable of binding to CD45.

[0105] The CD45 binding domain thus comprises or consist of a polypeptide sequence derived from an antibody capable of binding CD45. Suitably, the CD45 binding domain comprises or consists of a polypeptide sequence(s) derived from YTH24.5 (i.e. comprising CDRs as shown as SEQ ID NO: 1-6).

[0106] According to the present invention the present CAR comprises a CD45 binding domain which comprises:

[0107] (a) a heavy chain variable region (VH) which comprises:

[0108] CDR1 - DYYIH (SEQ ID NO: 1)

[0109] CDR2 - YINPKSGFTNYNEKFRR (SEQ ID NO: 2)

[0110] CDR3 - RTGVIPMDA (SEQ ID NO: 3); and

[0111] (b) a light chain variable region (VL) which comprises:

[0112] CDR1 - RSSQSFVSSDGNTYLN (SEQ ID NO: 4)

[0113] CDR2 - KVSNRLS (SEQ ID NO: 5)

[0114] CDR3 - GQASKIPLT (SEQ ID NO: 6).

[0115] In one embodiment, the CD45 binding domain comprises CDRs consisting of the following sequences:

[0116] VH CDR1 - DYYIH (SEQ ID NO: 1)

[0117] VH CDR2 - YINPKSGFTNYNEKFRR (SEQ ID NO: 2)

[0118] VH CDR3 - RTGVIPMDA (SEQ ID NO: 3) VL CDR1 - RSSQSFVSSDGNTYLN (SEQ ID NO: 4)

[0119] VL CDR2 - KVSNRLS (SEQ ID NO: 5)

[0120] VL CDR3 - GQASKIPLT (SEQ ID NO: 6).

[0121] Suitably, one or more of the CDRs may comprise one, two or three amino acid mutations. The CD45 binding domain according to the invention which comprises CDRs as described herein maintains the capacity to bind CD45.

[0122] Suitably the CD45 binding domain may be based on an antibody or fragment thereof.

[0123] As used herein, “antibody” means a protein or polypeptide having an antigen binding site or antigen-binding domain which comprises at least one complementarity determining region CDR. The antibody or fragment thereof may comprise 3 CDRs and have an antigen binding site which is equivalent to that of a domain antibody (dAb). The antibody or fragment thereof may comprise 6 CDRs and have an antigen binding site which is equivalent to that of a classical antibody molecule. The remainder of the polypeptide may be any sequence which provides a suitable scaffold for the antigen binding site and displays it in an appropriate manner for it to bind the antigen. The antibody or fragment thereof may be a whole immunoglobulin molecule or a part thereof such as a Fab, F(ab)’2, Fv, single chain Fv (ScFv) fragment. The antibody or fragment thereof may be a bifunctional antibody. The antibody or fragment thereof may be non-human, chimeric, humanised or fully human. The antibody or fragment thereof may be a monoclonal antibody or a polyclonal antibody. Preferably, the antibody or fragment thereof is a ScFv.

[0124] The antigen-binding domain of the present CAR may be a single chain variable fragments (scFvs) or a fragment antigen-binding region (Fab).

[0125] The CD45 binding domain may comprise a VH region having the sequence shown as SEQ ID NO: 7 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto (CDR sequences are shown in bold and underline).

[0126] The CD45 binding domain may comprise a VL region having the sequence shown as SEQ ID NO: 8 or a variant of having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto (CDR sequences are shown in bold and underline).

[0127] The CD45 binding domain may comprise a VH region having the sequence shown as SEQ ID NO: 7 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and a VL region having the sequence shown as SEQ ID NO: 8 or a variant of having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto (CDR sequences are shown in bold and underline).

[0128] Suitably, the CD45 binding domain may comprise a VH region having the sequence shown as SEQ ID NO: 7; and a VL region having the sequence shown as SEQ ID NO: 8.

[0129] VH (SEQ ID NO: 7):

[0130] QVNLLQSGAALVKPGASVKLSCKASSYTFTDYYIHWVKQSHGKTLEWIGYINPKSGFTNYN

[0131] EKFRRKATLTVDKSTNTAYMDISRLTSEDSATYYCTRRTGVIPMDAWGQGASVTVSS

[0132] VL (SEQ ID NO: 8): DVVMTQTPVSLSVSLGGQVSISCRSSQSFVSSDGNTYLNWYLQKPGQSPQLLIYKVSNRL SGVPDRFSGSGSGTDFTLKISRVEHDDLGVYYCGQASKIPLTFGSGTKLEIK

[0133] Suitably, the VH domain and VL domain may be connected by a peptide linker. Suitable peptide linkers are known in the art. The linker may comprise the sequence: SGGGGSGGGGSGGGGS (SEQ ID NO: 13).

[0134] Preferably, the VH domain and VL domain may be connected by a peptide linker, wherein the heavy chain variable region is positioned to the N-terminal side of the linker, which is positioned to the N-terminal side of the light chain variable region.

[0135] In some embodiments, the VH domain and VL domain are connected by a linker, wherein the light chain variable region is position to the N-terminal side of the linker, which is positioned to the N-terminal side of the heavy chain variable region.

[0136] Suitably, the CD45 binding domain comprises the amino acid sequence shown as SEQ ID NO: 9 or a variant thereof having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto (CDR sequences are shown in bold and underline). A variant CD45 binding domain according to the present invention maintains the capacity to bind CD45 and provides the same CD45 binding characteristics and properties as the CD45 binding domain shown as SEQ ID NO: 9.

[0137] SEQ ID NO: 9:

[0138] QVNLLQSGAALVKPGASVKLSCKASSYTFTDYYIHWVKQSHGKTLEWIGYINPKSGFTNYN EKFRRKATLTVDKSTNTAYMDISRLTSEDSATYYCTRRTGVIPMDAWGQGASVTVSSSGG GGSGGGGSGGGGSDWMTQTPVSLSVSLGGQVSISCRSSQSFVSSDGNTYLNWYLQKP GQSPQLLIYKVSNRLSGVPDRFSGSGSGTDFTLKISRVEHDDLGVYYCGQASKIPLTFGSG TKLEIK

[0139] In an alternative embodiment, the present invention further provides any aspect as described herein in respect of the CAR comprising a CD45 binding domain derived from YTH24.5, wherein the CD45 binding domain of the CAR instead comprises a CD45 binding domain derived from a YTH54.12 antibody (i.e comprising CDR regions comprising sequences of SEQ ID NOs: 29-34).

[0140] Suitably, the YTH54.12 CD45 binding domain comprises:

[0141] (a) a heavy chain variable region (VH) which comprises:

[0142] CDR1 - DYYMA (SEQ ID NO: 29)

[0143] CDR2 - SMSFAGSSTYYGDSVKG (SEQ ID NO: 30)

[0144] CDR3 - MYTTDYYLYWYFDF (SEQ ID NO: 31); and

[0145] (b) a light chain variable region (VL) which comprises:

[0146] CDR1 - KASKSISNYLA (SEQ ID NO: 32)

[0147] CDR2 - SGSTLQS (SEQ ID NO: 33)

[0148] CDR3 - QQYDEKPLT (SEQ ID NO: 34).

[0149] In one aspect, the CD45 binding domain comprises CDRs consisting of the following sequences:

[0150] VH CDR1 - DYYMA (SEQ ID NO: 29)

[0151] VH CDR2 - SMSFAGSSTYYGDSVKG (SEQ ID NO: 30)

[0152] VH CDR3 - MYTTDYYLYWYFDF (SEQ ID NO: 31)

[0153] VL CDR1 - KASKSISNYLA (SEQ ID NO: 32)

[0154] VL CDR2 - SGSTLQS (SEQ ID NO: 33)

[0155] VL CDR3 - QQYDEKPLT (SEQ ID NO: 34).

[0156] Suitably, the CD45 binding domain may comprise a VH region with at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 35; and a VL region with at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 36.

[0157] Suitably, the CD45 binding domain may comprise a VH region shown as SEQ ID NO: 35; and a VL region shown SEQ ID NO: 36. SEQ ID NO: 35 (YTH54.12 VH region): EVQLVESGGGLVQPGGSMKLSCAASGFTFSDYYMAWVRQAPKKGLEWVASMSFAGSST YYGDSVKGRFTISRDNAKTTLYLQMNSLRSEDTATYYCARMYTTDYYLYWYFDFWGPGTM VTVSS

[0158] SEQ ID NO: 36 (YTH54.12 VL region):

[0159] DVQMTQSPSYLAASPGESVSISCKASKSISNYLAWYQQKPGEANKILIYSGSTLQSGTPS RFSGSGSGTDFSLTIRNLEPEDFAVYYCQQYDEKPLTFGSGTKLEIK

[0160] The term “polypeptide” is used in the conventional sense to mean a series of amino acids, typically L-amino acids, connected one to the other, typically by peptide bonds between the a- amino and carboxyl groups of adjacent amino acids. The term “polypeptide” is used interchangeably with the terms “amino acid sequence”, “peptide” and / or “protein”. The term “residues” is used to refer to amino acids in an amino acid sequence.

[0161] The term "variant" refers to a polypeptide that has an equivalent function to the amino acid sequences described herein, but which includes one or more amino acid substitutions, insertions or deletions.

[0162] Thus, a CD45 binding domain may comprise a variant of a CD45 binding domain sequence as described herein having a least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.

[0163] The terms “selectively binds / selectively binding” and “specifically binds / specifically binding” may be used interchangeably herein. Selectively binds / specifically binding may also be used interchangeable with the term “targeting”.

[0164] “Heavy chain variable region” or “VH” refers to the fragment of the heavy chain of an antigenbinding domain or antibody that contains three CDRs interposed between flanking stretches known as framework regions, which are more highly conserved than the CDRs and form a scaffold to support the CDRs. “Light chain variable region” or “VL” refers to the fragment of the light chain of an antigen-binding domain or antibody that contains three CDRs interposed between framework regions.

[0165] “Complementarity determining region” or “CDR” with regard to antigen-binding domain or antibody or antigen-binding fragment thereof refers to a highly variable loop in the variable region of the heavy chain of the light chain of an antibody. CDRs can interact with the antigen conformation and largely determine binding to the antigen (although some framework regions are known to be involved in binding). The heavy chain variable region and the light chain variable region each contain 3 CDRs (heavy chain CDRs 1 , 2 and 3 and light chain CDRs 1 , 2 and 3, numbered from the amino to the carboxy terminus).

[0166] It may be possible to introduce one or more mutations (substitutions, additions or deletions) into each CDR without negatively affecting CD45-binding activity. Each CDR may, for example, have one, two or three amino acid mutations. The CD45 binding domain comprising the one or more of the CDRs which comprise one, two or three amino acid mutations may suitably maintain the capacity to bind CD45.

[0167] The CDRs of the variable regions of a heavy and light chain of an antigen-binding domain or antibody can be predicted from the heavy and light chain variable region sequences of the antibody, using prediction software available in the art, e.g. using the Abysis algorithm, or using the IMGT / V-QUEST software, e.g. the IMGT algorithm (ImMunoGeneTics) which can be found at www.IMGT.org, (see for example Lefranc et al, 2009 NAR 37:D1006-D1012 and Lefranc 2003, Leukemia 17: 260-266). CDR regions identified by either algorithm are considered to be equally suitable for use in the invention. CDRs may vary in length, depending on the antigen-binding domain or antibody from which they are predicted and between the heavy and light chains. Thus, the three heavy chain CDRs of an intact antigen-binding domain or antibody may be of different lengths (or may be of the same length) and the three light chain CDRs of an intact antigen-binding domain or antibody may be of different lengths (or may be of the same length). A CDR for example, may range from 2 or 3 amino acids in length to 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. Particularly, a CDR may be from 3-14 amino acids in length, e.g. at least 3 amino acids and less than 15 amino acids.

[0168] Suitably, the CDRs of the CD45 binding domain may be identified by any suitable method known in the art, for example using any suitable antibody numbering scheme. Suitably, the CDRs are identified using the Kabat numbering system (Kabat et al., U.S. Department of Health and Human Services, 1991). Suitably, the CDRs may be identified using any other suitable antibody numbering system known in the art, for example the Chothia numbering scheme (Chothia C, Lesk AM. J Mol Biol. (1987) 196:901-17), or the IMGT numbering scheme (Giudicelli V, et al. Nucleic Acids Res. (1997) 25:206-11 ; Lefranc MP. Immunol Today (1997) 18:509). The skilled person will appreciate that these different CDR labelling systems can give slightly different results, but in each case the CDRs can be easily identified by the skilled person. The CDR sequences set out in SEQ ID NOs 1-6 are the HCDR1-3 and LCDR1-3 sequences of SEQ ID NOs 7 and 8 as defined using the Kabat numbering scheme. As used herein, “variant” is synonymous with “mutant” and refers to a polynucleotide or amino acid sequence which differs in comparison to the corresponding wild-type sequence. The term “wild-type” is used to mean a gene or protein having a polynucleotide or amino acid sequence respectively, which is identical with the native gene or protein respectively.

[0169] The sequence may have one or more deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent molecule. These sequences are encompassed by the present invention. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues as long as the activity is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0170] Methods for determining binding specificity include, but are not limited to, ELISA, western blot, immunohistochemistry, flow cytometry, Forster resonance energy transfer (FRET), phage display libraries, yeast two-hybrid screens, co-immunoprecipitation, bimolecular fluorescence complementation and tandem affinity purification. Binding affinity can also be determined using methods such as fluorescence quenching, isothermal titration calorimetry.

[0171] Specific binding to CD45 may be determined using assays which are known in the art. For example, by an ELISA or binding affinity assay using a CD45 peptide. Antigen binding domains which specifically bind CD45 may be generated by immunising a rat or a mouse, for example, using a CD45 peptide. Such a strategy will result in the generation of antibodies specific against the peptide used as the immunogen.

[0172] The capacity of the CD45 binding domain to bind to CD45 may be assessed by determining the binding affinity. A quantitative assessment or measurement of binding affinity (e.g. establishing a KD value) may be determined or measured using methods know in the art, such as by surface plasmon resonance, for example by using the Biacore® system. In addition to the equilibrium dissociation constant (KD), the association rate constant (Ka (1 / Ms)), and the dissociation rate constant (Kd (1 / s)) may also be determined.

[0173] Surface Plasmon Resonance (SPR) experiments may be performed with a Biacore T200, for example.

[0174] The variants encompassed by the invention have a binding activity that is essential unaltered or improved compared to the corresponding, unaltered polypeptide. In the context of a CD45 binding molecule described above, in order to stimulate cell activation, the CD45 binding domain may bind to its cognate antigen (CD45) with a certain binding profile (for example, with a required binding affinity). Suitably, in order to stimulate T cell activation, the CD45 binding domain may bind to its cognate antigen (CD45) with a certain binding profile (for example, with a required binding affinity).

[0175] Spacer domain

[0176] The CAR may comprise an extracellular spacer domain which connects the antigen-binding domain to the transmembrane domain. The spacer domain may also be referred to as a hinge, linker or stalk. The spacer domain may allow the antigen-binding domain to adopt orientations that allow binding to the target. Spacer domains may be of a length and / or flexibility to help improve CAR binding and signalling efficiency. Spacer domains of appropriate length may optimise the immune synapse distance, which can be important for efficiency of endodomain signalling and effectiveness of T cell cytotoxic mechanisms such as lytic granule delivery. Spacer domains with high flexibility may improve efficiency of recognition of sterically hindered targets (J. Jayaraman et al., eBiomedicine. 2020;58:102931).

[0177] The spacer sequence may, for example, comprise a CD8 stalk, a CD28 stalk, an lgG1 Fc region or an lgG1 hinge, or a combination thereof. The spacer may alternatively comprise an alternative sequence which has similar length and / or domain spacing properties as a CD8 stalk, a CD28 stalk, an lgG1 Fc region or an lgG1 hinge. The spacer sequence may, for example, comprise a CD8 ectodomain, a CD28 ectodomain, a CD2 ectodomain, CD34 ectodomain or COMP.

[0178] A human I gG 1 spacer may be altered to remove Fc binding motifs.

[0179] An exemplary amino acid sequence for the spacer of the present invention is given below:

[0180] SEQ ID NO: 14 (CD8 stalk domain)

[0181] PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0182] A variant spacer sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 14.

[0183] Without wishing to be bound by theory, the CAR of the present invention may, for example, comprise a spacer which provides an optimal immune synapse distance when expressed in a T cell and when recognising its target, and has sufficient flexibility to allow efficient binding of the binder of the CAR to bind to the target.

[0184] Transmembrane domain

[0185] The transmembrane domain is a region of the CAR that anchors the CAR to the cell membrane, and connects the extracellular antigen-binding domain, optionally via the spacer, to the endodomain.

