Engineered trispecific tancar expressing cells targeting cd33, cd123 and cll1 and uses thereof in cancer therapy

Abstract

The present invention relates to a cell which expresses a tri-specific chimeric antigen receptor (CAR) with multiple antigen specificities. The cell targets multiple antigens characteristic of acute myeloid leukemia (AML), specifically CD33, CD123, and CLL1.

Description

[0001] CELL

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a cell which expresses a tri-specific chimeric antigen receptor (CAR) with multiple antigen specificities. The cell targets multiple antigens characteristic of acute myeloid leukemia (AML), specifically CD33, CD123, and CLL1

[0004] BACKGROUND TO THE INVENTION

[0005] Acute Myeloid Leukemia

[0006] Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the uncontrolled clonal proliferation of myeloid precursors in the bone marrow and blood, resulting in accumulation of leukemic blasts and severe impairment of normal hematopoiesis. AML is the most common acute leukemia in adults and has the highest death rate of all leukemias. An estimated 20830 people in the Unites States were predicted be diagnosed with AML and 10460 deaths were projected to occur from AML in 2015. Gains in long-term survival in AML over the last decade have remained modest.

[0007] Current induction chemotherapy can produce initial complete remission in almost 70% of young adult patients. However, 43% of patients will eventually relapse, and 18% never attain complete remission at frontline induction treatment. The 5-year overall survival rate for patients with AML after the first relapse ranges from 7 to 12%. Allogeneic hematopoietic stem cell transplantation (alloHSCT) provides the best chance to cure a patient with relapsed or refractory AML. It is the preferred treatment route following a second remission and can lead to 5-year disease-free survival in 40-50% of patients.

[0008] Using current treatment strategies, the second complete remission rate is achieved in only about half of relapsed patients who previously attained a complete remission that lasted longer than 6 months and is only 20% or fewer of patients with primary refractory disease and those with an initial complete remission lasting less than 6 months. In addition, the considerable complications of conventional salvage chemotherapy may worsen the performance status and organ function of the patient and decrease the chance of a successful allo HSCT. There is therefore a need for improved therapeutic approaches to treat AML.

[0009] Chimeric Antigen Receptors Chimeric antigen receptors (CARs) graft the specificity of a monoclonal antibody onto the effector function of a T-cell. CAR T-cell therapy directed against CD19 has been highly effective in B-cell malignancies, although CD19 negative escape is a cause of relapse in a considerable portion of patients. CAR T-cell therapy against AML is in early clinical testing. Despite several preclinical studies demonstrating cytotoxic potential of various AML antigen-targeting CAR T cells, translation to the clinic has been slow. This highlights inherent challenges in developing CAR-related treatment strategies for patients with AML. To date, a small number of patients with relapsed / refractory AML have been treated with CAR T-cell immunotherapies via early phase clinical trials, as shown Table 1.

[0010] Table 1 : Current phase I clinical trials of CAR T-cell immunotherapy for patients with relapsed / refractory AML

[0011] DESCRIPTION OF THE FIGURES

[0012] Figure 1 - Schematic diagram illustrating hematopoiesis in humans.

[0013] Figure 2 - Different binding domain formats of chimeric antigen receptors Fab CAR format; (b) dAb CAR format; (c) scFv CAR format

[0014] Figure 3 - Schematic of tri-specific CAR and triple compound CAR. Here the trispecific CAR format is labelled as a TanCAR.

[0015] Figure 4 - Dilution of cell trace violet in different CAR constructs.

[0016] Figure 5 - A - Fold expansion of cells transduced with different CAR constructs. All measurements are normalised to the result for the FMC63 CAR. B - Interferongamma production of cells transduced with various CAR constructs.

[0017] Figure 6 - 24 hour cytotoxicity assay. Cells transduced with various CAR constructs were exposed to either SupT1-NT cells (A) or M0LM14 cells (B) at an effectortarget (E:T) ratio of 1:4. Figure 7 -24 hour co-culture IL-2 production assay. Cells transduced with various CAR constructs were co-cultured either SupT1-NT cells (A) or M0LM14 cells (B) at an effectortarget (E:T) ratio of 1:4.

[0018] Figure 8 - 24 hour cytotoxicity assay conducted with single-antigen expressing M0LM14 cells. Each gene was knocked down in the target cell using CRISPR. An effectortarget (E:T) ratio of 1 :4 was used in each case. Results were normalised to the FMC63 CAR results.

[0019] Figure 9 - Bioluminescence burden of mice injected with luciferase expressing M0LM14 AML cells, followed by cells expressing various CAR constructs. The single CARs and the tri-specific CAR (labelled TanCAR) were able to control the tumour burden in this model.

[0020] Figure 10 - Comparison of survival of mice injected with luciferase expressing MOLM14 AML cells, followed by cells expressing various CAR constructs. At the end of the experiment, there were 4 / 5 mice in the tri-specific CAR (labelled TanCAR) and CD123 CAR groups alive, 3 / 5 in the CD33 CAR group and 1 / 5 of the triple compound group alive, with all of the mice receiving NT cells or CLL1CAR T cells only surviving to 22 and 25 days respectively.