[0186] T ransmembrane domains are protein sequences that are thermodynamically stable in the lipid bilayer of a cell membrane. They are most simply comprised of a protein sequence that crosses the cell membrane only once. Other modalities are possible, such as proteins that cross the membrane multiple times. For use in CARs, a simple transmembrane domain that crosses the cell membrane a single time is most commonly used. The most typical form is an a helix comprising several hydrophobic residues. Transmembrane domains for use in CARs can be sourced from any transmembrane protein. Suitability of a protein sequence as a transmembrane domain can be determined by the person skilled in the art using the DeepTMHM algorithm (https: / / dtu.biolib.com / DeepTMHMM) to determine the membrane spanning section of a transmembrane domain. Alternatively, as prediction of a hydrophobic a helix’s ability to form a transmembrane domain is possible due to its relatively simple structure, artificially designed transmembrane domains may be used. Such synthetic transmembrane components are described in e.g. US 7052906 B1.

[0187] Example transmembrane domains used in CARs in the art are, the CD28 TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41 ; Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426- 35; Casucci et al, Blood, 2013, Nov 14;122(20):3461-72), the 0X40 TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41), the 41 BB TM region (Brentjens et al, CCR, 2007, Sep 15; 13(18 Pt 1):5426-35), the CD3 TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41 ; Savoldo B, Blood, 2009, Jun 18;113(25):6392-402), and the CD8a TM region (Maher et al, Nat Biotechnol, 2002, Jan;20(1):70-5.; Imai C, Leukemia, 2004, Apr;18(4):676-84; Brentjens et al, CCR, 2007, Sep 15; 13(18 Pt 1):5426-35; Milone et al, Mol Ther, 2009, Aug; 17(8): 1453- 64.).

[0188] In the CAR of the present invention, the transmembrane domain may be derived from a CD8 transmembrane domain, a CD28 transmembrane domain, or any other transmembrane domain which provides good receptor stability.

[0189] The transmembrane domain may be derived from CD8, which gives good receptor stability. The transmembrane domain may be derived from any type I transmembrane protein. The transmembrane domain may be a synthetic sequence predicted to form a hydrophobic helix.

[0190] As used herein, the term "derived from" refers to the origin or source, and may include naturally occurring, recombinant, unpurified, or purified molecules. The term "derived from" encompasses the terms "originated from," "obtained from," "obtainable from," "isolated from," and "created from."

[0191] The CAR of the present invention may comprise an exemplary transmembrane domain with an amino acid sequence of SEQ ID NO: 18, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 18.

[0192] SEQ ID NO: 18 (CD8 transmembrane domain):

[0193] IYIWAPLAGTCGVLLLSLVIT

[0194] The CAR of the present invention may comprise an exemplary spacer + transmembrane domain with an amino acid sequence of SEQ ID NO: 15, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 15.

[0195] SEQ ID NO: 15 (CD8 spacer + transmembrane domain):

[0196] PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLL SLVIT

[0197] Endodomain

[0198] The endodomain or cytoplasmic domain refers to the intracellular portion of the CAR which comprises one or more intracellular signalling domains, and optionally one or more costimulatory domains that induce T cell activatory signalling upon CAR target binding. Upon CAR target binding, the intracellular domains activate downstream signalling processes.

[0199] So-called first-generation CARs use, for example, the CD3 signalling domain in the absence of any co-stimulatory domains. Upon target binding, this domain transmits an activation signal to the T cell via its three ITAM domains. The CD3 signalling domain alone may not provide a fully competent activation signal for sustained signalling and T cell persistence and proliferation. Second- and third-generation CARs add a single or multiple co-stimulatory domain(s), respectively, to provide enhanced signalling and T cell activation.

[0200] In the CAR of the present invention the endodomain may comprise a CD3 signalling domain. Further intracellular signalling domains are known in the art, for example, FcyRIII, FcsRI, the cytoplasmic tail of an Fc receptor and immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors.

[0201] The CAR of the present invention may comprise a CD3 intracellular signalling domain. An exemplary amino acid sequence of a CD3 intracellular signalling domain is given by SEQ ID NO: 11. The CAR of the present invention may comprise an intracellular signalling domain comprising an amino acid sequence of SEQ ID NO: 11 or a variant sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto, provided that the sequence provides an effective intracellular T cell signalling domain.

[0202] SEQ ID NO: 11 (CD3 intracellular signalling domain):

[0203] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0204] The endodomain of the CAR of the present invention may further comprise one or more costimulatory signalling domains. The costimulatory domain may comprise a CD28 costimulatory domain. The endodomain of the CAR of the present invention may comprise a CD3 signalling domain and a CD28 co-stimulatory domain.

[0205] Other co-stimulatory endodomains are known in the art, for example, CD28, CD137 (4-1 BB), CD134 (0X40), DapIO, CD27, CD2, CD5, ICAM-1 , LFA-1 , Lek, TNFR-I, TNFR-II, Fas, CD30 and CD40.

[0206] The endodomain of the CAR of the present invention may comprise a costimulatory domain selected from the group consisting of the endodomains of CD28, 4-1 BB, 0X40, CD27 and ICOS. The endodomain of the CAR of the present invention may comprise a CD3 signalling domain and one or more costimulatory domains selected from the group consisting of the endodomains of CD28, 4-1 BB, 0X40, CD27 and ICOS.

[0207] In some embodiments, the endodomain of the CAR of the present invention does not comprise a 4-1 BB endodomain.

[0208] The CAR of the present invention may comprise a CD28 co-stimulatory signalling domain. An exemplary amino acid sequence of a CD28 co-stimulatory signalling domain is given by SEQ ID NO: 10. The CAR of the present invention may comprise a co-stimulatory signalling domain comprising an amino acid sequence of SEQ ID NO: 10 or a variant sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto, provided that the sequence provides an effective intracellular co-stimulatory T cell signalling domain. SEQ ID NO: 10 (CD28 co-stimulatory signalling domain):

[0209] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0210] The CAR of the present invention may comprise an endodomain which consists of a CD3 intracellular signalling domain and an CD28 co-stimulatory signalling domain. For example, the present CAR may comprise an endodomain which consists of SEQ ID NO: 16. The CAR of the present invention may comprise an endodomain which consists of SEQ ID NO: 16 or a variant sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto, provided that the sequence provides an effective intracellular co-stimulatory T cell signalling domain.

[0211] CD3 intracellular signalling domain and an CD28 co-stimulatory signalling domain [SEQ ID NO: 16]

[0212] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0213] CAR structure

[0214] The skilled person would understand that linkers may be used between any of the domains of the CAR as necessary. Suitable linker sequences are known in the art.

[0215] In one embodiment, the CAR has the general structure, from N terminus to C terminus:

[0216] CD45 binding domain - optional spacer domain - transmembrane domain - endodomain.

[0217] In one embodiment, the CAR has the general structure, from N terminus to C terminus:

[0218] CD45 binding domain - optional spacer domain - transmembrane domain - one or more costimulatory domain - intracellular signalling domain.

[0219] In one embodiment, the CAR has the general structure, from N terminus to C terminus:

[0220] CD45 binding domain - CD8 stalk domain - CD8 transmembrane domain - CD28 intracellular costimulatory domain - CD3 intracellular signalling domain.

[0221] The CAR of the present invention may comprise or consist of an amino acid sequence of SEQ ID NO: 12, or a variant thereof with at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 12.

[0222] SEQ ID NO: 12 (CD45CAR): QVNLLQSGAALVKPGASVKLSCKASSYTFTDYYIHWVKQSHGKTLEWIGYINPKSGFTNYNE KFRRKATLTVDKSTNTAYMDISRLTSEDSATYYCTRRTGVIPMDAWGQGASVTVSSSGGGG SGGGGSGGGGSDVVMTQTPVSLSVSLGGQVSISCRSSQSFVSSDGNTYLNWYLQKPGQSPQL LIYKVSNRLSGVPDRFSGSGSGTDFTLKISRVEHDDLGVYYCGQASKIPLTFGSGTKLEIKSDP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLV ITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0223] A variant sequence may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 12 to 15, provided that the CAR is able to i) bind CD45, and ii) induce cytotoxic immune cell signalling.

[0224] Signal peptide

[0225] The CAR of the present invention may, when initially expressed in a cell, additionally comprise a signal peptide at the N-terminus.

[0226] Signal peptides, also known as leader peptides or signal sequences, are short peptides that are able to influence the targeting of a protein within a cell, for example targeting a protein to a secretion pathway.

[0227] The CAR of the present invention may comprise a signal peptide. The CAR of the present invention may comprise a signal peptide at its N-terminus.

[0228] The signal peptide may be derived from a naturally occurring signal peptide from a native protein. The signal peptide may be a synthetic signal peptide (Park et al., 2022, Applied Microbiology and Biotechnology 106, 3571-3582, https: / / doi.org / 10.1007 / s00253-022-11955- 6). The signal peptide may be derived from a murine or human native protein. In some embodiments, the signal peptide is derived from a murine IgGK signal sequence, or the signal peptide is derived from a human immunoglobulin heavy chain signal sequence.

[0229] The CAR of the present invention may comprise a signal peptide derived from a human gene. Suitably, the signal peptide is derived from the human IGHV signal peptide. The CAR of the present invention may comprise a signal peptide with an amino acid sequence of SEQ ID NO: 23, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0230] SEQ ID NO: 23 (human IGHV signal peptide)

[0231] MEFGLSWVFLVALLRGVQ

[0232] Suicide gene Since T cells engraft and are autonomous, a means of selectively deleting CAR T cells in recipients of CAR T cells is desirable, due to side effects or unacceptable toxicity. In particular for CD45CAR-T cells, successful myeloablation may be followed by infusion of a hematopoietic stem cell graft, cells of which express CD45 on their cell surface. To avoid killing of the incoming graft, in some embodiments of the invention, the CD45CAR-T cells comprise a suicide gene.

[0233] The earliest clinical experience with suicide genes is with the Herpes Virus Thymidine Kinase (HSV-TK) which renders T cells susceptible to Ganciclovir. HSV-TK is a highly effective suicide gene. However, pre-formed immune responses may restrict its use to clinical settings of considerable immunosuppression such as haploidentical stem cell transplantation. Inducible Caspase 9 (iCasp9) is a suicide gene constructed by replacing the activating domain of Caspase 9 with a modified FKBP12. iCasp9 is activated by an otherwise inert small molecular chemical inducer of dimerization (CID), such as Rimiducid. iCasp9 has been recently tested in the setting of haploidentical HSCT and can abort GvHD. Both iCasp9 and HSV-TK are intracellular proteins, so when used as the sole transgene, they have been coexpressed with a marker gene to allow selection of transduced cells.

[0234] The suicide gene may be an inducible suicide gene, such as an iCaspase 9 gene, thymidine kinase, cytosine deaminase (CD), or cytochrome P450. Other inducible suicide switches are known in the art. WO2016 / 135470 describes a suicide gene which also comprises Caspase 9 but can be induced to dimerise using rapamycin or a rapamycin analog, termed Rapcasp9 or Rapacasp9. WO2013 / 153391 describes a marker / suicide gene known as RQR8 which can be detected with the antibody QBEndlO and expressing cells lysed with the therapeutic antibody Rituximab.

[0235] Without being bound by theory, cells expressing constructs comprising iCaspase 9 are able to be eliminated by activation of the caspase 3 apoptotic pathway when a small molecule is administered. In certain embodiments, the suicide gene is iCaspase 9, and the small molecule is Rimiducid. Rimiducid is a lipid-permeable tacrolimus analogue and a protein dimerizer. Elimination of cells expressing such an iCaspase 9 can be accomplished by provision of Rimiducid to the cell.

[0236] Suitably, a CAR of the present invention may be expressed with a suicide gene. The suicide gene may be selected from any one of iCasp9, thymidine kinase, cytosine deaminase (CD), cytochrome P450, Rapcasp9, or RQR8.

[0237] Suitably, the suicide gene may be iCasp9. An iCasp9 may comprise the sequence shown as SEQ ID NO: 21 or a variant thereof having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity.

[0238] SEQ ID NO: 21 :

[0239] MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQ MSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGFGDVGALESLRGNADLA YILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELA RQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNI FNGTSCPSLGGKPKLFFIQACG GEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRD PKSGSWYVETLDDI FEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSVDYPYDVP DYALD

[0240] Suitably, the suicide gene is expressed from a separate expression construct to the present CAR. Suitably, the suicide gene is expressed from the genome of a cell comprising the CAR of the present invention. Suitably, the suicide gene is expressed from the same expression construct as the CAR, for example by using a self-cleaving peptide between the sequences, or a polycistronic sequence. Suitably, transcription of the expression construct produces a polypeptide which comprises the CAR and a suicide gene joined by a cleavage site. The cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into the CAR and the suicide gene without the need for any external cleavage activity.

[0241] Various self-cleaving sites are known, including the Foot-and-Mouth disease virus (FMDV) 2A peptide and similar sequence (Donnelly et al, Journal of General Virology (2001), 82, 1027- 1041), or the 2A-like sequence from Thosea asigna virus. Suitably, the nucleic acid of the vector of the present invention comprises a T2A peptide sequence of SEQ ID NO: 27, or a variant sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the peptide sequence retains self-cleaving activity.

[0242] SEQ ID NO: 27 (T2A peptide sequence)

[0243] GSGEGRGSLLTCGDVEENPGP

[0244] Suitably, the expression construct comprising the CAR and a suicide gene is arranged wherein the CAR is to the N-terminus of the suicide gene (for example iCaspase 9) and the cleavage site (for example a self-cleaving T2A cleavage site) is between them. In alternative configurations, the suicide gene (for example iCaspase 9) is to the N-terminus of the CAR, with the cleavage site (for example a self-cleaving T2A site) between them.

[0245] Gene editing system Gene editing systems are known in the art. A gene editing system may be a protein, or combination of a protein and nucleic acid. Common gene editing systems include, CRISPR / Cas systems, TALENs, and ZFNs. CRISPR / Cas systems include one or more nucleic acid molecules that guide a protein to a target sequence to enable gene editing. The one or more nucleic acid molecules may be a crRNA and tracrRNA, or a gRNA. The protein may be a nuclease of the Cas family, for example Cas9. The protein may also be a nuclease- dead Cas enzyme, or a nickase.

[0246] Gene editing systems may be used in the present invention to create a knock-out cell. The gene editing system may be used to introduce a transgene to the genome of a cell. Suitably, one or more gene editing systems may be used to knock-out one or more genes and introduce one or more transgenes into a cell.

[0247] Suitably, the gene editing system may be used for reducing or eliminating expression of endogenous CD45 in a cell. In one embodiment, the gene editing system may be used for knocking out, knocking down or mutating an endogenous gene encoding for CD45 in a cell. Suitably, the gene editing system may be used for knocking out, knocking down or mutating a PTPRC gene.

[0248] Suitably, the gene editing system for reducing or eliminating expression of endogenous CD45 in a cell is a CRISPR / Cas9 gene editing system. Suitably, the gene editing system comprises a Cas9 enzyme and one or more gRNAs. Optionally, the gene editing system further comprises a donor nucleic acid. Suitably, the gene editing system comprises one or more gRNAs that enable targeting of the PTPRC gene by the Cas9 enzyme. Suitable techniques and gene editing systems for this purpose are described in Chen, et al. 2018 (Chen et al. J Immunol 2018 201 (5): 1586-1598).

[0249] Suitably, a gRNA shown as SEQ ID NO: 19 may be used to knock out CD45 in a cell, such as a cell according to the present invention.

[0250] GUAUUUGUGGCUUAAACUCU (SEQ ID NO: 19)

[0251] The present invention also provides a method of reducing or eliminating expression of endogenous CD45 in a cell using a CRISPR / Cas9 gene editing system comprising a gRNA according to SEQ ID NO: 19.

[0252] Suitably, the gene editing system may be used for reducing or eliminating expression of an endogenous TCR in a cell. In one embodiment, the gene editing system may be used for knocking out, knocking down or mutating an endogenous gene encoding for T cell receptor alpha constant (TRAC) in a cell. Suitably, the gene editing system may be used for knocking out, knocking down, or mutating a TRAC gene in a cell.

[0253] The present invention also provides a method of reducing or eliminating expression of endogenous TRAC in a cell using a CRISPR / Cas9 gene editing system comprising a gRNA according to SEQ ID NO: 20.

[0254] Suitably, the gene editing system for reducing or eliminating expression of endogenous TCR in a cell is a CRISPR / Cas9 gene editing system. Suitably, the gene editing system comprises a Cas9 enzyme and one or more gRNAs. Optionally, the gene editing system further comprises a donor nucleic acid. Suitably, the gene editing system comprises one or more gRNAs that enable targeting of the TRAC gene by the Cas9 enzyme. Suitable techniques and gene editing systems for this purpose are described in Georgiadis, et al. 2018 (Georgiadis et al. Mol Ther. 26(5): 1215-1227).

[0255] Suitably, a gRNA shown as SEQ ID NO: 20 may be used to knock out TRAC in a cell, such as a cell according to the present invention.