[0021] Figure 11 - Triple CAR T-cells show potent in vitro responses which are equivalent or greater than those of single CAR-expressing T-cells. MOLM14 expresses CD33, CD123 and CLL1 naturally, SupT1-CD123 / CD33 / CLL1 is a T-cell line engineered to express all 3 antigens at high level. FMC63CAR is an irrelevant, CD19 targeting CAR, NT = non-transduced T-cells. A) Cytotoxicity: E:T 1:4, 24 hour co-culture. B) In vitro proliferation assay E:T 1 :10 and 7 day co-culture. C-D) IFN-gamma and IL-2 production at E:T 1 :4, 24 hour co-culture Significance is indicated for comparison to NT condition

[0022] Figure 12 - Triple CAR T-cells show potent in vitro responses which are equivalent or greater than those of single CAR-expressing T-cells. MOLM14 expresses CD33, CD123 and CLL1 naturally, SupT1-CD123 / CD33 / CLL1 is a T-cell line engineered to express all 3 antigens at high level. FMC63CAR is an irrelevant, CD19 targeting CAR, NT = non-transduced T-cells. A) Cytotoxicity: E:T 1:4, 24 hour co-culture. B) In vitro proliferation assay E:T 1 :10 and 7 day co-culture. C-D) IFN-gamma and IL-2 production at E:T 1 :4, 24 hour co-culture Significance is indicated for comparison to

[0023] NT condition

[0024] Figure 13 - Improved in vivo anti-tumour efficacy of triple compound CAR T-cells against antigenically-heterogeneous MOLM14 AML compared to single CAR T-cells. To model sub-clonal heterogeneity and emergence of antigen-loss AML variants, CRISPR gene disruption of CD33, CD123 and CLL1 of parental MOLM14 was carried out, creating 3 KO cell lines each lacking one antigen. These were mixed 1 :1 :1 and injected at a total of 1x10A6 cells into mice. A) BLI was carried out to assess tumour trajectory. B) Absolute count of M0LM14 cells in spleen I BM / extra medullary sites. C) Survival of cohorts. Median OS was 29-31 days (recipients of NT cells and single CARs) but recipients of triple CAR T cells remained alive to end of experiment (D40, p=0.003 compared to NT).

[0025] SUMMARY OF ASPECTS OF THE INVENTION

[0026] In a first aspect, the present invention provides a cell expressing a tri-specific chimeric antigen receptor (CAR) with multiple antigen specificities. The cell targets multiple antigens characteristic of acute myeloid leukemia (AML), specifically CD33, CD123, and CLL1. The tri-specific CAR comprises:

[0027] (i) an anti-CD33 antigen binding domain;

[0028] (ii) an anti-CLL1 antigen binding domain;

[0029] (iii) an anti-CD123 antigen binding domain;

[0030] (iv) a spacer derived from CD8;

[0031] (v) a transmembrane domain; and

[0032] (vi) an intracellular signalling domain.

[0033] One or more, or all, of the antigen binding domains may comprise a domain antibody (dAb) antigen binding domain.

[0034] An anti-CD33 domain antibody (dAb) antigen binding domain may comprise the following complementarity determining regions:

[0035] (i) CDR1 - GRTFSMHS (SEQ ID No. 1); CDR2 - VTWSGDTF (SEQ ID No. 2); CDR3 - KDDPYRPAYDY (SEQ ID No. 3);

[0036] (ii) CDR1 - GRTFSSYV (SEQ ID No. 4); CDR2 - ISWSGGST (SEQ ID No. 5); CDR3 - AAMELRGGSYNYASSRQYDY (SEQ ID No. 6); (iii) CDR1 - EIAFSNFN (SEQ ID No. 7); CDR2 - ISSHGDTNY (SEQ ID No. 8); CDR3 - NANDPFLSVSDF (SEQ ID No. 9);

[0037] (iv) CDR1 - GSIFSINA (SEQ ID No. 10); CDR2 - ISWSGGST (SEQ ID No. 5); CDR3 - AAISGWGRSIRVGERYEYDY (SEQ ID No. 11);

[0038] (v) CDR1 - GRTSSSST (SEQ ID No. 12); CDR2 - ITLSGGST (SEQ ID No. 13); CDR3 - AARRWSNNRGGYDRAGYDY (SEQ ID No. 14); or

[0039] (vi) CDR1 - GRTFSSYA (SEQ ID No. 15); CDR2 - ITWSGGST (SEQ ID No. 16); CDR3 - AMLLRGGLYDYTDYILYNY (SEQ ID No. 17).

[0040] An anti-CD33 domain antibody (dAb) antigen binding domain may comprise one of the sequences shown as SEQ ID No. 18, 19, 20, 21, 22 or 23.