[0256] UCUCUCAGCUGGUACACGGC (SEQ ID NO: 20)

[0257] Suitably, the gene editing system may be used for introducing a transgene into a cell. In one embodiment, the gene editing system may be used to introduce an inducible suicide gene into a cell. In one embodiment, the suicide gene is an iCaspase 9 gene.

[0258] Suitably, the gene editing system for introducing the inducible suicide gene into a cell is a CRISPR / Cas9 gene editing system. Suitably, the gene editing system comprises a Cas9 enzyme, one or more gRNAs and a donor nucleic acid. Suitably, the donor nucleic acid comprises a nucleic acid encoding the inducible suicide gene. Suitably, the donor nucleic acid comprises nucleotide sequences with homology to flanking regions of the target insertion site. Suitably, the gene editing system comprises one or more gRNAs that enable targeting of a specific genomic locus by the Cas9 enzyme.

[0259] In some embodiments, a gene editing system may be used for reducing or eliminating expression of a target gene and simultaneously introducing a transgene into a cell. Suitably, a gene editing system may be used for knocking out a PTPRC and / or TRAC gene and introducing a nucleic acid encoding an inducible suicide gene. Suitably, the gene editing system comprises one or more gRNAs that enable targeting of the PTPRC or TRAC locus, and a donor nucleic acid comprising a nucleic acid encoding an iCaspase 9. In some embodiments, a gene editing system is delivered to a cell using a vector. In some embodiments, a gene editing system is delivered to a cell using electroporation. In some embodiments, the gene editing system is a CRISPR / Cas9 gene editing system, and the gRNA and Cas9 are delivered to a cell using electroporation. Electroporation methods are known in the art, for example the MaxCyte® electroporation system.

[0260] Nucleic acid

[0261] The present invention further provides nucleic acid sequences encoding a CAR as defined above, and fragments or variants thereof.

[0262] The nucleic acid sequence may be an RNA or DNA sequence or a variant thereof. The term "polynucleotide" includes an RNA or DNA sequence. It may be single or double stranded. It may, for example, be genomic, recombinant, mRNA or cDNA.

[0263] The nucleic acid encoding the CAR may comprise a polynucleotide sequence of SEQ ID NO: 22, or a variant thereof with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a polynucleotide sequence of SEQ ID NO: 22.

[0264] The nucleic acid encoding the CAR may consist of a polynucleotide sequence of SEQ ID NO: 22, or a variant thereof with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a polynucleotide sequence of SEQ ID NO: 22.

[0265] SEQ ID NO: 22 (YTH24.5 CD45 CAR1 sequence)

[0266] CAAGTTAATTTGTTGCAATCTGGAGCCGCACTTGTTAAACCGGGAGCTTCTGTCAAGTT GAGTTGTAAAGCAAGCTCCTACACATTTACCGATTATTACATACACTGGGTTAAGCAGTC TCACGGCAAAACTTTGGAGTGGATCGGATATATAAACCCCAAATCAGGCTTTACGAACT ACAACGAGAAATTCCGAAGGAAGGCAACTTTGACGGTCGATAAATCCACTAATACGGCC TACATGGACATAAGTAGATTGACATCTGAGGACAGTGCAACGTATTACTGCACTCGCCG AACCGGGGTTATTCCGATGGATGCCTGGGGACAGGGAGCGAGTGTGACTGTGTCCTC ATCAGGCGGAGGCGGGTCTGGAGGAGGCGGCAGCGGCGGCGGCGGGTCAGACGTA GTTATGACCCAAACACCCGTCTCACTGAGTGTCTCTCTCGGCGGCCAAGTGTCTATATC CTGTCGGAGTTCTCAATCATTTGTTTCCTCCGATGGGAATACGTACCTCAACTGGTATCT GCAAAAACCGGGACAATCTCCTCAACTCCTTATCTATAAGGTGTCCAACAGATTGAGTG GGGTCCCAGACCGCTTCAGTGGGTCTGGGTCAGGTACTGACTTTACGCTTAAAATTTCA AGGGTCGAGCACGACGATCTTGGCGTATATTACTGCGGGCAAGCTAGTAAAATCCCCC TGACCTTCGGTAGTGGGACGAAACTCGAAATCAAGTCGGATCCCACCACCACCCCAGC CCCACGGCCACCTACCCCTGCCCCAACCATCGCCAGCCAGCCCCTGAGCCTGCGGCC TGAAGCCTGCAGGCCTGCCGCCGGAGGAGCCGTGCACACAAGGGGCCTGGACTTCGC CTGCGACATCTATATCTGGGCCCCCCTGGCCGGGACATGCGGGGTGCTGCTGCTGTC CCTGGTGATTACACTGTATTGCCGCTCCAAGAGGAGCAGGCTCCTGCACAGTGACTAC ATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCC CCACCACGCGACTTCGCAGCCTATCGCTCCCGGGTGAAGTTCTCTCGCTCTGCCGATG CCCCAGCCTATCAGCAGGGCCAGAATCAGCTGTACAATGAACTGAACCTGGGCAGGCG GGAGGAGTACGACGTGCTGGATAAGCGGAGAGGCAGAGACCCCGAGATGGGCGGCA AACCACGGCGCAAAAATCCCCAGGAGGGACTCTATAACGAGCTGCAGAAGGACAAAAT GGCCGAGGCCTATTCCGAGATCGGCATGAAGGGAGAGAGAAGACGCGGAAAGGGCCA CGACGGCCTGTATCAGGGATTGTCCACCGCTACAAAAGATACATATGATGCCCTGCAC ATGCAGGCCCTGCCACCCAGA

[0267] The nucleic acid encoding the CAR may comprise a polynucleotide sequence encoding a signal peptide. Suitably, the polynucleotide sequence encoding a signal peptide encodes a signal peptide with an amino acid sequence of SEQ ID NO: 23, or a variant thereof with at least 80% sequence identity to SEQ ID NO: 23. Suitably, the polynucleotide encoding a signal peptide comprises or consists of a polynucleotide sequence of SEQ ID NO: 24, or a variant thereof with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 24.

[0268] SEQ ID NO: 24 (polynucleotide sequence encoding for human IGHV signal peptide)

[0269] ATGGAGTTTGGGCTGTCCTGGGTTTTTTTGGTGGCGCTGTTGCGCGGTGTCCAG

[0270] The present invention provides a nucleic acid encoding the CAR of the present invention with a signal peptide. The nucleic acid encoding the CAR with a signal peptide may comprise a polynucleotide sequence of SEQ ID NO: 25, or a variant thereof with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a polynucleotide sequence of SEQ ID NO: 25.

[0271] The nucleic acid encoding the CAR with a signal peptide may consist of a polynucleotide sequence of SEQ ID NO: 25, or a variant thereof with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a polynucleotide sequence of SEQ ID NO: 25.

[0272] SEQ ID NO: 25 (YTH24.5 CD45 CAR1 sequence including human IGHV signal peptide) ATGGAGTTTGGGCTGTCCTGGGTTTTTTTGGTGGCGCTGTTGCGCGGTGTCCAGCAAG TTAATTTGTTGCAATCTGGAGCCGCACTTGTTAAACCGGGAGCTTCTGTCAAGTTGAGT TGTAAAGCAAGCTCCTACACATTTACCGATTATTACATACACTGGGTTAAGCAGTCTCAC GGCAAAACTTTGGAGTGGATCGGATATATAAACCCCAAATCAGGCTTTACGAACTACAA CGAGAAATTCCGAAGGAAGGCAACTTTGACGGTCGATAAATCCACTAATACGGCCTACA TGGACATAAGTAGATTGACATCTGAGGACAGTGCAACGTATTACTGCACTCGCCGAACC GGGGTTATTCCGATGGATGCCTGGGGACAGGGAGCGAGTGTGACTGTGTCCTCATCA GGCGGAGGCGGGTCTGGAGGAGGCGGCAGCGGCGGCGGCGGGTCAGACGTAGTTAT GACCCAAACACCCGTCTCACTGAGTGTCTCTCTCGGCGGCCAAGTGTCTATATCCTGTC GGAGTTCTCAATCATTTGTTTCCTCCGATGGGAATACGTACCTCAACTGGTATCTGCAA AAACCGGGACAATCTCCTCAACTCCTTATCTATAAGGTGTCCAACAGATTGAGTGGGGT CCCAGACCGCTTCAGTGGGTCTGGGTCAGGTACTGACTTTACGCTTAAAATTTCAAGGG TCGAGCACGACGATCTTGGCGTATATTACTGCGGGCAAGCTAGTAAAATCCCCCTGAC CTTCGGTAGTGGGACGAAACTCGAAATCAAGTCGGATCCCACCACCACCCCAGCCCCA CGGCCACCTACCCCTGCCCCAACCATCGCCAGCCAGCCCCTGAGCCTGCGGCCTGAA GCCTGCAGGCCTGCCGCCGGAGGAGCCGTGCACACAAGGGGCCTGGACTTCGCCTG CGACATCTATATCTGGGCCCCCCTGGCCGGGACATGCGGGGTGCTGCTGCTGTCCCT GGTGATTACACTGTATTGCCGCTCCAAGAGGAGCAGGCTCCTGCACAGTGACTACATG AACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCA CCACGCGACTTCGCAGCCTATCGCTCCCGGGTGAAGTTCTCTCGCTCTGCCGATGCCC CAGCCTATCAGCAGGGCCAGAATCAGCTGTACAATGAACTGAACCTGGGCAGGCGGG AGGAGTACGACGTGCTGGATAAGCGGAGAGGCAGAGACCCCGAGATGGGCGGCAAAC CACGGCGCAAAAATCCCCAGGAGGGACTCTATAACGAGCTGCAGAAGGACAAAATGGC CGAGGCCTATTCCGAGATCGGCATGAAGGGAGAGAGAAGACGCGGAAAGGGCCACGA CGGCCTGTATCAGGGATTGTCCACCGCTACAAAAGATACATATGATGCCCTGCACATGC AGGCCCTGCCACCCAGA

[0273] The present invention further provides a nucleic acid encoding the CD45 binding domain as defined above. Suitably, the nucleic acid may comprise a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9, or a sequence with at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto, wherein said polynucleotide sequence comprises or consists of the polynucleotide sequence of SEQ ID NO: 22, or a variant thereof with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 22, wherein the variant sequence encodes the amino acid of SEQ ID NO: 9, or a sequence with at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.

[0274] SEQ ID NO: 26 (nucleotide sequence encoding CD45 binding domain) CAAGTTAATTTGTTGCAATCTGGAGCCGCACTTGTTAAACCGGGAGCTTCTGTCAAGTT GAGTTGTAAAGCAAGCTCCTACACATTTACCGATTATTACATACACTGGGTTAAGCAGTC TCACGGCAAAACTTTGGAGTGGATCGGATATATAAACCCCAAATCAGGCTTTACGAACT ACAACGAGAAATTCCGAAGGAAGGCAACTTTGACGGTCGATAAATCCACTAATACGGCC TACATGGACATAAGTAGATTGACATCTGAGGACAGTGCAACGTATTACTGCACTCGCCG AACCGGGGTTATTCCGATGGATGCCTGGGGACAGGGAGCGAGTGTGACTGTGTCCTC ATCAGGCGGAGGCGGGTCTGGAGGAGGCGGCAGCGGCGGCGGCGGGTCAGACGTA GTTATGACCCAAACACCCGTCTCACTGAGTGTCTCTCTCGGCGGCCAAGTGTCTATATC CTGTCGGAGTTCTCAATCATTTGTTTCCTCCGATGGGAATACGTACCTCAACTGGTATCT GCAAAAACCGGGACAATCTCCTCAACTCCTTATCTATAAGGTGTCCAACAGATTGAGTG GGGTCCCAGACCGCTTCAGTGGGTCTGGGTCAGGTACTGACTTTACGCTTAAAATTTCA AGGGTCGAGCACGACGATCTTGGCGTATATTACTGCGGGCAAGCTAGTAAAATCCCCC TGACCTTCGGTAGTGGGACGAAACTCGAAATCAAG

[0275] In one aspect of the present invention, there is also provided a nucleic acid encoding a gene editing system as defined above. Suitably, the gene editing system is a CRISPR / Cas gene editing system. Suitably, the nucleic acid encoding the CRISPR / Cas gene editing system may comprise a nucleic acid sequence encoding a Cas protein, one or more gRNAs, and optionally a donor nucleic acid sequence.

[0276] In one embodiment, there is provided a nucleic acid encoding a gene editing system for reducing or eliminating expression of endogenous CD45 in a cell and introducing the inducible suicide gene (e.g. iCaspase9) into the same cell. Suitably, the nucleic acid encoding the gene editing system may comprise a nucleic acid sequence encoding a Cas protein, one or more gRNAs that enable targeting of the PTPRC gene by the Cas9 enzyme, and a donor nucleic acid encoding an inducible suicide gene (i.e. iCaspase 9).

[0277] Due to the redundancy of the genetic code, variations in nucleic acid sequences are possible that encode for the same polypeptide. These sequences are encompassed by the present invention. Therefore, multiple polynucleotides are envisaged, each with a different nucleic acid sequence but which encodes a polypeptide according to the invention or a further polypeptide as described herein. It is possible to design and produce such nucleic acid sequences without difficulty.

[0278] The polynucleotide may be codon optimised for production in the host cell of choice.

[0279] “variant” is synonymous with “mutant” and refers to a polynucleotide or amino acid sequence which differs in comparison to the corresponding wild-type sequence. The term “wild-type” is used to mean a gene or protein having a polynucleotide or amino acid sequence respectively, which is identical with the native gene or protein respectively.

[0280] Suitably, the nucleic acid sequence may be operably linked to a heterologous sequence, such as a promoter or regulatory sequence, forming an expression cassette.

[0281] The expression cassette may comprise one or more control sequences. Control sequences are sequences that control and regulate transcription and, where appropriate, the translation of the CAR of the present invention, and include promoter sequences, transcriptional regulators encoding sequences, ribosome binding sequences (RBS) and / or transcription terminating sequences. The expression cassette may additionally include an enhancer, which may be adjacent to or distant from the promoter sequence and can function to increase transcription from the same. The expression control sequence may be functional in prokaryotic cells or in eukaryotic cells and organisms, such as mammalian cells. The expression cassette may comprise a promoter.

[0282] Any suitable promoter may be used, the selection of which may be readily made by the skilled person. The promoter sequence may be constitutively active (i.e. operational in any host cell background), or alternatively may be active only in a specific host cell environment, thus allowing for targeted expression of the nucleotide of interest (e.g. CAR) in a particular cell type (e.g. a tissue-specific promoter). The promoter may show inducible expression in response to presence of another factor, for example a factor present in a host cell. In any event, where the vector is administered for therapy, it is preferred that the promoter should be functional in the target cell background. In general, it is advantageous to employ a strong promoter functional in eukaryotic cells. The strong promoter may be, but not limited to, the immediate early cytomegalovirus promoter (CMV-IE) of human or murine origin, or optionally having another origin such as the rat or guinea pig.

[0283] In some embodiments, the promoter is a constitutive promoter.

[0284] In one embodiment, the nucleotide sequence encoding the CAR is operably linked to one or more promoter(s).

[0285] In one embodiment, the nucleotide sequences encoding the CAR and any other polynucleotide of interest are operably linked to one or more promoter(s).

[0286] In one embodiment, the nucleotide sequences encoding the CAR and any other polynucleotide of interest are operably linked to the same promoter. The nucleotide sequences encoding the CAR and any other polynucleotide of interest may share a promoter such that their expression may be regulated by a single regulatory sequence. In one embodiment, the nucleotide sequences encoding the CAR and polynucleotide of interest are independently operably linked to one or more promoter(s).

[0287] The nucleotide sequences encoding the polynucleotide of interest and the CAR may each be operably linked to a separate promoter such that their expression may be independently regulated by independent regulatory sequences. In one embodiment, the nucleotide sequences encoding the polynucleotide of interest and the CAR are operably linked to separate promoter(s).

[0288] In one embodiment, the polynucleotide of interest and the CAR are encoded in opposing directions.

[0289] In one embodiment, the polynucleotide of interest and the CAR are encoded in opposing directions and are independently operably linked to separate promoters.

[0290] In one embodiment, the polynucleotide of interest and the CAR are encoded in the same direction.

[0291] In one embodiment, the promoter is selected from the group consisting of: a cytomegalovirus promoter (CMV), a human phosphoglycerate kinase promoter (PGK), an EF-1a promoter and an inducible NFAT promoter.

[0292] In one embodiment, the promoter is a cytomegalovirus (CMV) promoter.

[0293] In another embodiment, the promoter is a minimal cytomegalovirus (mCMV or minCMV) promoter (mCMV, see, for example, Amendola Nat Biotech. 2005;23:108-116).

[0294] In one embodiment, the promoter is human phosphoglycerate kinase (PGK) promoter.

[0295] In one embodiment, the promoter is an EF-1a promoter.