[0041] An anti-CLL-1 domain antibody (dAb) antigen binding domain may comprise the following complementarity determining regions:

[0042] (i) CDR1 - GFTFGNHD (SEQ ID No. 48); CDR2 - IDSGGNVI (SEQ ID No. 49); CDR3 - ATDLDSGAESLESVY (SEQ ID No. 50);

[0043] (ii) CDR1 - GFAFGSAD (SEQ ID No. 51); CDR2 - IDSGGNTQ (SEQ ID No. 52); CDR3 - TDLDPTTDSLENVY (SEQ ID No. 53);

[0044] (iii) CDR1 - GRTFSAYF (SEQ ID No. 54); CDR2 - INWNGDSS (SEQ ID No. 55); CDR3 - AADTHGAVGLGSERLYDY (SEQ ID No. 56);

[0045] (iv) CDR1 - GIGVSSTG (SEQ ID No. 57); CDR2 - IDRDGTT (SEQ ID No. 58); CDR3 - TVVGDYY (SEQ ID No. 59);

[0046] (v) CDR1 - GFIFGNYD (SEQ ID No. 60); CDR2 - ISSGGNDI (SEQ ID No. 61); CDR3 - AADLDPGTDSLDNIH (SEQ ID No. 62); or

[0047] (vi) CDR1 - GFTLDYYA (SEQ ID No. 63); CDR2 - ISSSDGST (SEQ ID No. 64); CDR3 - AEAVYYAGVCVAMYDS (SEQ ID No. 65).

[0048] An anti-CLL-1 domain antibody (dAb) antigen binding domain may comprise one of the sequences shown as SEQ ID No. 66, 67, 68, 69, 70 or 71.

[0049] An anti-CD123 domain antibody (dAb) antigen binding domain may comprise the following complementarity determining regions:

[0050] (i) CDR1 - GRSINTYA (SEQ ID No. 24); CDR2 - INYNSRYT (SEQ ID No. 25); CDR3 - AATSYYPTDYDVASRVATWPS (SEQ ID No. 26);

[0051] (ii) CDR1 - GISLNA (SEQ ID No. 27); CDR2 - IKIGGVS (SEQ ID No. 28); CDR3 - NTYPPYLNGMDY (SEQ ID No. 29); (iii) CDR1 - GRSFNTDA (SEQ ID No. 30); CDR2 - ISWDGTRT (SEQ ID No. 31); CDR3 - AAEPQKAWPIGTSAAGFRS (SEQ ID No. 32);

[0052] (iv) CDR1 - GSSISV (SEQ ID No. 33); CDR2 - ISWSDGNT (SEQ ID No. 34); CDR3 - AVEPRGWPKGHRY (SEQ ID No. 35);

[0053] (v) CDR1 - GSSFSINV (SEQ ID No. 36); CDR2 - ISWSDGST (SEQ ID No. 37); CDR3 - AVEPRGWPKGHRY (SEQ ID No. 38); or

[0054] (vi) CDR1 - GSIFRINA (SEQ ID No. 39); CDR2 - VNWIGGTT (SEQ ID No. 40); CDR3 - SATDKGGSSRY (SEQ ID No. 41).

[0055] An anti-CD123 domain antibody (dAb) antigen binding domain may comprise one of the sequences shown as SEQ ID No. 42, 43, 44, 45, 46, 47, or 103.

[0056] An anti-FLT3 domain antibody (dAb) antigen binding domain may comprise the following complementarity determining regions:

[0057] (i) CDR1 - GIFKTNY (SEQ ID No. 72); CDR2 - FTNDGST (SEQ ID No. 73); CDR3 - YGLGH (SEQ ID No. 74);

[0058] (ii) CDR1 - GTISSIRY (SEQ ID No. 75); CDR2 - ITSSGNT (SEQ ID No. 76); CDR3 - YTMGY (SEQ ID No. 77);

[0059] (iii) CDR1 - GIFSTNY (SEQ ID No. 78); CDR2 - FTNDGGT (SEQ ID No. 79); CDR3 - CGLGH (SEQ ID No. 80);

[0060] (iv) CDR1 - GSISSIRY (SEQ ID No. 81); CDR2 - ITSSGST (SEQ ID No. 82); CDR3 - YTMGY (SEQ ID No. 83); or

[0061] (v) CDR1 - GIFSTNH (SEQ ID No. 84); CDR2 - FTNDGST (SEQ ID No. 85); CDR3 - YGLGH (SEQ ID No. 86).

[0062] An anti-FLT3 domain antibody (dAb) antigen binding domain may comprise one of the sequences shown as SEQ ID No. 87, 88, 89, 90 or 91.

[0063] The three antigen binding domains may be presented in any order relative to the membrane. Preferably, the anti-CD123 binding domain is the binding domain most proximal to the membrane and the anti-CLL1 binding domain is most distal from the membrane, with the anti-CD33 binding domain juxtaposed between the two. However, other orders for the binding domains are contemplated herein.

[0064] The intracellular signalling domain may comprise domains capable of transmitting one or more signals to the cell. For example, the ITAM-containing CD3-zeta endodomain may be used to transmit an activation signal to the cell after antigen is bound. Co- stimulatory endodomains can also be used to provide signals that promote T-cell proliferation and survival. There are two main types of co-stimulatory signals: those that belong the Ig family (CD28, ICOS) and the TNF family (0X40, 41 BB, CD27, GITR etc). Preferably the intracellular signalling endodomain comprises 4-1 BB and CD3

[0065] The tri-specific CAR may comprise the sequence shown in SEQ ID No. 96, or a variant having at least 80, 85, 90, 95, 98 or 99% sequence identity thereto.

[0066] In a second aspect, the present invention provides a nucleic acid construct which encodes tri-specific CAR as define in the first aspect.

[0067] In a third aspect, the present invention provides a method for making a cell according to the first aspect of the invention which comprises the step of transducing or transfecting a cell with a nucleic acid construct according to the second aspect of the invention.

[0068] In a fourth aspect, the present invention provides a vector comprising a nucleic acid construct according to the second aspect of the invention.

[0069] In a fifth aspect there is provided a pharmaceutical composition which comprises a plurality of cells according to the first aspect of the invention, together with a pharmaceutically acceptable carrier, diluent or excipient.

[0070] In a sixth aspect, there is provided a method for treating cancer which comprises the step of administering a pharmaceutical composition according to the fifth aspect of the invention to a subject.

[0071] The cancer may be acute myeloid leukemia (AML).

[0072] The method may also involve the step of subsequently administering an allogeneic transplant to the subject.

[0073] In a seventh aspect, there is provided a pharmaceutical composition according to the fifth aspect of the invention for use in treating cancer. In an eighth aspect, there is provided the use of a cell according to the first aspect of the invention in the manufacture of a pharmaceutical composition for treating cancer.

[0074] AML blast phenotype is much more heterogenous than that of acute lymphoblastic leukemia (ALL) blasts. Myelopoiesis is driven by stem cells which stochastically and in response to cues either replenish their compartment or differentiate. Akin to normal myeloid stem cells, AML stem cells propagate or differentiate to cause bone-marrow replacement with a range of cells at different differentiation states. AML stem cells can occur along the range of myelopoiesis and consequently have different surface antigen profile. Further, in a given patient, there may be stem cell nexi or a hierarchy of different stem cells at different points in ontogeny all contributing to the disease burden (Figure 1).

[0075] In order to treat the maximum number of patients, the present inventors have found that AML targeting by chimeric antigen receptors requires targeting of multiple antigens simultaneously along the myeloid lineage.

[0076] The tri-specific CAR of the present invention, incorporating a CD8-derived spacer domain, provides an advantage over prior art systems by offering lower basal activation compared to the hinge spacer. Furthermore, while targeting a single antigen may fail to eliminate the disease-relevant stem cell compartment, targeting multiple myeloid antigens simultaneously, however, means that the treatment is universal across AMLs. Immunotherapy using CAR-T cells targeting multiple antigens eradicates the disease stem cell compartment irrespective of the number and position of stem cell compartments. Targeting multiple antigens also reduces the likelihood of escape by antigen down-regulation.

[0077] FURTHER ASPECTS

[0078] The present invention also provides a cell composition comprising CAR-expressing cells expressing multiple CARs (also termed a compound CAR).

[0079] The composition of cells may express: an anti-CD33 chimeric antigen receptor (CAR); an anti-CLL1 CAR; and an anti-CD123 CAR. More specifically, the composition of cells may express a triple compound CAR with the binder / spacer combination: aCD33 / CD8, aCD123 / CD28, and aCLL1 / HNG. The triple compound CAR of the invention may be expressed in a cell using cleavage site as described elsewhere in the application. In particular, 2A self-cleaving peptides may be used.

[0080] The cells of the invention may also express the sort-suicide gene RQR8, which is described in WO2013 / 153391 (the contents of which are incorporated herein by reference).

[0081] The nucleic acid sequences, nucleic acid constructs, vectors and kits of vectors and methods described below may be used to make the cells of the cell composition of this aspect of the invention.

[0082] The cell composition may be used in a method for treating a disease, as described below.

[0083] Yet further aspects of the invention are summarised in the following numbered paragraphs:

[0084] A1. A cell comprising: an anti-CD33 chimeric antigen receptor (CAR) having a CD8 spacer; an anti-CLL1 CAR having a hinge spacer; and an anti-CD123 CAR having a CD28 spacer.

[0085] A2. A cell according to paragraph A1 , wherein one or more CAR(s) comprise(s) a domain antibody (dAb) antigen binding domain.

[0086] A3. A cell according to any of paragraphs A1 and A2, wherein each CAR comprises a domain antibody (dAb) antigen binding domain.

[0087] A4. A cell according to any preceding paragraph wherein the anti-CD33 CAR has the amino acid sequence shown in SEQ ID NO: 104.

[0088] A5. A cell according to any preceding paragraph wherein the anti-CLL1 CAR has the amino acid sequence shown in SEQ ID NO: 105.