[0296] In one embodiment, the promoter is an an inducible NFAT promoter. The inducible module may be composed of a synthetic NFAT response element usually comprising repetitions of the consensus NFAT binding site placed upstream of a minimal promoter.

[0297] Vector

[0298] The present invention also provides a vector which comprises a polynucleotide according to the present invention. For example, the vector of the invention may comprise a polynucleotide comprising a nucleic acid sequence that encodes a CAR of the invention. Such a vector may be used to introduce the nucleic acid sequence into a host cell so that it expresses and produces a CAR of the invention.

[0299] The vector may be any agent capable of delivering or maintaining nucleic acid in a host cell, and includes viral vectors, plasmids, naked nucleic acids, nucleic acids complexed with polypeptide or other molecules and nucleic acids immobilised onto solid phase particles. The vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector.

[0300] In certain embodiments, the vector is a lentiviral vector.

[0301] The vector may comprise the nucleic acid sequence encoding the CAR according to the invention, operably linked to a heterologous sequence, such as a promoter or regulatory sequence. In general, it is advantageous to employ a strong promoter functional in eukaryotic cells. The strong promoter may be, but not limited to, the eukaryotic translation elongation factor 1 apromoter (EF-1 a) of human or murine origin, or optionally having another origin such as the rat or guinea pig.

[0302] In more general terms, the promoter has either a viral, or a cellular origin. A strong viral promoter other than EF-1 a that may be usefully employed in the practice of the invention is the early / late promoter of the SV40 virus, the immediate early cytomegalovirus promoter (CMV-IE) or the LTR promoter of the Rous sarcoma virus. A strong cellular promoter that may be usefully employed in the practice of the invention is the promoter of a gene of the cytoskeleton, such as e.g. the desmin promoter (Kwissa et al., 2000), or the actin promoter (Miyazaki et al., 1989).

[0303] The promoter may be constitutive promoter. The promoter may be a tissue specific promoter. Suitably, the promoter is an EF-1 a promoter.

[0304] The vector may be capable of transfecting or transducing a cell. In one aspect, the vector may be capable of transfecting or transducing a T cell. In one aspect, the vector may be capable of transfecting or transducing an NK cell.

[0305] The vector may be monocistronic or bicistronic. In some embodiments the vector comprises the nucleic acid sequence encoding the CAR according to the invention, and a nucleic acid sequence encoding a further transgene. In some embodiments, the vector comprises a nucleic acid sequence encoding a suicide gene, such as iCasp9. The nucleic acid may produce a polypeptide which comprises the CAR and a suicide gene joined by a cleavage site. The cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into the CAR and the suicide gene without the need for any external cleavage activity.

[0306] Various self-cleaving sites are known, including the Foot-and-Mouth disease virus (FMDV) 2A peptide and similar sequence (Donnelly et al, Journal of General Virology (2001), 82, 1027- 1041), or the 2A-like sequence from Thosea asigna virus. Suitably, the nucleic acid of the vector of the present invention comprises a self-cleaving T2A peptide encoded by a nucleic acid comprising a polynucleotide sequence of SEQ ID NO: 28, or a variant polynucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the encoded self-cleaving T2A peptide retains self-cleaving functionality.

[0307] SEQ ID NO: 28 (polynucleotide sequence encoding T2A peptide sequence)

[0308] GGCAGCGGCGAGGGCAGAGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCC AGGCCCC

[0309] Suitably, the vector is arranged wherein the nucleic acid encoding the CAR is to the 5’ direction of a nucleic acid encoding a suicide gene (for example iCaspase 9) and a nucleic acid encoding a cleavage site (for example a self-cleaving T2A cleavage site) is between them. In alternative configurations, the nucleic acid encoding a suicide gene (for example iCaspase 9) is to the 5’ of the nucleic acid encoding the CAR, with a nucleic acid encoding a cleavage site (for example a self-cleaving T2A site) between them.

[0310] The vector may also comprise a gene editing system as defined above. Suitably, the vector may comprise a nucleic acid encoding a gene editing system as described above. Suitably, the vector may comprise one or more nucleic acids encoding one or more gene editing systems in addition to a nucleic acid encoding the CAR.

[0311] Cell

[0312] The present invention further provides a cell comprising a CAR as defined above.

[0313] The present invention further provides a cell comprising a CAR, nucleic acid or vector as defined above

[0314] The present invention further provides a population of cells comprising a CAR as defined above. The cell(s) may comprise a CAR polypeptide of the present invention, a polynucleotide encoding a CAR of the present invention, and / or a vector comprising a polynucleotide encoding a CAR of the present invention.

[0315] Suitably, the host cell(s) may be a mammalian cell.

[0316] The cell(s) may be an immune cell. The cell(s) may be a lymphocyte. The cell(s) may be a T cell, a natural killer (NK) cell, or a natural killer T (NKT) cell. T cells, also referred to as T cells or T lymphocytes, are lymphocytes that play a key role in adaptive immunity. They are distinguishable from other lymphocytes such as NK cells and B cells by the expression of the cell-surface T cell receptor (TCR). NKT cells express cell-surface T cell receptor (TCR) but share some properties and activities of NK cells.

[0317] There are many subsets of T cells, with a variety of activities and functions. T cells may be divided by the expression of CD4 or CD8. CD4+ T cells are known as T helper cells, and comprise multiple subsets including TH1 , TH2, TH9, TH17, TH22, T follicular helper (Tfh), and T regulatory (Treg) cells. CD8+ T cells are known as cytotoxic T cells or cytotoxic T-lymphocytes (CTLs), and comprise subsets including Tc1 , Tc2, Tc9, Tc17, and Tc22. T cell subsets show large heterogeneity and may have significant overlap with other subsets.

[0318] Conventional T helper cells (CD4+) promote the activity of other immune cells, including, among others, CD8+ T cells, NK cells, and B-cells. The type of immune cell activated depends on the subset of T helper cell activated, which will depend on the type of pathogen being responded to. CD4+ T cells are activated when they recognise peptide antigen presented by MHC class II molecules on the surface of antigen presenting cells (APCs).

[0319] Regulatory T cells (Tregs) are a subset of CD4+ T cells that function to supress the immune response, by inhibiting T cell proliferation and activation. They are important in inhibiting T cell activation towards the end of an immune response, and in inhibiting autoreactive T cells that escaped negative selection in the thymus. However, Tregs can also act as a barrier to effective antitumor immune response by preventing T cell activity in tumour sites.

[0320] Cytolytic T cells (CTLs) are CD8+ T cells that can directly destroy target body cells that are infected or malignant. CTLs identity such cells and are activated when they recognise peptide antigen presented by MHC Class I molecules on the surface of nucleated body cells. CTLs induce apoptosis by multiple mechanisms, including releasing perforin-containing granules that create pores in target cell membranes, and releasing granzymes which trigger apoptosis via a caspase cascade. CTLs also release pro-inflammatory cytokines which contribute to the local immune response including via macrophage activation. Memory T cells may be either CD4+ or CD8+. Memory T cells are a subpopulation of T cells that persist long-term after an immune response to a particular antigen has resolved. The subsequent activation of the immune response to a repeat occurrence of the antigen is rapid due to the quick expansion of this memory T cell population. Human memory cells are distinguishable by the typical expression of CD45RO on the cell surface. A further subset of human memory cells, termed stem memory cells, are distinguishable by the expression of CD45RA on the cell surface rather than CD45RO.

[0321] Gamma delta (yb) T cells are an unconventional T cell that are found at lower abundance than the “classical” T cells. The “classical” T cells express heterodimeric T cell receptors (TCRs) comprising a and p TCR chains. The gamma delta T cells express heterodimeric T cell receptors formed instead from y and 5 TCR chains. Gamma delta T cells are activated in a non-MHC restricted manner, where the yb TCR may be able to variably recognise markers of cell stress, including metabolites, heat shock proteins or lipids and glycolipids presented by the MHC-related CD1 protein depending on the gamma and delta chains present.

[0322] Natural killer T (NKT) cells are T lymphocytes that function similarly to both innate natural killer (NK) cells and adaptive T cells. NKTs express a T cell receptor, which depending on the NKT subtype can be semi-invariant comprising Valpha14-Jalpha18 chains paired with Vbeta8.2, Vbeta7, or Vbeta2 in type I, or invariant, NKTs, or comprise a more diverse range of T cell receptors in type II, or variant, NKTs. These cells can recognise glycolipid antigen presented by CD1d molecules.

[0323] Cytokine-Induced Killer (CIK) cells are a heterogenous population of cytotoxic T lymphocytes, which express CD3 and CD56. These cells do not require antigen-specific stimuli for activation and show non-MHC-restricted cytotoxicity (Cappuzello E. et al., Cytokine & Growth Factor Reviews. 2017;36:99-105).

[0324] The cell provided by the present invention may be any of the T cell types described above.

[0325] The cell of the present invention may be a natural killer (NK) cell.

[0326] Natural killer (NK) cells are a cell of the innate immune system. NK cells can destroy cells that are infected or malignant in a peptide-MHC independent manner. NK cells are able to identify cells that have altered cell surface protein expression, including down-regulation of MHC class I molecules on the cell surface. An inhibitory signal is generated when NK cells recognise MHC class I via KIR family receptors. In the absence or reduction of this signal, and presence of activating signals from activation receptors, NK cells are activated and cytolysis of the target cell occurs. The cell of the present invention may be any cell type mentioned above. Suitably, the cell of the present invention is a cytolytic immune cell. In one embodiment, the cell of the present invention is a T cell. In another embodiment, the cell of the present invention is a natural killer (NK) cell.

[0327] The cell(s) may comprise an inducible suicide switch as described above. Suitably, the cell(s) may comprise a nucleic acid encoding an inducible suicide gene. Suitably, the cell(s) may comprise a suicide gene selected from the group consisting of iCasp9, thymidine kinase, cytosine deaminase (CD), cytochrome P450, Rapcasp9, or RQR8. Suitably, the cell(s) comprises a nucleic acid encoding iCasp9. Suitably, the cell(s) expresses iCasp9.

[0328] The cell(s) may be able to be eliminated in response to an externally provided stimulus. Suitably, cell(s) comprising an inducible suicide switch may be eliminated on provision of an activator of said inducible suicide switch. For example, wherein the cell(s) comprise an inducible Caspase 9 gene, such as for iCasp9 or Rapcasp9, the cell(s) may be eliminated on provision of a small molecule such as Rimiducid or Rapamycin. Suitably, the externally provided stimulus may be provided to the cell(s) in vitro or in vivo.

[0329] The cell(s) may be modified such that it is not bound by the CAR of the present invention. The cell(s) may have reduced expression of CD45. The cell(s) may lack expression of CD45. The cell(s) may be CD45 knock-down or knock-out cells. Suitably, the cell(s) may be CD45 knockout cells. Suitably, the CD45 knock-out cells may be generated through use of a gene editing system. Suitably, the gene editing system is a CRISPR-based gene editing system. Suitably, the gene editing system is a CRISPR / Cas9 gene editing system, comprising one or more gRNA specific for the PTPRC locus.

[0330] The present inventors surprisingly showed that the CD45CAR-T cells of the present invention retained functional activity even when CD45 was knocked-out. Should it be desirable to reestablish function of CD45 in a cell of the invention wherein CD45 had been knocked-out, but to maintain lack of binding of the CAR of the present invention to the cell, the cell may be modified to express a variant CD45 which is not bound by the CAR of the present invention. Modification of CD45 to abolish CAR binding is demonstrated in e.g. WO2023 / 091954. Suitably, the cell(s) may be modified to express a variant CD45 that is not bound by the CAR of the present invention but which retains CD45 activity. Suitably, a nucleic acid encoding a variant CD45 may be introduced through gene editing techniques. Alternatively, the cell of the invention may not have CD45 knocked-out as described above, instead, the endogenous CD45 gene may be modified to a variant form not bound by the CAR of the invention through gene editing techniques. Suitably, the gene editing techniques involve the use of a gene editing system, such as CRISPR / Cas9. The gene editing may utilise a gRNA of SEQ ID NO: 19.

[0331] For CAR-T cells derived from a third party and administered to a subject, the T cell receptor from the donor may be able to bind epitopes on the subject’s cells. Accordingly, there may be off-target effects from cytotoxicity to a subject’s cells that are not CD45+(i.e. non-target cells).

[0332] The cell(s) of the invention may be modified to reduce non-target activity. The cell(s) of the invention may be modified to prevent binding to non-target cells in a subject. The cell(s) may be modified so as to prevent graft vs host disease (GVHD). The cell(s) may have reduced expression of TCR. The cell(s) may lack expression of TCR. The cell(s) may have reduced expression or lack expression of a TCR subunit. The cell(s) may be TRAC knock-down or knock-out cells. Suitably, the cell(s) may be TRAC knock-out cells. Suitably, the TRAC knockout cells may be generated through use of a gene editing system. Suitably, the gene editing system is a CRISPR-based gene editing system. Suitably, the gene editing system is a CRISPR / Cas9 gene editing system, comprising one or more gRNA specific for the TRAC locus. The gene editing may utilise a gRNA of SEQ ID NO: 20.

[0333] The cell of the present invention may suitably be a cytotoxic T-cell, wherein the cell:

[0334] (i) comprises a CAR according to the present invention,

[0335] (ii) comprises an inducible suicide gene,

[0336] (iii) has reduced or eliminated expression of CD45, optionally wherein the cell is a PTPRC knock-out cell, and / or

[0337] (iv) has reduced or eliminated expression of TCR, optionally wherein the cell is a TRAC knock-out cell.

[0338] The present invention also provides a method of making the cell of the invention. The method may comprise introducing the nucleotide and / or vector of the invention into the cell, for example by transfection or transduction.

[0339] The cell 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). The cell may be from a peripheral blood mononuclear cell (PBMC) sample from the patient or a donor. Alternatively, cells may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to, for example, T or NK cells. Alternatively, an immortalized T cell line which retains its lytic function and could act as a therapeutic may be used.

[0340] The cell may be activated and / or expanded prior to being transduced with the CAR-encoding nucleic acid, for example by treatment with an anti-CD3 monoclonal antibody.

[0341] The sample may be obtained from a subject to be treated with the cell of the present invention, or the sample may be obtained from a third party not requiring treatment with the cell of the present invention. Suitably, the sample is obtained from a third party not requiring treatment with the cell of the present invention. Suitably, the cell is allogenic.

[0342] In one aspect of the invention, there is provided a method of making the cell as described above.

[0343] The cell of the present invention may be generated by a method comprising the following steps:

[0344] (i) isolation of a cell-containing sample from a subject, or, provision of a cell-containing sample,

[0345] (ii) transduction or transfection of the cell-containing sample with one or more vector of the invention, to provide a population of engineered cells, and, optionally,

[0346] (iii) supplying one or more gene editing systems to the cell-containing sample, such that the one or more gene editing systems are introduced to the cell(s) of the cellcontaining sample, to provide a population of further engineered cells.

[0347] Suitably, the one or more vector of step (ii) comprises a nucleic acid encoding the CAR of the invention as defined above. In some aspects, the one or more vector of step (ii) further comprises an inducible suicide gene. In some aspects, step (ii) comprises transducing or transfecting a single vector of the invention. Suitably, step (ii) comprises transducing or transfecting a single vector of the invention, wherein the vector is bicistronic and wherein the vector comprises a nucleic acid encoding the CAR as defined above and an inducible suicide gene.

[0348] Suitably, the one or more gene editing system in step (iii) are introduced to the cell(s) through the use of electroporation. In some embodiments, the one or more gene editing system may alternatively be introduced to the cell(s) through the use of vectors comprising nucleic acids encoding the one or more gene editing system. Suitably, the one or more gene editing system in step (iii) comprises a gene editing system for reducing or abolishing expression of CD45 in the cell and / or a gene editing system for reducing or abolishing TCR expression in a cell. Suitable gene editing systems for these purposes are described above. Briefly, a gene editing system for reducing or abolishing expression of CD45 may knockdown or knockout a PTPRC gene. A gene editing system for reducing or abolishing expression of TCR may knockdown or knockout a gene encoding a subunit of the TCR, for example TRAC.

[0349] Suitably, the order of steps (ii) and (iii) may be reverse. Suitably, steps (ii) and (iii) may be carried out separately or simultaneously. Suitably, wherein at more than one gene editing system is supplied to the cell-containing sample, step (iii) may be carried out multiple times wherein each gene editing system is supplied to the cell-containing sample and introduced to the cell(s) separately. Suitably, wherein at more than one gene editing system is supplied to the cell-containing sample, step (iii) may be carried out simultaneously for all gene editing systems.

[0350] The cell(s) may be cultured prior to, or after, introduction of the one or more vector of the present invention. The cell(s) may be cultured prior to, or after, introduction of the one or more gene editing system of the present invention. The population of engineered cells may be further cultured after transduction or transfection. The population of engineered cells may be further separated from the cell-containing sample, using any suitable method known in the art. The population of engineered cells may be further treated to activate the cells to provide more effective function (e.g. anti-tumour and cytolytic activity), for example by treatment with an anti-CD3 monoclonal antibody. The steps may be performed in a closed and sterile cell culture system.