[0089] A6. A cell according to any preceding paragraph wherein the anti-CD123 CAR has the amino acid sequence shown in SEQ ID NO: 106. A7. A cell according to paragraph A1 , wherein the anti-CD33 CAR has the amino acid sequence shown in SEQ ID NO: 104, the anti-CLL1 CAR has the amino acid sequence shown in SEQ ID NO: 105, and the anti-CD123 CAR has the amino acid sequence shown in SEQ ID NO: 106.

[0090] A8. A nucleic acid construct encoding: an anti-CD33 chimeric antigen receptor (CAR) having a CD8 spacer; an anti-CLL1 CAR having a hinge spacer; and an anti- CD123 CAR having a CD28 spacer.

[0091] A9. A method for making a cell according to paragraph A1 which comprises the step of transducing or transfecting a cell with a nucleic acid construct according to paragraph A7.

[0092] A10. A vector comprising a nucleic acid construct according to paragraph A8.

[0093] A11. A kit of vectors, which comprises:

[0094] (i) a first vector which comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR) which binds CD33 and has a CD8 spacer;

[0095] (ii) a second vector which comprises a nucleic acid sequence encoding a CAR which binds CLL1 and has a hinge spacer; and

[0096] (iii) a third vector which comprises a nucleic acid sequence encoding a CAR which binds CD123 and has a CD28 spacer.

[0097] A12. A pharmaceutical composition which comprises a plurality of cells according any of paragraphs A1 to A7, together with a pharmaceutically acceptable carrier, diluent or excipient.

[0098] A13. A method for treating cancer which comprises the step of administering a pharmaceutical composition according to paragraph A12 to a subject.

[0099] A14. A method according to paragraph A13, wherein the cancer is acute myeloid leukemia (AML).

[0100] A15. A method according to paragraph A14, which also involves the step of subsequently administering an allogeneic transplant to the subject. A16 A pharmaceutical composition according to paragraph A12 for use in treating cancer.

[0101] A17. The use of a cell according to any of paragraphs A1 to A7 in the manufacture of a pharmaceutical composition for treating cancer.

[0102] A18. The cell of any preceding paragraph, wherein the cell is a T cell or a natural killer (NK) cell.

[0103] DETAILED DESCRIPTION

[0104] CHIMERIC ANTIGEN RECEPTORS

[0105] The present invention relates to a cell which expresses a chimeric antigen receptors at the cell surface.

[0106] A classical chimeric antigen receptor (CAR) is a chimeric type I trans-membrane protein which connects an extracellular antigen-recognizing domain (binder) to an intracellular signalling domain (endodomain). The binder is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats which comprise an antibody-like antigen binding site. A spacer domain is usually necessary to isolate the binder from the membrane and to allow it a suitable orientation. A common spacer domain used is the Fc of I gG 1. More compact spacers can suffice e.g. the stalk from CD8a and even just the lgG1 hinge alone, depending on the antigen. A trans-membrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.

[0107] Early CAR designs had endodomains derived from the intracellular parts of either the y chain of the FCER1 or CD3 Consequently, these first generation receptors transmitted immunological signal 1 , which was sufficient to trigger T-cell killing of cognate target cells but failed to fully activate the T-cell to proliferate and survive. To overcome this limitation, compound endodomains have been constructed: fusion of the intracellular part of a T-cell co-stimulatory molecule to that of CD3 results in second generation receptors which can transmit an activating and co-stimulatory signal simultaneously after antigen recognition. The co-stimulatory domain most commonly used is that of CD28. This supplies the most potent co-stimulatory signal - namely immunological signal 2, which triggers T-cell proliferation. Some receptors have also been described which include TNF receptor family endodomains, such as the closely related 0X40 and 41 BB which transmit survival signals. Even more potent third generation CARs have now been described which have endodomains capable of transmitting activation, proliferation and survival signals.

[0108] When a CAR binds the target-antigen, this results in the transmission of an activating signal to the T-cell on which it is expressed. Thus, the CAR directs the specificity and cytotoxicity of the T cell towards tumour cells expressing the targeted antigen.

[0109] CARs typically comprise: (i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iii) an intracellular domain which comprises or associates with a signalling domain.

[0110] A CAR may have the general structure:

[0111] Antigen binding domain - spacer domain - transmembrane domain - intracellular signalling domain (endodomain).

[0112] Antigen binding domain

[0113] The antigen binding domain is the portion of the chimeric receptor which recognizes antigen. In a classical CAR, the antigen-binding domain comprises a single-chain variable fragment (scFv) derived from a monoclonal antibody (see Figure 2c). CARs have also been produced with domain antibody (dAb) or VHH antigen binding domains (see Figure 2b); or in a Fab CAR format (Figure 2a). A FabCAR comprises two chains: one having an antibody-like light chain variable region (VL) and constant region (CL); and one having a heavy chain variable region (VH) and constant region (CH). One chain also comprises a transmembrane domain and an intracellular signalling domain. Association between the CL and CH causes assembly of the receptor.

[0114] The antigen binding domain(s) of the CAR may be a single domain binder, also known as a “dAb”, “VHH”, “domain antibody” or “nanobody”.

[0115] A conventional IgG molecule is comprised of two heavy and two light chains. Heavy chains comprise three constant domains and one variable domain (VH); light chains comprise one constant domain and one variable domain (VL). The naturally functional antigen binding unit is formed by noncovalent association of the VH and the VL domain. This association is mediated by hydrophobic framework regions. A single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. The first single-domain antibodies were engineered from heavy-chain antibodies found in camelids which lack the light chain and the CH1 domain of a classical antibody. These heavy chain antibodies comprise a single, antigen binding domain, the VHH domain. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, ‘immunoglobulin new antigen receptor’), from which single-domain antibodies called VNAR fragments can be obtained. An alternative approach is to split the dimeric variable domains from common immunoglobulin G (IgG) from humans or mice into monomers. Although most research into single-domain antibodies is currently based on heavy chain variable domains, Nanobodies derived from light chains have also been shown to bind specifically to target epitopes.

[0116] A single-domain antibody can be obtained by immunization of dromedaries, camels, llamas, alpacas or sharks with the desired antigen and subsequent isolation of the mRNA coding for heavy-chain antibodies. By reverse transcription and polymerase chain reaction, a gene library of single-domain antibodies may be produced. Screening techniques like phage display and ribosome display help to identify the clones binding the antigen. Alternatively, single-domain antibodies can be made from common murine or human IgG with four chains.

[0117] The present invention relates to the targeting of multiple antigens using a tri-specific CAR structure similar to a TanCAR, as described in more detail below. Domain antibody antigen binding domains are particularly suited for such approaches because they are discrete and do not have a tendency to concatenate. They are also less complex, meaning that expression and folding are less likely to be compromised and that the sequence coding for such a tri-specific CAR requires less space on a viral vector genome.

[0118] TANCARs and TRI-SPECIFIC CARs

[0119] The cell of the present invention comprises a tri-specific CAR structure (Figure 3).

[0120] Bispecific CARs known as tandem CARs or TanCARs have been developed to target two or more cancer specific markers simultaneously. In a TanCAR, the extracellular domain comprises two antigen binding specificities in tandem, joined by a linker. The two binding specificities (scFvs) are thus both linked to a single transmembrane portion: one scFv being juxtaposed to the membrane and the other being in a distal position. When a TanCAR binds either or both of the target antigens, this results in the transmission of an activating signal to the cell it is expressed on.

[0121] Grada et al (2013, Mol Ther Nucleic Acids 2:e105) describes a TanCAR which includes a CD19-specific scFv, followed by a Gly-Ser linker and then a HER2-specific scFv. The HER2-scFv was in the juxta-membrane position, and the CD19-scFv in the distal position. The TanCAR was shown to induce distinct T cell reactivity against each of the two tumour restricted antigens. This arrangement was chosen because the respective lengths of HER2 (632 aa / 125A) and CD19 (280aa, 65A) lends itself to that spatial arrangement. It was also known that the HER2 scFv bound the distal- most 4 loops of HER2.

[0122] A tri-specific CAR structure can be created by adding another antigen binding domain to a TanCAR, via a suitable linker. The cell of the invention comprises a tri-specific CAR comprising three antigen binding specificities in series. The tri-specific CAR binds a combination of CD33, CD123, and CLL-1. In some cases herein this tri- specific CAR format may also be referred to as a TanCAR.

[0123] The antigen binding domains may be in any order in the molecule. For example, the CD33-binding antigen binding domain may be juxtaposed to the membrane and the CD123-binding antigen binding domain may be distal to the membrane, with the CLL- 1 antigen binding domain between the two; or the CD123-binding antigen binding domain may be juxtaposed to the membrane and the CLL-1 -binding antigen binding domain may be distal to the membrane, with the CD33-binding domain between the two. All possible orders are contemplated herein.

[0124] A tri-specific CAR of the invention may comprise the sequence shown in SEQ ID No. 96, or a variant having at least 80, 85, 90, 95, 98 or 99% sequence identity thereto.

[0125] SEQ ID No. 96 (Tri-specific CAR)

[0126] QVQLQQSGGGLVQPGGSLRLSCVGSGFIFGNYDMSWVRQAPGKEVEFVAGISSG GNDIVYEDAVKGRFSISRDNARNTVYLDMASVKPEDAGVYYCAADLDPGTDSLDNIH HGQGTQVFVSSGGGGSGGGGSGGGGSQVQLQQSGGGLVQAGGSLRLSCAASGS IFSINAMGWFRQAPGKEREFVAAISWSGGSTYYADFVKGRFTISRDNAKNTVYLQM NSLKPEDTAIYYCAAISGWGRSIRVGERYEYDYWGQGTQVTVSSGGGGSGGGGSG GGGSQVQLQESGGGLVQAGESLRLTCAVSGISLNAMGWYRQAPGKQLREWVAVIK IGGVSNYAVSVKGRFTISRDNAKNTIYLQMNSLKPEDTGVYYCNTYPPYLNGMDYW GKGTLVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD IYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR

[0127] The nucleic acid sequence of an example of a TanCAR construct, including RQR8 suicide gene, is given in SEQ ID NO: 114. This TanCAR construct includes the CD123 binder F8, described elsewhere in this document. The nucleic acid of a second TanCAR construct, including the CD123 binder B12, is provided in SEQ ID NO: 115.