[0351] The cell of the present invention may be used for adoptive cell transfer. As used herein the term “adoptive cell transfer” refers to the administration of a cell population to a patient. The cell may be isolated from a subject and one or more vector of the invention may be introduced by a method described herein before the cell is administered to the patient.

[0352] Adoptive cell transfer may be allogenic or autologous. By “autologous cell transfer” it is to be understood that the starting population of cells is obtained from the same subject as that to which the transduced cell population is administered. Autologous transfer is advantageous as it avoids problems associated with immunological incompatibility and is available to subjects irrespective of the availability of a genetically matched donor. By “allogeneic cell transfer” it is to be understood that the starting population of cells is obtained from a different subject as that to which the transduced cell population is administered. Optionally, the donor will be genetically matched to the subject to which the cells are administered to minimise the risk of immunological incompatibility. Alternatively, the donor may be mismatched and unrelated to the patient. Suitable doses of transduced cell populations are such as to be therapeutically and / or prophylactically effective. The dose to be administered may depend on the subject and condition to be treated, and may be readily determined by a skilled person.

[0353] Pharmaceutical composition

[0354] The present invention also relates to a pharmaceutical composition comprising a therapeutic agent of the invention, such as a nucleotide, vector, cell, or population of cells of the present invention.

[0355] The pharmaceutical composition may comprise, in addition to the therapeutic agent, a pharmaceutically acceptable carrier, diluent or excipient. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as (or in addition to) the carrier, excipient or diluent, any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s), and other carrier agent(s). The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides or compounds. Such a formulation may, for example, be in a form suitable for intravenous administration.

[0356] Kit

[0357] The present invention relates to kits comprising products of the present invention.

[0358] In one aspect of the invention, there is provided a kit for use in a method of manufacture of a cell, population of cells or pharmaceutical composition of the present invention. Suitably, such a kit comprises a nucleic acid or a vector encoding the CAR of the present invention. In certain embodiments, the kit comprises a nucleic acid or a vector encoding the CAR of the present invention and an inducible suicide gene. In further embodiments, the kit further comprises one or more gene editing systems. Suitably, the one or more gene editing system are for reducing or abolishing expression of CD45 in a cell, and / or reducing or abolishing expression of TCR in a cell. In certain embodiments, the kit further comprises a cell, optionally wherein the cell is an immune cell.

[0359] In one aspect of the invention, there is provided a kit comprising a cell of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0360] Subject and use The CAR, nucleotide, vector, cell, population of cells, or pharmaceutical composition may be for use as a medicament. The CAR, nucleotide, vector, cell, population of cells, or pharmaceutical composition may be for use in treating or preventing cancer in a subject in need thereof.

[0361] The subject may be a patient. The patient may be a human patient. The patient may be a nonhuman animal.

[0362] The primary therapeutic applications of the CAR and cells of the invention include in the treatment of cancer, particularly haematological cancers, and in conditioning subjects for bone marrow or haematopoietic stem cell transplant and gene therapy.

[0363] As used herein, the term "conditioning" may be understood to mean the process of preparing a subject for transplantation with a preparation containing haematopoietic stem cells, or for gene therapy, by selectively depleting (i.e., by cell killing) the subject's autologous haematopoietic stem cells, haematopoietic progenitor cells, and / or leukocytes, to provide a niche for engraftment of the transplanted cells.

[0364] The CAR cells of the invention exert targeted cell killing activity, for example through cell- mediated cytotoxicity mechanisms, of cells expressing CD45 (i.e., CD45-positive cells). Typically such cells are those of the haematopoietic system.

[0365] The subject may be a patient that requires haematopoietic stem cell transplantation (SCT) or haematopoietic cell gene therapy. The subject may be a patient that requires myeloablation in order to create a niche for the incoming stem cell graft or gene therapy. The subject may be a patient who is unwilling or unable to receive systemic conditioning techniques previously used for this purpose, such as extensive chemotherapy or radiotherapy. The subject may be a patient who is chemorefractory (i.e. cannot be treated with chemotherapy, the subject has a cancer that is not responsive to chemotherapy, such as traditional chemotherapies). In some aspects, the subject is contraindicated for chemotherapy and / or radiotherapy. In some aspects, the subject has an immunodeficiency, optionally a congenital immunodeficiency or an acquired immunodeficiency. In some aspects, the subject has pre-existing organ toxicity (i.e. is too ill for conventional conditioning regimes), or has a DNA or telomere repair disorder precluding conventional chemo / radiotherapy conditioning.

[0366] The subject may have, may have been diagnosed with, or may be suspected of having, a disease or disorder that can be treated by haematopoietic stem cell transplant. In some aspects, the subject may have, may have been diagnosed with, or may be suspected of having, a disease or disorder that can be treated by gene therapy, optionally with haematopoietic stem cell transplant. In some aspects, the subject has, has been diagnosed with, or is suspected of having, cancer, preferably a haematological cancer. In some aspects, the subject has, has been diagnosed with, or is suspected of having, acute myeloid leukaemia, acute lymphoblastic leukaemia, chronic lymphocytic leukaemia, chronic myeloid leukaemia, myelodysplasia, multiple myeloma, non-Hodgkin's lymphoma and Hodgkin's disease.

[0367] The subject may be a patient that requires treatment for a haematological cancer. The subject may be a patient who is at risk of developing cancer.

[0368] In some aspects the malignant disease or disorder is caused by CD45-expressing cells. In some aspects, the malignant disease or disorder is cancer. In preferred aspects, the malignant disease or disorder is a haematological cancer. In some aspects, the malignant disease or disorder is selected from the group consisting of acute myeloid leukaemia, acute lymphoblastic leukaemia, chronic lymphocytic leukaemia, chronic myeloid leukaemia, myelodysplasia, myeloproliferative disease, multiple myeloma, non-Hodgkin's lymphoma and Hodgkin's disease. In some aspects, the malignant disease or disorder is a high risk or refractory haematological malignancy. In some aspects, the high risk or refractory haematological malignancy may be AML, T-ALL or multiple myeloma.

[0369] The patient may be diagnosed with a cancer by any suitable means known to those of skill in the art. For example, patients may be diagnosed by imaging, such as computerised tomography (CT) scan, magnetic resonance imaging (MRI), positron emission tomography (PET) scan, ultrasound or X-ray, or they may be diagnosed with a biopsy, a physical exam, or via blood tests, such as blood smear, complete blood count, or analysis of circulating cancer biomarkers.

[0370] The patient may have been previously determined to be at risk of developing a cancer. The increased risk may have been determined by genetic screening and / or by reviewing the patient’s family history. The patient may have been determined to express one or more genetic markers indicative of an increased risk of developing a cancer.

[0371] Suitably, a person skilled in the art will be aware of genetic risk factors (e.g. genetic markers) associated with increased risk of developing a cancer. The skilled person may use any suitable method or technique known in the art to determine whether the subject has an increased risk of developing a cancer.

[0372] The subject may have previously received treatment for the cancer. The subject may be in remission from the cancer. The subject may be resistant to chemotherapy. The present invention provides methods of treating haematological cancer. In some embodiments, the method comprises administering a cell or pharmaceutical composition of the invention, to a subject in need thereof.

[0373] In some aspects methods may further comprise administering one or more other therapeutic agents. For example, the methods may further comprise administering one or more anticancer agents. In some aspects the methods further comprise administering to the subject radiation and / or chemotherapy. In some aspects, the methods further comprise administering to the subject one or more of enasidenib, gilteritinib, ivosidenib, midostaurin, fludarabine, cyclophosphamide, rituximab, bendamustine, chlorambucil, ibrutinib, idelalisib, obinutuzumab, ofatumumab, prednisolone, brentuximab vedotin, lenalidomide, pomalidomide, carfilzomib, daratumumab, thalidomide, panobinostat, bortezomib, all-trans retinoic acid, arsenic trioxide, idarubicin, daunorubicin, cytarabine, azacitidine, mitoxantrone, cytarabine, etoposide, gemtuzumab, 5- azacytidine, hydroxyurea, midostaurin , vincristine, steroids, doxorubicin, asparaginase, ifosfamide, methotrexate, nelarabine, melphalan, bendamustine, carmustine (bis-chloroethylnitrosourea, BCNll), cis platin, carboplatin, busulphan, treosulphan, thiotepa, or total body irradiation. In some preferred aspects, the methods further comprise administering to the subject one or more cancer treatment(s) selected from: daunorubicin, idarubicin, mitoxantrone, cytarabine, etoposide, fludarabine, gemtuzumab, 5-azacytidine, hydroxyurea, midostaurin, vincristine, steroids, doxorubicin, asparaginase, cyclophosphamide, ifosfamide, methotrexate, nelarabine, daratumumab, melphalan, thalidomide, lenolidamide, bortezimib, pomalidomide, carfolizimib, bendamustine, carmustine (bis-chloroethylnitrosourea, BCNll), cis platin, carboplatin, rituximab, ofatumumab, obinutuzumab, ibrutinib, idelasalib, and brentuximab. In some aspects, the methods further comprise administering to the subject one or more conditioning agent(s) selected from: busulphan, treosulphan, thiotepa, and total body irradiation. In such aspects, the further therapeutic agents may be formulated within the same pharmaceutical composition as the cells of the invention; or preferably, the further therapeutic agents may be administered in a separate formulation. The further therapeutic agents may be administered concurrently with the cells or pharmaceutical compositions of the invention. The cells or pharmaceutical compositions of the present invention may be administered before, after or concurrently with the one or more further agents.

[0374] In some aspects, the subject has been diagnosed with, or is suspected of having, a non- malignant disease, disorder or condition. In some aspects, the subject has been diagnosed with, or is suspected of having a disorder or disease selected from the group consisting of: severe aplastic anaemia or other bone marrow failure disorder (such as Fanconi anaemia, dyskeratosis congenita, Shwachman-Diamond Syndrome, severe congenital neutropenia, Diamond-Blackfan anaemia), a primary immunodeficiency (such as SCID (e.g., newborn SCID), chronic granulomatous disease, Wiskott-Aldrich syndrome, CD40 ligand deficiency, XLP, MHC Class II deficiency, primary haemophagocytic lymphohistiocytosis), a haemoglobinopathy, preferably a transfusion dependent haemoglobinopathy (such as sickle cell disease, - thalassaemia major), a genetic metabolic disease (such as Hurler's syndrome, X-linked adrenoleukodystrophy, alphamannosidosis, osteopetrosis, metachromatic leukodystrophy, Sanfilippo disease), or an autoimmune disorder (such as multiple sclerosis, systemic sclerosis, juvenile inflammatory arthritis, systemic lupus erythematosus). The subject may be a patient that requires haematopoietic SCT or haematopoietic gene therapy for treating a non-malignant disease or disorder.

[0375] In some aspects the non-malignant disease, disorder or condition is caused by CD45- expressing cells. In some aspects, the non-malignant disease, disorder or condition may be selected from the group consisting of: severe aplastic anaemia or other bone marrow failure disorder (such as Fanconi anaemia, dyskeratosis congenita, Shwachman Diamond Syndrome, severe congenital neutropenia, Diamond-Blackfan anaemia), a primary immunodeficiency (such as SCID, newborn SCID, chronic granulomatous disease, Wiskott- Aldrich syndrome, CD40 ligand deficiency, XLP, MHC Class II deficiency, primary haemophagocytic lymphohistiocytosis), a transfusion dependent haemoglobinopathy (such as sickle cell disease, p-thalassaemia major), a genetic metabolic disease (such as Hurler's syndrome, X-linked adrenoleukodystrophy, alpha mannosidosis, osteopetrosis, metachromatic leukodystrophy, Sanfilippo disease) or an autoimmune disorder (such as multiple sclerosis, systemic sclerosis, juvenile inflammatory arthritis, systemic lupus erythematosus). In some preferred aspects, the methods described herein of preparing a subject for transplantation of haematopoietic stem cells, and the methods of engrafting stem cells in a subject, may be useful in the treatment of a Fanconi anaemia, chronic granulomatous disease and / or Hurler's syndrome. In some most preferred aspects, the methods described herein of preparing a subject for transplantation of haematopoietic stem cells, and the methods of engrafting stem cells in a subject, may be useful in the treatment of sickle cell anaemia, p- thalassaemia, and / or newborn SCID.

[0376] In some aspects, the present invention provides a method of preparing a subject for transplantation of haematopoietic stem cells, the method comprising administering a cell of the invention or a pharmaceutical composition of the invention, to a subject in need thereof. In some aspects, the present invention provides a cell of the invention or a pharmaceutical composition of the invention for use in a method of preparing a subject for transplantation of haematopoietic stem cells, the method comprising administering said cell or pharmaceutical composition to a subject in need thereof. Preparing a subject for transplantation of haematopoietic stem cells may comprise, or consist essentially of, conditioning the subject for engraftment of haematopoietic stem cells.

[0377] The cell or pharmaceutical composition of the invention typically result in cell killing of CD45- positive cells. It is usually advantageous, before transplantation with a preparation containing haematopoietic stem cells or gene therapy, to reduce as far as possible the number of immunological effector cells in the subject's body, preferably to eliminate them entirely. Thus, the cell or pharmaceutical composition of the invention provide broad spectrum cell killing of CD45-positive cells, including T-cells, NK cells, lymphocytes and monocytes. This may minimise graft rejection or graft versus host disease following transplantation.

[0378] As discussed above, in some aspects, preparing a subject for transplantation with haematopoietic stem cells may comprise conditioning the subject for engraftment of haematopoietic stem cells. Thus, it may mean the process of selectively depleting (i.e., by cell killing) the subject's autologous haematopoietic stem cells and / or leukocytes, to provide a niche for engraftment of transplanted haematopoietic stem cells. In some aspects the methods of the invention may be myeloablative, non-myeloablative or reduced intensity, preferably the methods of the invention provide reduced toxicity myeloablative conditioning. Typically, the subject's autologous haematopoietic stem cells comprise a defect or mutation, which results in a disorder or disease in the subject. Thus, depleting or substantially eliminating the subject's autologous haematopoietic stem cells and replacing them with corrected or healthy haematopoietic stem cells provides a treatment for the disease.

[0379] In some aspects, the haematopoietic stem cells for transplantation into the subject are allogeneic. The term "allogeneic" may be understood in the context of the invention to mean a donor's haematopoietic stem cells, i.e., the haematopoietic stem cells are isolated or derived from a donor, typically a human donor. The haematopoietic stem cells are not the subject's own haematopoietic stem cells, i.e., they are not derived from the subject. In some aspects, the allogeneic haematopoietic stem cells for transplantation into the subject are from a healthy donor, i.e., a donor not having the same disease as the subject, or preferably a donor not having any disease. In preferred aspects, the allogeneic haematopoietic stem cells for transplantation into the subject are from a healthy, H LA-matched donor. As used herein, the term "H LA-matched" is used to mean, that the human leukocyte antigen (HLA) types of the donor haematopoietic stem cells are a close match with the subject's HLA types. In some aspects, the donor and subject have at least 6, preferably at least 8, and most preferably at least 10 matching HLA markers. In some aspects the donor may be haploidentical with the subject (i.e., exactly half of the HLA markers match). In some aspects, the donor may be a sibling, parent or child of the subject.