[0128] OR GATES

[0129] “Logic Gate” CAR combinations are described in WO2015 / 075469, WO2015 / 075470, WO2015 / 075470, and WQ2020 / 035676. A CAR logic gate is a CAR combination which, when expressed by a cell, such as a T cell, is capable of detecting a particular pattern of expression of at least two target antigens. If the at least two target antigens are arbitrarily denoted as antigen A and antigen B, the three possible options are as follows:

[0130] “OR GATE” - T cell triggers when either antigen A or antigen B is present on the target cell

[0131] “AND GATE” - T cell triggers only when both antigens A and B are present on the target cell

[0132] “AND NOT GATE” - T cell triggers if antigen A is present alone on the target cell, but not if both antigens A and B are present on the target cell

[0133] Engineered T cells expressing these CAR combinations can be tailored to be exquisitely specific for cancer cells, based on their particular expression (or lack of expression) of two or more markers.

[0134] An “OR Gate” comprises two or more CARs each directed to a distinct target antigen expressed by a target cell. The advantage of an OR gate is that the effective targetable antigen is increased on the target cell, as it is effectively antigen A + antigen B. This is especially important for antigens expressed at variable or low density on the target cell, as the level of a single antigen may be below the threshold needed for effective targeting by a CAR-T cell. Also, it avoids the phenomenon of antigen escape. For example, some lymphomas and leukemias become CD19 negative after CD19 targeting: using an OR gate which targets CD19 in combination with another antigen provides a “back-up” antigen, should this occur.

[0135] The cell of the present invention may express a triple OR gate comprising three CARs (also termed a compound CAR or triple compound CAR). For example, the cell may express: a CAR which binds CD33, a CAR which binds CLL-1 ; and a CAR which binds CD123.

[0136] In the triple compound CARs of the invention, one of more CAR(s) may be a dAb CAR. In particular, all of the CARs of the cell may be dAb CARs.

[0137] In a preferred triple compound CAR of the present invention, the binder / spacer combination may be aCD33 / HNG, aCD123 / CD8, and aCLL1 / CD28 as shown in figure 3. Another preferred triple compound CAR of the present invention has the binder / spacer combination: aCD33 / CD8, aCD123 / CD28, and aCLL1 / HNG.

[0138] The triple compound CAR of the invention may be expressed in a cell using cleavage site as described elsewhere in the application. In particular, 2A self-cleaving peptides may be used.

[0139] A preferred CD33 CAR for use in a triple compound CAR has the following sequence, or a variant having at least 80, 85, 90, 95, 98 or 99% sequence identity thereto:

[0140] SEQ ID NO: 104 (CD33 CAR)

[0141] QVQLQQSGGGLVQAGGSLRLSCAASGSIFSINAMGWFRQAPGKEREFVAAISWSG GSTYYADFVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAAISGWGRSIRVGERY EYDYWGQGTQVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR A preferred CD123 CAR for use in a triple compound CAR has the following sequence, or a variant having at least 80, 85, 90, 95, 98 or 99% sequence identity thereto:

[0142] SEQ ID NO: 105 (CD123 CAR)

[0143] QVQLQESGGGLVQAGESLRLTCAVSGISLNAMGWYRQAPGKQLREWVAVIKIGGV SNYAVSVKGRFTISRDNAKNTIYLQMNSLKPEDTGVYYCNTYPPYLNGMDYWGKGT LVTVSSDPAKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPKDPKACDIYI WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR

[0144] A preferred CLL1 CAR for use in a triple compound CAR has the following sequence, or a variant having at least 80, 85, 90, 95, 98 or 99% sequence identity thereto:

[0145] SEQ ID NO: 106 (CLL1 CAR)

[0146] QVQLQQSGGGLVQPGGSLRLSCVGSGFIFGNYDMSWVRQAPGKEVEFVAGISSG GNDIVYEDAVKGRFSISRDNARNTVYLDMASVKPEDAGVYYCAADLDPGTDSLDNIH HGQGTQVFVSSDPAEPKSPDKTHTCPPCPKDPKACDIYIWAPLAGTCGVLLLSLVIT LYCKRGRKKLLYI FKQPFM RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0147] The nucleic acid sequences of repeated structures, such as the CD8 transmembrane region, 41 BB endodomain, and CD3zeta endodomain, may be codon wobbled. A codon wobbled signal peptide may also be included in the construct.

[0148] The amino acid sequence of a preferred triple compound CAR construct, including signal peptides, RQR8 safety switch and 2A sequences is given in SEQ ID NO: 112, or a variant having at least 80, 85, 90, 95, 98 or 99% sequence identity thereto.