[0380] In the methods of the invention, in some aspects, said transplantation of allogeneic haematopoietic stem cells may be for treating a malignant disease or disorder. In some such aspects, the transplantation of allogeneic haematopoietic stem cells may be for treating a disease or disorder selected from the group consisting of: acute myeloid leukaemia, acute lymphoblastic leukaemia, chronic myeloid leukaemia, chronic lymphocytic leukaemia, myelodysplasia, myeloproliferative diseases, non-Hodgkin's lymphoma and Hodgkin's disease. Alternatively, in some other aspects, said transplantation of allogeneic haematopoietic stem cells may be for treating a non-malignant disease or disorder. In some such aspects, the transplantation of allogeneic haematopoietic stem cells may be for treating a disease or disorder selected from the group consisting of: severe aplastic anaemia and other bone marrow failure disorders, a primary immunodeficiency, primary 1 haemophagocytic lymphohistiocytosis, a haemoglobinopathy, and a genetic metabolic disease. In some aspects, the transplantation of allogeneic haematopoietic stem cells may be for treating (i) a bone marrow failure disorder such as idiopathic severe aplastic anaemia, Fanconi anaemia, dyskeratosis congenita, severe congenital neutropenia, Shwachman-Diamond Syndrome, or Diamond Blackfan anaemia; (ii) a primary immunodeficiency such as SCID (e.g., newborn SCID), chronic granulomatous disease, Wiskott-Aldrich syndrome, CD40 ligand deficiency, XLP, MHC Class II deficiency, or primary haemophagocytic lymphohistiocytosis; (iii) a haemoglobinopathy such as sickle cell disease, p- thalassaemia major; or (iv) a genetic metabolic disease such as Hurler syndrome, X-linked adrenoleukodystrophy, alpha mannosidosis, or osteopetrosis. In some aspects, the transplantation of allogeneic haematopoietic stem cells may be for treating a disease or disorder selected from the group consisting of: severe aplastic anaemia and other bone marrow failure disorders, a haemoglobinopathy, a primary immunodeficiency, primary haemophagocytic lymphohistiocytosis, a genetic metabolic disease, Fanconi anaemia, dyskeratosis congenita, severe congenital neutropenia, Shwachman-Diamond Syndrome, Diamond-Blackfan syndrome, SCID (e.g., newborn SCID), chronic granulomatous disease, Wiskott-Aldrich syndrome, CD40 ligand deficiency, XLP, MHC Class II deficiency, primary haemophagocytic lymphohistiocytosis, sickle cell disease, - thalassaemia major, Hurler syndrome, alpha mannosidosis, X-linked adrenoleukodystrophy and osteopetrosis. In such aspects, the subject is a human (e.g., a patient) in need of treatment for one or more of said diseases or disorders, i.e., a subject having, or suspected of having, one or more of said diseases or disorders. In some aspects, the subject has been diagnosed as having one or more of said diseases or disorders, and is therefore in need of treatment as described herein. In some aspects, the allogeneic haematopoietic stem cells for transplantation into the subject are genetically-modified. In such aspects, the transplantation of genetically-modified allogeneic haematopoietic stem cells may be for gene therapy of the subject. Thus, the invention further provides a method of preparing a subject for transplantation of allogeneic genetically-modified haematopoietic stem cells for gene therapy, the method comprising administering a CAR cell according to invention. Typically, the subject's autologous haematopoietic stem cells comprise a deficiency, disease or mutation that results in a disease or disorder. The genetically-modified allogeneic haematopoietic stem cells for transplantation may have been isolated or derived from the donor and treated ex vivo, for example by gene therapy, e.g., by transduction with a viral vector carrying a gene for a desired expression product, or through gene / base editing using for example TALENS or CRISPR technology. Following conditioning with the CAR cell of the invention, genetically-modified allogeneic haematopoietic stem cells can be transplanted into the subject, which may be useful for gene therapy for treating a genetic haematological disease or disorder, a primary immunodeficiency or a genetic metabolic disorder. For example, following conditioning with the antibodies, antibody drug conjugates or pharmaceutical compositions of the invention, genetically- modified allogeneic haematopoietic stem cells can be transplanted into the subject for treating Fanconi anaemia, where autologous HSCs are difficult to harvest, or for treating metachromatic leukodystrophy (MLD), where overexpression may be beneficial.

[0381] In some aspects, the haematopoietic stem cells for transplantation into the subject are autologous. The term "autologous" may be understood in the context of the invention to mean the subject's own haematopoietic stem cells, i.e. , haematopoietic stem cells isolated or derived from the subject. In some aspects, the haematopoietic stem cells for transplantation into the subject may be the subject's own haematopoietic stem cells. In the methods of the invention, in some aspects, said transplantation of autologous haematopoietic stem cells may be for treating a malignant disease or disorder. In some such aspects, the transplantation of autologous haematopoietic stem cells may be for treating a disease or disorder selected from the group consisting of: multiple myeloma, non Hodgkin's lymphoma, and Hodgkin's disease. In some other aspects, the transplantation of autologous haematopoietic stem cells may be for treating an autoimmune disease or disorder. In some such aspects, the transplantation of autologous haematopoietic stem cells may be for treating a disease or disorder selected from the group consisting of: multiple sclerosis, systemic sclerosis, juvenile inflammatory arthritis, and systemic lupus erythematosus. In some aspects, the transplantation of autologous haematopoietic stem cells may be for treating a disease or disorder selected from the group consisting of: multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, an autoimmune disease or disorder, multiple sclerosis, systemic sclerosis, juvenile inflammatory arthritis and systemic lupus erythematosus.

[0382] In some aspects, the haematopoietic stem cells for transplantation into the subject are autologous and genetically-modified. In such aspects, the transplantation of genetically- modified autologous haematopoietic stem cells may be for gene therapy of the subject. Thus, the invention further provides a method of preparing a subject for transplantation of autologous genetically-modified haematopoietic stem cells for gene therapy, the method comprising administering a cell of the invention or a pharmaceutical composition of the invention. Typically, the subject's autologous haematopoietic stem cells comprise a deficiency, disease or mutation that results in a disease or disorder. The genetically-modified autologous haematopoietic stem cells for transplantation may have been isolated or derived from the subject and treated ex vivo, for example by gene therapy, to correct the deficiency, disease or mutation, so that the haematopoietic stem cells no longer result in the disease or disorder. In some aspects, the genetically-modified haematopoietic stem cells for transplantation comprise, or consist essentially of, autologous haematopoietic stem cells (i.e. , haematopoietic stem cells isolated from the subject to be treated), which have been genetically modified, e.g., by transduction with a viral vector carrying a gene for a desired expression product, or through gene / base editing using for example TALENS or CRISPR technology. For example, one or more viral vector(s) comprising a gene encoding the adenosine deaminase (ADA) or p-globin genes can be used in a known manner to insert these genes into haematopoietic stem cells isolated from a subject having an ADA deficiency or a haemoglobinopathy, respectively. Similarly, gene / base editing using CRISPR technology maybe used to silence the BCL11A repressor gene to enable expression of y-globin in haematopoietic stem cells isolated from a subject having sickle cell disease and / or p-thalassaemia. Following conditioning with the cell or pharmaceutical composition invention, transplantation of such gene-corrected (i.e., genetically modified) autologous shaematopoietic stem cells can be curative for these disorders.

[0383] Thus, in some aspects, the haematopoietic stem cells for transplantation into the subject may be genetically-modified autologous haematopoietic stem cells. In the methods of the invention, in some aspects, said transplantation of genetically-modified autologous haematopoietic stem cells may be for gene therapy. In some aspects, the transplantation of genetically-modified autologous haematopoietic stem cells may be for gene therapy for treating a genetic haematological disease or disorder, a primary immunodeficiency or a genetic metabolic disorder. In some aspects, the transplantation of genetically-modified autologous haematopoietic stem cells may be for gene therapy for treating (i) a genetic haematological disease or disorder selected from a haemoglobinopathy, a transfusion dependent haemoglobinopathy, such as sickle cell disease and p-thalassaemia major, and Fanconi anaemia; (ii) a primary immunodeficiency selected from SCID (e.g., newborn SCID), chronic granulomatous disease, Wiskott-Aldrich syndrome and primary HLH; or (iii) a genetic metabolic disorder selected from Hurler's syndrome, Sanfilippo disease, X- adrenoleukodystrophy, and metachromatic leukodystrophy. In some aspects, the transplantation of genetically-modified autologous haematopoietic stem cells may be for treating a disease or disorder selected from the group consisting of: a genetic haematological disease or disorder, a primary immunodeficiency, a genetic metabolic disorder, sickle cell disease, p- thalassaemia major, Fanconi anaemia, primary HLH, SCID (e.g., newborn SCID), chronic granulomatous disease, Wiskott-Aldrich syndrome, Hurler's syndrome, Sanfilippo disease, X-adrenoleukodystrophy, and metachromatic leukodystrophy.

[0384] In some aspects, the haematopoietic stem cells are comprised within a composition. Thus, in some aspects, the methods or medical uses of the invention are for preparing a subject for transplantation of with a composition comprising or consisting essentially of haematopoietic stem cells. The composition may be a pharmaceutical composition and may comprise a pharmaceutically acceptable carrier, as described herein. In some aspects, the subject is, or is intended to be, subsequently administered haematopoietic stem cells, a population of haematopoietic stem cells, or a composition comprising haematopoietic stem cells, wherein the haematopoietic stem cells are allogeneic, autologous or genetically-modified autologous haematopoietic stem cells as described herein. In some aspects, the methods or medical uses of the invention further comprise administering to the subject haematopoietic stem cells, which may be allogeneic, autologous or genetically-modified autologous haematopoietic stem cells as described herein. In some aspects, the methods or medical uses of the invention further comprise administering to the subject a population of haematopoietic stem cells, which may be allogeneic, autologous or genetically modified autologous haematopoietic stem cells as described herein. In some aspects, the methods or medical uses of the invention further comprise administering to the subject a composition comprising haematopoietic stem cells and optionally a pharmaceutically acceptable carrier, wherein the haematopoietic stem cells are allogeneic, autologous or genetically-modified autologous haematopoietic stem cells as described herein. In seem aspects, the administered haematopoietic stem cells engraft in a target tissue of the subject. Preferably, said target tissue is bone marrow.

[0385] In some aspects, the present invention provides a method of engrafting stem cells in a subject, the method comprising (a) administering an effective amount of a cell, population of cells or a pharmaceutical composition of the invention, to the subject; and (b) administering a stem cell population to the subject (preferably to a target tissue of the subject), wherein the administered stem cell population engrafts in a target tissue of the subject. In some aspects, the present invention provides a method of engrafting stem cells in a subject, the method comprising administering an effective amount of a cell, population of cells or a pharmaceutical composition of the invention, to the subject; wherein the subject is (or is intended to be) subsequently administered a stem cell population (preferably to a target tissue of the subject), wherein the administered stem cell population will engraft in a target tissue of the subject. In some aspects, the present invention provides a cell, population of cells or a pharmaceutical composition of the invention for use in a method of engrafting stem cells in a subject, the method comprising (a) administering an effective amount of the cell, population of cells or pharmaceutical composition of the invention, to the subject; and (b) administering a stem cell population to the subject (preferably to a target tissue of the subject), wherein the administered stem cell population engrafts in a target tissue of the subject. Preferably, the stem cells are haematopoietic stem cells, which may be allogeneic, autologous or genetically-modified autologous haematopoietic stem cells as described herein. Preferably, the target tissue is bone marrow. A method of engrafting stem cells in a subject may describe the process of depleting or substantially eliminating (e.g., by cell killing) a subject's autologous stem cells, which may comprise a mutation or defect that is causing a disease or disorder in the subject, to make space in the bone marrow stem cell niche for replacement of the subject's autologous stem cells with healthy stem cells. Said healthy stem cells may be from a healthy donor or may be produced from stem cells that have been isolated from the subject and treated by gene therapy to correct the defect or mutation that is causing the disease or disorder in the subject, as described above. The stem cells are typically haematopoietic stem cells. The stem cell population may comprise or consist essentially of haematopoietic stem cells. In some aspects, the stem cell population comprises exogenous stem cells, e.g., isolated from a healthy donor. In some aspects, the stem cell population comprises the subject's autologous stem cells, e.g., that have been genetically modified to correct a disease or genetic defect. The stem cell population typically comprises healthy or corrected stem cells, preferably healthy or corrected haematopoietic stem cells. The healthy stem cells, preferably healthy haematopoietic stem cells are typically from a healthy donor, as described above. The corrected stem cells, preferably corrected haematopoietic stem cells are typically from the subject (i.e. , are autologous) and treated ex vivo with gene therapy to correct the defect or mutation that is causing the disease or disorder in the subject, as described above. The healthy or corrected stem cells are typically transplanted into the subject and engraft in the target tissue of the subject. The target tissue may be any tissue to which the stem cell population may be targeted. The target tissue is preferably bone marrow. Thus, the healthy or corrected stem cells are typically haematopoietic stem cells and once transplanted, integrate into the subject's haematopoietic system, in the bone marrow.

[0386] As used herein the term "stem cells" refers to undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell. Typically, stem cells are the earliest type of cell in a cell lineage and are defined by their ability to form multiple cell types (multi potency) and their ability to self-renew. As used herein, "haematopoietic stem cells" refers to stem cells that can differentiate into the hematopoietic lineage and give rise to all blood cell types such as white blood cells and red blood cells, including myeloid (e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid lineages (e.g., T-cells, B-cells, NK-cells). Human hematopoietic stem cells can be identified, for example by cell surface markers such as CD34+, CD90+, CD49f+, CD38- and CD45RA-.

[0387] As used herein the term "effective amount" may be understood to mean and amount of the cell, population of cells or pharmaceutical composition of the invention that is sufficient to have the desired effect. Typically it is an amount sufficient to deplete or substantially eliminate the subject's autologous haematopoietic stem cell population. The skilled practitioner is readily capable of determining an effective amount.

[0388] In some aspects, the stem cell population is administered to the target tissue of the subject after the cell or pharmaceutical composition has cleared or dissipated from the subject's target tissue. This prevent or reduces a cytotoxic effect on the administered stem cell population. Accordingly, in some aspects, the stem cell population is administered to the subject after the number of the cells, or concentration of the pharmaceutical composition in the subject's target tissue has been reduced to an undetectable concentration. The period of time necessary to clear the cells, population of cells or pharmaceutical composition from the subject's target tissue may be determined using routine means available to one of skill in the art, for example, by detecting the number of cells or concentration of the pharmaceutical composition in the subject's target tissue. In some aspects, the stem cell population is administered to the target tissue of the subject after the cells, population of cells or pharmaceutical composition has substantially cleared from the subject's target tissue. In some aspects "substantially cleared" means that the level of cells or pharmaceutical composition of the invention remaining in the target tissue of the subject does not induce significant cell death in the transplanted stem cell population. For example, the stem cell population may be administered to the target tissue of the subject at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 11 , 12, 13, 14, 15, 18, 21 or more days, preferably at least 1 day, most preferably at least 2 days, after the administration of the cell or pharmaceutical composition of the invention. Too long a period between administration of cells, population of cells or pharmaceutical composition of the invention and administration of the new stem cell population would undesirably expose the subject to prolonged duration of neutropenia. In some aspects, the stem cell population may be administered to the subject, e.g., to the target tissue of the subject, from 6 to 72 hours, from 1 to 5 days, from 1 to 7 days, from 1 to 10 days, from 1 to 14 days, from 1 to 21 days, or from 1 to 30 days; preferably from about 1 to about 7 days, after the administration of the cell or pharmaceutical composition of the invention. In some aspects, the methods of the invention further include administration of an inducer of an inducible suicide gene to the subject. For example, wherein the cell of the invention comprises an inducible iCasp9 suicide gene, Rimiducid may be administered to the subject following administration of the cell, population of cells or pharmaceutical composition of the invention. Suitably, the inducer may be administered in an amount effective to induce cell death of at least 90%, at least 95%, at least 98%, at least 99%, or of 100% of the cells of the invention present in the subject. The skilled practitioner would be capable of determining an appropriate dosage of the inducer to achieve this effect. Suitably, the inducer may be administered from 6 to 72 hours, from 1 to 5 days, from 1 to 7 days, from 1 to 10 days, from 1 to 14 days, from 1 to 21 days, or from 1 to 30 days; preferably from about 1 to about 7 days, after the administration of the cell or pharmaceutical composition of the invention. Suitably, the inducer is administered prior to the administration of the stem cell population. Suitably, the inducer may also be administered upon detection of unacceptable levels of toxicity of the cells, population of cells or pharmaceutical composition of the present invention in the subject.

[0389] In some aspects, the methods or medical uses of the invention result in conditioning of a subject's target tissues and engraftment of stem cells and achieve at least about 5-100% donor chimerism, preferably at least about 50-100%, most preferably at least about 80-100% donor chimerism (i.e. , percentage of the cells derived from the donor) in the subject's target tissue (e.g., bone marrow) four months post-administration of the stem cell population to the subject. Preferably, the donor chimerism is complete, i.e., at least 95% donor chimerism. In some aspects, the donor chimerism is stable high-level mixed chimerism, i.e., at least 50% donor chimerism, which is typically sufficient for cure. Most preferably, the donor chimerism is in both myeloid and lymphoid lineages. The level of engraftment needed may depend on the clinical scenario. For haematological malignancies at least about 50-100%, preferably at least about 80-100%, and most preferably complete donor chimerism, is the aim. For non-malignant disorders, whilst complete donor chimerism is still preferable, mixed chimerism (e.g., 30-70%, preferably 50-70% donor chimerism) is often curative. The level of donor chimerism needed to be curative may depend on the disease. For example, for haemoglobinopathies stable, 30% donor chimerism in myeloid lineage may be curative; for primary immunodeficiencies even 10- 20% donor chimerism may be sufficient. In some aspects, the methods or medical uses of the invention that comprise transplantation of genetically-modified autologous haematopoietic stem cells to the subject, for example for gene therapy, may result in a viral copy number of 0.1-10 copies / cell, preferably 0.5-4 copies / cell and most preferably 1-2 copies / cell, in the relevant cell lineage (e.g., myeloid and / or lymphoid) in the blood. In some aspects, the methods of the invention result in conditioning of a subject's target tissues and engraftment of stem cells and achieve an engraftment rate of at least 50%, preferably at least 60%, most preferably at least 80%, of subjects treated according to the methods of the invention. The methods and compositions described herein may provide an enhanced or improved engraftment efficiency, i.e., the efficiency with which an administered stem cell population (e.g., HSCs) engrafts in the conditioned target tissue of the subject (e.g., bone marrow).

[0390] The method of treating or preventing a cancer or non-malignant disease disorder or condition may involve:

[0391] (i) isolating an immune cell-containing sample from a subject;

[0392] (ii) transducing or transfecting said cell or population of said cells in the sample with a nucleic acid or vector of the invention, and optionally gene editing said cell or population of cells with a gene editing system of the invention; and

[0393] (iii) administering the cell or population of cells from step (ii) to a subject.

[0394] The subject of step (i) and step (iii) may be the same or different. Wherein the subject of step (i) and step (iii) are different, the subjects may be a H LA-matched.

[0395] The method may further comprise additional steps of expanding the cell or population of cells from step (ii) to increase their number. The method may further comprise selecting cells for expression of the CAR of the present invention and / or other cell markers indicative of transduction / transfection efficacy, cell lineage, identity and / or activation status. The method may further comprise activation of the cell or cell population from step (ii) to improve their efficacy in tumour killing.

[0396] Administration

[0397] The nucleotide, vector, cell, population of cells, and / or pharmaceutical composition of the invention may be administered by a variety of routes that make the agent bioavailable. For example, the agent can be administered parenterally, intraperitoneally, intravenously, subcutaneously, transcutaneously, intramuscularly, and / or via local delivery for example by catheter or stent. The appropriate dosage of a cell, population of cells or pharmaceutical composition of the invention in the methods of treatment and medical uses described herein will depend for example on the disease to be treated, the subject group and individual subject requirements, but a skilled person would be readily capable of determining a suitable dosage regime.

[0398] Variants, derivatives, analogues, homologues and fragments

[0399] In addition to the specific proteins and polynucleotides mentioned herein, the present invention also encompasses the use of variants, derivatives, analogues, homologues and fragments thereof.

[0400] In the context of the present invention, a variant of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question substantially retains at least one of its endogenous functions. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue present in the naturally-occurring protein.

[0401] The term “derivative” as used herein, in relation to proteins or polypeptides of the present invention includes any substitution of, variation of, modification of, replacement of, deletion of and / or addition of one (or more) amino acid residues from or to the sequence providing that the resultant protein or polypeptide substantially retains at least one of its endogenous functions.

[0402] The term “analogue” as used herein, in relation to polypeptides or polynucleotides includes any mimetic, that is, a chemical compound that possesses at least one of the endogenous functions of the polypeptides or polynucleotides which it mimics.

[0403] Proteins used in the present invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine. A substitution may involve replacement of an amino acid for a similar amino acid (a conservative substitution). A similar amino acid is one which has a side chain moiety with related properties as grouped together, for example as shown below:

[0404] (i) basic side chains: lysine (K), arginine (R), histidine (H); (ii) acidic side chains: aspartic acid (D) and glutamic acid (E);

[0405] (iii) uncharged polar side chains: asparagine (N), glutamine (Q), serine (S), threonine (T) and tyrosine (Y); or

[0406] (iv) non-polar side chains: glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W) and cysteine (C). Variant sequences may comprise amino acid substitutions, additions, deletions and / or insertions.

[0407] Conservative substitutions, additions or deletions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

[0408] The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue), e.g. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur e.g. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids, such as ornithine.

[0409] The term “variant” as used herein may mean an entity having a certain homology with the wild type amino acid sequence or the wild type nucleotide sequence. The term “homology” can be equated with “identity”.

[0410] Preferably, reference to a sequence which has a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence which has the stated percent identity over the entire length of the SEQ ID NO referred to.

[0411] Identity comparisons can be conducted by eye or, more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percentage homology or identity between two or more sequences.

[0412] Percentage homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0413] Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the nucleotide sequence may cause the following codons to be put out of alignment, thus potentially resulting in a large reduction in percent homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local homology.

[0414] However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0415] Calculation of maximum percentage homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al. (1984) Nucleic Acids Res. 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid - Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. Another tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174: 247-50; FEMS Microbiol. Lett. (1999) 177: 187-8).

[0416] Although the final percentage homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see the user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

[0417] Once the software has produced an optimal alignment, it is possible to calculate percentage homology, preferably percentage sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0418] “Fragments” are also variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion of a full- length polypeptide or polynucleotide.

[0419] Such variants may be prepared using standard recombinant DNA techniques such as site- directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5' and 3' flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.

[0420] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0421] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0422] The terms "comprising", "comprises" and "comprised of' as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of' also include the term "consisting of.

[0423] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0424] 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.

[0425] EXAMPLES

[0426] Example 1 - CD45CAR design and generation of CRISPR-Cas9-edited DKO CD45 CAR T cells.

[0427] Gene-edited CAR T cells from healthy donor peripheral blood mononuclear cells (PBMCs) were generated using reagents and methods compatible with subsequent Good Manufacturing Practice (GMP) process development (Figure 3). PBMCs were stimulated with CD3 / CD28 beads or TransAct to activate T cells, followed by knockout CD45 using Cas9 / guide RNA ribonucleoprotein (RNP) delivered using the MaxCyte electroporation system (Chen et al.; 2018; J Immunol. 201 (5): 1586-1598 - CD45 guide RNA sequence GUAUUUGUGGCUUAAACUCU (SEQ ID NO: 19). In later experiments, CD45 and TRAC were knocked out (Georgiadis et al., 2018; Molecular therapy : the journal of the American Society of Gene Therapy. 26(5): 1215-1227 - TRAC guide RNA sequence - UCUCUCAGCUGGUACACGGC (SEQ ID NO: 20). For CRISPR / Cas9 mediated knock-outs, T cells were washed with PBS and resuspended in OPTIMEM media at 1x108cells / ml. The following CRISPR reagents were then added Alt-R S.p. HiFi CAS9 nuclease version 3 (2.8pM; IDT), CD45 guide RNA (3.8pM; Synthego) and / or TRAC guide RNA (1.7pM; Synthego). Electroporation was carried out using expanded T cell 3 setting on a Maxcyte electroporator. T cells were rested for 15mins immediately post-electroporation before being re-suspended at 2.5x106cells / ml in RPMI media supplemented with 10% FBS and 40 units / ml interleukin-2.

[0428] Unedited or gene-edited T cells were then transduced with lentivirus delivering different CD45 CAR constructs based on YTH24.5 and YTH54.12 antibodies (Figure 4A) or CD19 CAR. The CD45 CARs were designed with a CD28 co-stimulatory domain. The CD19 CAR was used as 1) a control CAR targeting an unrelated antigen, CD19, with known cytotoxic activity and 2) a CAR which was no fratricide activity. Successful knockout of CD45 in CRISPR edited cells, but not in unedited cells, and expression of a ~60 KDa CAR protein was confirmed by western blotting (Figure 4B). All CD45 CAR constructs except CD45 CAR2 were expressed well. Flow cytometry also confirmed the expression of the CARs on the cell surface (Figure 4C). Optimisation experiments indicated that CD45CAR1 , derived from the YTH24.5 clone and with the CD45 binding domain in the VH / VL orientation, resulted in the highest lentivirus titres and had the highest in vitro activity, therefore all subsequent experiments were carried out with the CD45 CAR1 construct.

[0429] Unedited and DKO CAR T cells were subsequently generated. During the manufacture process, enrichment of CD45' CD45CAR T cells was observed which corresponded with increasing loss of the CD45+fraction from 23.1 % at day 6 to 0.67% on day 10 (Figure 5). This is considered to be a result of fratricide of the CD45+cells by the DKO CD45CAR T cells. This marked decrease in CD45+cells was not seen in untransduced (CAR-) DKO or unedited CAR- cells or CD19 CAR+cells.

[0430] Inference of CRISPR edits (ICE) and CRISPR Off-target Sites with Mismatches, Insertions, and Deletions (COSMID) analysis was carried on cells from day 10 of manufacture. Over 90% gene editing of CD45 in DKO CD45CAR T cells was achieved (Figure 6A). This was less efficient for TRAC (Figure 6B), but flow cytometry confirmed efficient knockdown of both CD45 and TCRap at the protein level in DKO CD45CAR T cells (Figure 6C). COSMID analysis was carried out to determine the off-target potential of the CRISPR-Cas system targeting CD45 and TRAC loci. Setting a threshold of 3 mismatches and 1 insertion and 1 deletion, a list of potential lead candidates was generated. For the CD45 gRNA, COSMID analysis confirmed that the gRNA was specific, reporting 3 genomic loci with a score less than 2 and with 2-3 mismatches. This in silico analysis showed 12 other potential off-targets (score between 2-26) with just 1 gene, NKAIN2, associated with mutagenic potential. Similarly, COSMID analysis for the TRAC gRNA also confirmed that the gRNA was specific, reporting 1 genomic locus with a score less than 2 and with 3 mismatches. 11 other potential off-targets sites were identified (score between 3-23) of which only 1 gene, GCAWKR was associated with mutagenic potential. These results, indicate a safe profile for the CD45 and TRAC gRNAs.

[0431] Example 2 - In vitro functional assessment of CD45 CAR1 T cells

[0432] To select the most suitable target cells for subsequent in vitro functional assays, the CD45 antigen density on a series of human AML and ALL leukaemia cell lines was assessed (Figure 7). Jurkat cells expressed the highest levels of CD45 with -179,000 CD45 molecules per cell. SupT1 CD19, MOLM14 and OCIM1 expressed medium-high levels of between -40,000 to 77,000 molecules / cell. HL-60 and MV4-11 were AML cell lines which expressed medium-low levels of CD45 (10,000 to 13,000 molecules / cell), whilst the T-ALL cell line, J45.01 , Jurkat CD45KO and SupT1CD45KO expressed low levels of CD45 (550-2000 molecules / cell). Nalm6 and HEK293T (non-haemopoietic cell line) were negative for CD45, showing only background levels of staining.

[0433] Functional assays were carried to assess the functionality of DKO CD45CAR T cells compared to unedited CD45 CAR T cells. 51 Cr cytotoxicity assays showed that only DKO CD45CAR1 , but not unedited (WT) or DKO untransduced (UT) or unedited CD45CAR1 T cells could induce specific cytotoxicity against CD45+ SupT 1 target cells even at a 1 : 1 effectortarget (E:T) ratio, but not CD45- SupT1 CD45KO cells (Figure 8). CD19CAR T cells were only able to specifically CD19 expressing SupT 1 CD19 cells and that this was independent of CD45 / TRAC knockout. Similarly, DKO CD45CAR1 T cells showed specific proliferation in response to CD45-expressing targets in 3H-thymidine incorporation assays (Figure 9A-9C) as well as specific production of interferon gamma (IFN-y) (Figure 9D-10F). Similar data was seen with DKO CD45CAR1 cells generated from multiple donors. The lack of proliferation and IFN- y production of the DKO CAR T cells in response to PHA is further evidence of successful TRAC knockout which ablates the response to PHA. Interestingly, unedited CD45CAR T cells which express CD45 proliferate and secrete IFN- y in absence of exogenous target cells. This is considered to be due to CD45CAR binding to CD45+ cells in the cultures which may occur before fratricide is observed.

[0434] Example 3 - Comparison of different CD45 CARs The in vitro function of CD45 CAR1 was compared against other CD45 CARs derived from other anti-CD45 antibodies (Table 1). These included the BC8 binder with either a 4-1 BB (Wellhausen et al.-, 2023; Sci Transl Med. 15(714):eadi1145) or a CD28 co-stimulatory domain, a comparative binder A in two scFv orientations and TanCARI , as well as a CAR1 T2A iCasp9 construct (CAR1 co-expressing inducible caspase 9).

[0435] Table 1. CD45 CAR constructs

[0436] Differences in transduction efficiency were adjusted for by addition of DKO UT cells. As shown in Figure 10A, DKO T cells transduced with CD45CAR1 and Binder A (VH / VL) showed markedly enhanced deletion of residual CD45+ cells at 7 days post-transduction compared to all other constructs, suggesting improved activity against CD45+ targets compared to the BC8- based CARs and Binder A (VL / VH). This was confirmed in flow-based cytotoxicity assays where DKO CD45 CAR1 T cells expressing CAR1 showed enhanced cytotoxicity against CD45+ MOLM14 and Jurkat cells particularly at low E:T ratios compared to the Binder A and BC8 based CAR T cells (Figure 10B). This effect was particularly pronounced in cell line targets expressing low CD45 antigen density. Similarly, DKO CD45 CAR1 T cells showed enhanced proliferation (Figure 10C) and showed secretion of IL-2 in response to CD45+ targets compared to CAR T cells expressing BC8-based CARs and Binder A-based CARs (Figure 10D). T cells transduced with a bicistronic lentiviral vector encoding CAR1 and iCasp9 also showed high level specific cytotoxicity against CD45+ targets.

[0437] Taken together, these results show the CD45CAR1 has improved functionality compared to other CD45 CARs tested.

[0438] Example 4 - DKO CD45CAR T cells target human clonogenic progenitors and HSCs

[0439] The activity of DKO CD45CAR1 T cells against human haemopoietic progenitors and HSCs was assessed. DKO CD45CAR1 T cells targeted human CD34+cells and inhibited colony formation at 1 :1 E:T ratios with more pronounced inhibition at 5:1 E:T ratio (Figure 11 A). NSG mice were subsequently transplanted with human CD34+cells and injected with 2x106unedited CD45 CAR1 or DKO CD45 CAR1 T cells on the following day (Figure 11 B). Engraftment of human CD45+cells in the bone marrow at 5 weeks post-CD34+cell transplant was almost completely ablated by DKO CD45CAR1 T cells (mean human CD45+= 0.04%) and significantly reduced by unedited CD45CAR1 cells (mean human CD45+= 8%) compared to PBS treatment (mean human CD45+= 78%) (Figure 11C). This also resulted in the complete absence of human HSCs in the bone marrow of mice treated with DKO CD45CAR1 T cells or unedited CD45CAR1 T cells compared to PBS control mice (Figure 11 D).

[0440] Example 5 - Assessment of the anti-leukaemic activity of DKO CD45CAR1 T cells in vivo.

[0441] The ability of DKO CD45CAR1 T cells to prevent development of AML in a xenogeneic MOLM14-Luciferase tumour model was then tested. As shown in Figure 12, DKO CD45CAR T cells but not DKO CD19CAR T cells effectively prevented development of AML, with complete deletion of AML from the marrow and improved survival in this model. This contrasts to the data of Wellhausen et al (as above) where CRISPR-Cas9 knockout of CD45 in CD19 CAR T cells prevented their function in vivo; confirming the data in present earlier examples that DKO CD45CAR1 T cells may have improved functionality compared to the other CD45CAR (e.g. BC8CAR) in the context of CD45 deficiency. Importantly, mice receiving DKO CD45CAR T cells showed no toxicity to the non-haemopoeitic tissues.

[0442] To test the efficacy of DKO CD45CAR T cells in a more clinically relevant setting, the ability to induce remission in mice with established AML was assessed. As shown in Figure 13, DKO CD45 CAR T cells were able to induce regression of AML and prolonged survival in an established MOLM14 tumour model whereas DKO CD19CAR T cells derived from the same donor did not, demonstrating the potent anti-leukaemic activity of DKO CD45CAR T cells in this disease.

[0443] Infusion of 2x106DKO CD45CAR1 T cells induced sustained disease control for at least 3 weeks, whereas treatment with 2x106CD19CAR T cells did not (Figure 13B and 13C; Figure 15B and 15C). CD19CAR T cell treated mice showed the same disease progression as control mice treated with PBS or untransduced DKO T cells (Figure 13B and 13C; Figure 15B and 15C). Furthermore, at a higher dose of 5x106DKO CD45CAR1 T cells, complete elimination of AML was observed by day 36 (Figure 15B and 15C).

[0444] The mice were re-challenged with tumour at 4 weeks after the initial DKO CD45CAR T infusion, and compared to a cohort of naive mice which received AML cells but no CAR T cells. In DKO CD45CAR T infused mice following rechallenge, tumour burden was reduced and progression was impaired, likely due to the persistence of DKO CD45CAR1 T cells, compared to the cohort of naive mice (Figure 15C). Both doses of DKO CD45CAR1 T cells induced prolonged survival of AML xenograft mice, whereas mice treated with DKO CD19CAR T cells did not and showed the same survival as mice treated with PBS and untransduced DKO T cells (Figure 13D; Figure 15D). Abundant levels of human T cells were detected in the bone marrow of mice treated with 2x106DKO CD45CAR1 T at 4 weeks after CAR T infusion (Figure 15E). However, almost no T cells were detected in mice treated with 5x106DKO CD19CAR T cells, despite these mice being culled at an earlier timepoint. Furthermore, AML cells were absent from bone marrow in 4 / 5 mice treated with 2x106DKO CD45CAR1 T cells (Figure 15F). These data suggest that DKO CD45CAR1 T cells can mediate regression of established AML and can persist for up to 4 weeks in vivo.

[0445] Example 6 - Deletion of DKO CD45CAR1 T cells using Rimiducid inducible caspase 9

[0446] Since DKO CD45CAR T cells were shown to efficiently delete human HSCs (Figure 11), treatment with them would be predicted to not only target leukaemic cells but also render the patient aplastic. If DKO CD45CAR1 T cells persisted this could potentially prevent engraftment of donor / gene corrected stem cells. In order to prevent this, a Rimiducid inducible caspase 9 suicide gene was co-expressed with CD45CAR1 using a bicistronic lentiviral vector to enable deletion of DKO CD45CAR1 T cells prior to SCT / gene therapy. As shown in Figure 14, DKO CD45CAR1 T cells co-expressing iCaspase 9 (CAR1 T2A iCasp9) were rapidly and efficiently deleted after treatment with Rimiducid in vitro.

[0447] 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.

[0448] Example 7 - DKO CD45CAR1 T cells have enhanced functionality compared to DKO BC8-based CAR T cells in vivo

[0449] The anti-tumour activity of DKO CD45CAR1 and DKO BC8-based CAR T cells in vivo was compared using a MOLM-14 xenogeneic AML model (Figure 16A). Both DKO CD45CAR1 and DKO BC8 (CD28) CAR T cells induced tumor regression, whereas DKO BC8 (41 BB) CAR T did not and showed the same disease course as mice treated with PBS and untransduced DKO T cells (Figure 16B and 16C). Tumour burden in DKO CD45CAR1 treated mice was lower than in mice treated with DKO BC8 (CD28) CAR T cells. Furthermore, DKO CD45CAR1 T cell treatment prolonged survival of AML xenograft mice compared to mice treated with BC8-based CAR T cells (Figure 16D). These data demonstrate that DKO CD45CAR1 T cells generated using the YTH24.5 clone, have improved functionality in vitro and in vivo when compared to the BC8-based CD45CAR T cells.

[0450] Example 8 - DKO CD45CAR1 T cells demonstrate potent activity against primary AML and CD34+stem / progenitor cells in vitro and in vivo

[0451] To further demonstrate that CD45 is a superior target for CAR T cells in AML, analysis of leukaemic stem cells (LSCs) from 15 high risk paediatric patients using single cell CITEseq showed that CD45 was expressed at high levels and almost all LSCs (Figure 2). In contrast, the expression of CD33, CD123 and CLL-01 , were expressed at lower levels and were expressed on a lower proportion of LSCs, between 0.7 to 0.9. These data suggest that targeting CD45 on bulk AML cells and on LSCs with DKO CD45CAR T cells could treat AML and potentially reduce occurrence of relapse.

[0452] In addition, DKO / CD45CAR1 were able to specifically kill primary AML and CD34+stem cells / progenitors cells in vitro. Three different adult de novo AML samples were co-cultured for 48h in vitro with DKO Unt, DKO CD45CAR1 and DKO CD19CAR. Only DKO CD45CAR1 cells showed significant and dose-dependent killing of primary AML cells, with 100% specific killing achieved at an EffectorTarget ratio of 1 :1 (Figure 17A). However DKO Unt and DKO CD19CAR T cells resulted in non- specific or background levels of killing. As CD45 is also on all healthy nucleated hematopoietic cells including HSCs, we assessed ability of DKO CD45CAR1 T cells to target CD45+healthy haematopoietic cells. Co-culture of DKO CD45CAR1 T cells with healthy donor CD34+stem / progenitor cells lead to a >90% reduction in the absolute number of CD34+cells (Figure 17B).

[0453] In a PDX model of AML in NSG mice, DKO CD45CAR1 T cells were able to significantly reduce the numbers of PDX (hCD45+ / CD33+cells) detected in the bone marrow at 5 weeks post-treatment (Figure 18B). A mean of 512 PDX cells were detected in the bone marrow of DKO CD45CAR1 treated mice compared to 3407 for DKO Unt and 6469 for DKO CD19CAR at 5 weeks post-treatment. These data demonstrate that DKO CD45AR1 T cells can target AML and normal haematopoietic cells and therefore can be used to treat AML and be used as a conditioning agent prior to stem cell transplant.

Claims

CLAIMS1. A chimeric antigen receptor (CAR) comprising a CD45 antigen binding domain comprising:(a) a heavy chain variable region that comprises:(i) a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 1,(ii) a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 2, and(iii) a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 3, and(b) a light chain variable region that comprises:(i) a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 4,(ii) a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 5, and(iii) a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 6.

2. The CAR according to claim 1 , wherein the heavy chain variable region comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 8.

3. The CAR according to claim 1 or 2, wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 8.

4. The CAR according to any previous claim, wherein the antigen binding domain is an scFv, wherein the heavy chain variable region and the light chain variable region are joined by a linker, optionally where the linker comprises an amino acid sequence of SEQ ID NO: 13.

5. The CAR according to claim 4, wherein the heavy chain variable region is positioned to the N-terminal side of the linker, which is positioned to the N-terminal side of the light chain variable region.

6. The CAR according to claim 4 or 5, wherein the scFv comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 9.

7. The CAR according to claim 4 or 5, wherein the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 9.

8. The CAR according to any previous claim, wherein the CAR further comprises a spacer domain and / or an intracellular costimulatory domain.

9. The CAR according to claim 8, wherein the spacer comprises a CD8 spacer, optionally wherein the CD8 spacer comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 14.

10. The CAR according to claim 9, wherein the CAR comprises a CD8 spacer and a CD8 transmembrane domain, optionally wherein the CD8 spacer is linked to the CD8 transmembrane domain and together comprise an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 15.

11. The CAR according to any of claims 8 to 10, wherein the intracellular costimulatory domain comprises a CD28 domain.

12. The CAR according to claim 11 , wherein the intracellular costimulatory domain comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 10.

13. The CAR according to claim 11 or 12, wherein the intracellular costimulatory domain comprises an amino acid sequence of SEQ ID NO: 10.

14. The CAR according to any previous claim, wherein the CAR comprises an intracellular signalling domain comprising a CD3 domain.

15. The CAR according to claim 14, wherein the intracellular signalling domain comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 11 .

16. The CAR according to claim 14 or 15, wherein the intracellular signalling domain comprises or consists of an amino acid sequence of SEQ ID NO: 11.

17. The CAR according to any of claims 14 to 16, wherein the intracellular signalling domain does not comprise a 4-1 BB costimulatory domain.

18. The CAR according to any of claims 11-17, wherein the CAR comprises an endodomain that comprises or consists of:(i) an intracellular costimulatory domain comprising an CD28 domain, and(ii) an intracellular signalling domain comprising a CD3 domain, optionally wherein the total endodomain comprises or consists of an amino acid sequence with at least 95% identity to SEQ ID NO: 16.

19. The CAR according to any previous claim, wherein the CAR comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 12.

20. The CAR according to any previous claim, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 12.

21. A nucleic acid encoding the CAR according to any one of claims 1-20.

22. The nucleic acid according to claim 21 , wherein the nucleic acid comprises a polynucleotide sequence with at least 75% sequence identity to SEQ ID NO: 22.

23. The nucleic acid according to claim 20 or 21, wherein the nucleic acid comprises a polynucleotide sequence of SEQ ID NO: 22.

24. A vector comprising the nucleic acid of any of claims 20-23.

25. The vector according to claim 24, wherein the vector is a viral vector, optionally wherein the vector is a lentiviral vector.

26. The vector according to claim 24 or 25, wherein the vector comprises a further nucleic acid encoding transgene.

27. The vector according to claim 26, wherein the transgene is an inducible suicide switch gene, optionally wherein the inducible suicide switch gene is iCaspase 9.

28. The vector according to any one of claims 24-27, wherein the vector further comprises one or more gene editing systems.

29. The vector according to claim 28, wherein the vector comprises one or more nucleic acids encoding one or more gene editing systems, wherein the one or more gene editing systems comprise:(i) a gene editing system for reducing or eliminating expression of endogenous CD45,(ii) a gene editing system for reducing or eliminating expression of endogenous TCR, and / or(iii) a gene editing system for the introduction of an inducible suicide switch gene. optionally wherein the inducible suicide switch gene is designed to be inserted at an endogenous CD45 gene locus.

30. A cell which comprises the CAR of any one of claims 1-20, the nucleic acid of any one of claims 21-23, and / or the vector of any one of claims 24-29.

31. The cell according to claim 30, wherein the cell is an effector immune cell, optionally wherein the cell is a T cell or NK cell.

32. The cell according to claim 30 or 31, wherein the cell is a cytotoxic immune cell.

33. The cell according to claim 32, wherein the cell is a first cell which is modified such that the cytotoxic effect of a second cell according to claim 32 against the first cell is reduced.

34. The cell according to any one of claims 30-33 wherein the cell is modified such that it is not bound by a CAR according to any of claims 1-20.

35. The cell according to any one of claims 30-34, wherein the cell has reduced expression or lacks expression of endogenous CD45.

36. The cell according to claim 35, wherein the cell is a CD45 knock-out cell.

37. The cell according to any of claims 30-36, wherein the cell has reduced expression or lacks expression of an endogenous T cell receptor (TCR).

38. The cell according to claim 37, wherein the cell is a T cell receptor alpha constant (TRAC) knock-out cell.

39. The cell according to any of claims 30-38, wherein the cell comprises an inducible suicide switch.

40. The cell according to any one of claims 30-39, wherein the cell comprises a rimiducid- inducible caspase 9 suicide gene.

41. A method of making a cell according to any of claims 30 to 40.

42. The method according to claim 41 , wherein the method comprises introducing the CAR according to any one of claims 1-20, the nucleic acid according to any one of claims 21-23, and / or the vector according to any one of claims 24-29 to a cell.

43. The method according to claim 41 or 42, wherein the method comprises introducing a vector according to any one of claims 24-29 to a cell.

44. The method according to any one of claim 41 or 42, wherein the method comprises introducing a vector according to claim 27 into the cell.

45. The method according to any one of claims 41-44, wherein the method comprises:(i) reducing or abolishing expression of CD45 in a cell, and / or(ii) reducing or abolishing expression of TCR in a cell.

46. The method according to claim 45, wherein the reducing or abolishing expression of CD45 is carried out by knock-down or knock-out of a PTPRC gene in a cell.

47. The method according to claim 45 or 46, wherein the reducing or abolishing expression of TCR is carried out by knock-down or knock-out of a gene encoding a subunit of the TCR in a cell, optionally wherein the gene is a TRAC gene.

48. The method according to claim 45 to 47, wherein the knock-down or knock-out is carried out by a gene editing system, optionally wherein the gene editing system is a CRISPR-based gene editing system, further optionally wherein the CRISPR-based gene editing system is a CRISPR / Cas9 gene editing system.

49. The method according to claim 48, wherein the gene editing system is introduced into the cell through use of a vector or electroporation, preferably wherein the gene editing system is introduced through use of electroporation.

50. A pharmaceutical composition comprising a plurality of cells according to any of claims 30-40.

51. A kit for the production of a cell according to any one of claims 30-40, wherein the kit comprises a vector according to any of claims 24-29 and optionally one or more gene editing systems.

52. A kit for the production of a cell according to any one of claims 30-40, wherein the kit comprises a vector according to any of claims 24-29 and:(i) a gene editing system for reducing or abolishing expression of CD45 in a cell, and / or(ii) a gene editing system for reducing or abolishing expression of TCR in a cell, optionally wherein the gene editing systems are suitable for introduction to a cell via electroporation.

53. A kit for the production of a cell according to any one of claims 30-40, wherein the kit comprises a vector according to any of claims 24-29 and optionally one or more vectors comprising a nucleic acid encoding one or more gene editing systems.

54. The kit according to claim 53, wherein the kit comprises:(i) a vector according to any of claims 24-27 and(ii) one or more vectors comprising:(a) a gene editing system for reducing or eliminating expression of endogenous CD45,(b) a gene editing system for reducing or eliminating expression of endogenous TCR, and / or(c) a gene editing system for the introduction of an inducible suicide switch gene.

55. The kit according to claim 54, wherein the kit comprises:(i) a vector according to any of claims 24-27,(ii) a vector comprising a gene editing system for reducing or eliminating expression of endogenous CD45(iii) a vector comprising a gene editing system for reducing eliminating expression of endogenous TCR, optionally wherein the gene editing system deletes the endogenous TRAC gene.

56. The kit according to claim 51 to 55, wherein the gene editing systems are CRISPR- based gene editing systems, optionally wherein the gene editing systems comprise an RNA-guided nuclease, one or more guide nucleic acids, and / or one or more donor nucleic acids.

57. The CAR according to any of claims 1-20, the nucleic acid according to any of claims 21-23, the vector according to any of claims 24-29, the cell according to any of claims30-40 or the pharmaceutical composition according to claim 50 for use as a medicament.

58. The CAR according to any one of claims 1-20, the nucleic acid according to any of claims 21-23, the vector according to any of claims 24-29, the cell according to any of claims 30-40 or the pharmaceutical composition according to claim 50 for use in myeloablation.

59. The CAR, nucleic acid, vector, cell or pharmaceutical composition for use according to claim 58, wherein the myeloablation is to eradicate the hematopoietic stem cells of a subject to create a niche for an incoming stem cell graft.

60. The CAR according to any of claims 1-20, the nucleic acid according to any of claims 21-23, the vector according to any of claims 24-29, the cell according to any of claims 30-40 or the pharmaceutical composition according to claim 50 for use in the treatment or prevention of cancer.61 . The CAR, nucleic acid, vector, cell or pharmaceutical composition for use according to claim 60, wherein the cancer is a haematological malignancy, optionally wherein the haematological malignancy is a high risk or refractory haematological malignacy.

62. The CAR, nucleic acid, vector, cell or pharmaceutical composition for use according to claim 61 , wherein the high risk or refractory haematological malignancy may be AML, T-ALL or multiple myeloma.

63. The CAR according to any one of claims 1-20, the nucleic acid according to any of claims 21-23, the vector according to any of claims 24-29, the cell according to one of claims 30-40 or the pharmaceutical composition according to claim 50 for use in the treatment or prevention of a non-malignant haematological disorder.

64. The CAR, nucleic acid, vector, cell or pharmaceutical composition for use according to claim 63 wherein the non-malignant haematological disorder is a genetic disorder of the haematopoietic system.

65. The CAR, nucleic acid, vector, cell or pharmaceutical composition for use according to claim 64 wherein the non-malignant haematological disorder is a haemoglobinopathy.

66. The CAR, nucleic acid, vector, cell or pharmaceutical composition for use according to claim 63 wherein the non-malignant haematological disorder is a bone marrow failure.Q7. The CAR according to any of claims 1-20, the nucleic acid according to any of claims 21-23, the vector according to any of claims 24-29, the cell according to any of claims 30-40 or the pharmaceutical composition according to claim 50 for use in a method of preparing a subject for a haematopoietic stem cell transplant.

68. The CAR, nucleic acid, vector, cell or pharmaceutical composition for use according to claim 67, wherein the haematopoietic stem cell transplant comprises stem cell from a healthy donor, and / or gene edited stem cells.

69. A method of treating or preventing cancer in a subject, wherein the method comprises administering the CAR according to any of claims 1-20, the nucleic acid according to any of claims 21-23, the vector according to any of claims 24-29, the cell according to any of claims 30-40 or the pharmaceutical composition according to claim 50 to the subject.

70. A method of treating or preventing a non-malignant haematological disorder, wherein the method comprises administering CAR according to any of claims 1-20, the nucleic acid according to any of claims 21-23, the vector according to any of claims 24-29, the cell according to any of claims 30-40 or the pharmaceutical composition according to claim 50 to the subject.

71. A method of myeloablation, wherein the method comprises administering the CAR according to any of claims 1-20, the nucleic acid according to any of claims 21-23, the vector according to any of claims 24-29, the cell according to any of claims 30-40 or the pharmaceutical composition according to claim 50 to the subject.

72. A method of eradicating or reducing haematopoietic stem cells in a subject, wherein the method comprises administering the CAR according to any of claims 1-20, the nucleic acid according to any of claims 21-23, the vector according to any of claims 24-29, the cell according to any of claims 30-40 or the pharmaceutical composition according to claim 50 to the subject.

73. The method according to claim 72, wherein the number of haematopoietic stem cells is reduced so as to allow engraftment of an incoming stem cell graft.

74. Use of the CAR according to any of claims 1-20, the nucleic acid according to any of claims 21-23, the vector according to any of claims 24-29, the cell according to any of claims 30-40 or the pharmaceutical composition according to claim 50 for the manufacture of a medicament for the treatment or prevention of cancer.

75. Use of the CAR according to any of claims 1-20, the nucleic acid according to any of claims 21-23, the vector according to any of claims 24-29, the cell according to any of claims 30-40 or the pharmaceutical composition according to claim 50 for the manufacture of a medicament for the treatment or prevention of a non-malignant haematological disorder.

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