[0149] SEQ ID NO. 112 (Triple Compound CAR)

[0150] MGTSLLCWMALCLLGADHADACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAK PTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPWRAEGRGSLLTC

[0151] GDVEENPGPMETDTLLLWVLLLWVPGSTGQVQLQQSGGGLVQPGGSLRLSCVGS GFIFGNYDMSWVRQAPGKEVEFVAGISSGGNDIVYEDAVKGRFSISRDNARNTVYL DMASVKPEDAGVYYCAADLDPGTDSLDNIHHGQGTQVFVSSDPAEPKSPDKTHTC PPCPKDPKACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQ EEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMETDTLLLWVLLLW VPGSTGQVQLQQSGGGLVQAGGSLRLSCAASGSIFSINAMGWFRQAPGKEREFVA AISWSGGSTYYADFVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAAISGWGRSI RVGERYEYDYWGQGTQVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPRATNFSLLKQAGDVEENPGPMETDTLLLWVLLLW VPGSTGQVQLQESGGGLVQAGESLRLTCAVSGISLNAMGWYRQAPGKQLREWVA VIKIGGVSNYAVSVKGRFTISRDNAKNTIYLQMNSLKPEDTGVYYCNTYPPYLNGMD YWGKGTLVTVSSDPAKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPKD PKACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR

[0152] An example nucleic acid sequence for the triple compound CAR construct provided in SEQ ID NO: 112 is provided in SEQ ID NO: 113. A variant of SEQ ID NO 113 having at least 80, 85, 90, 95, 98 or 99% sequence identity thereto is also contemplated herein.

[0153] TARGET ANTIGEN

[0154] The, or one of the, antigen binding domain(s) of the CAR may specifically bind one of the following target antigens: CD33, CD123, and CLL-1. Antigen binding domains targeting FLT-3 are also contemplated herein as an alternative.

[0155] CD33

[0156] CD33 is a myeloid differentiation antigen which is displayed on some normal B-cells and activated T- and natural killer cells but is not expressed on pluripotent hematopoietic stem cells or outside the hematopoietic system. It is found on at least a subset of blasts in nearly all acute myeloid leukemias (AMLs). With an average of 104 molecules / leukemic cell, CD33 is not highly abundant but levels vary considerably across individual patients.

[0157] The extracellular portion of CD33 contains two immunoglobulin domains and the intracellular portion contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The amino acid sequence of human CD33 is available from Uniprot Accession number P20138.

[0158] Several commercially available antibodies against CD33 are known, such as WM-53, P67.6, HIM3-4 (Thermofisher).

[0159] The present invention provides a domain antibody (dAb) which binds CD33 and comprises the following complementarity determining regions:

[0160] (i) CDR1 - GRTFSMHS (SEQ ID No. 1); CDR2 - VTWSGDTF (SEQ ID No. 2); CDR3 - KDDPYRPAYDY (SEQ ID No. 3 );

[0161] (ii) CDR1 - GRTFSSYV (SEQ ID No. 4); CDR2 - ISWSGGST (SEQ ID No. 5); CDR3 - AAMELRGGSYNYASSRQYDY (SEQ ID No. 6);

[0162] (iii) CDR1 - EIAFSNFN (SEQ ID No. 7); CDR2 - ISSHGDTNY (SEQ ID No. 8); CDR3 - NANDPFLSVSDF (SEQ ID No. 9);

[0163] (iv) CDR1 - GSIFSINA (SEQ ID No. 10); CDR2 - ISWSGGST (SEQ ID No. 5); CDR3 - AAISGWGRSIRVGERYEYDY (SEQ ID No. 11);

[0164] (v) CDR1 - GRTSSSST (SEQ ID No. 12); CDR2 - ITLSGGST (SEQ ID No. 13); CDR3 - AARRWSNNRGGYDRAGYDY (SEQ ID No. 14); or

[0165] (vi) CDR1 - GRTFSSYA (SEQ ID No. 15); CDR2 - ITWSGGST (SEQ ID No. 16); CDR3 - AMLLRGGLYDYTDYILYNY (SEQ ID No. 17).

[0166] The anti-CD33 dAb may comprise one of the sequences shown as SEQ ID No. 18, 19, 20, 21 , 22 or 23.

[0167] SEQ ID No. 18 (CD33 dAb P1.E4 - 44738)

[0168] QVQLESGGGLVQAGGSLRLSCAASGRTFSMHSMGWFRQAPGKEREFVAAVTWSG DTFAYADFVKGRFTISRGIAKNTLYLQMNSLKPEDTAVYYCAAKDDPYRPAYDYWG QGTQVTVSS

[0169] SEQ ID No. 19 (CD33 dAb P1.H3- 44739) QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYVMGWFRQAPGKEREFVAAISWS

[0170] GGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAAMELRGGSYNYAS

[0171] SRQYDYWGQGTQVTVSS

[0172] SEQ ID No. 20 (CD33 dAb P1.G8 - 44742)

[0173] QVQLQESGGGLVQTGGSLTLSCAASEIAFSNFNMGWYRQGSGKQRTLVAQISSHG DTNYLDSMKGRFTISRDNNKKTVYLQMNALKPEDTAVYYCNANDPFLSVSDFWGQ GTQVTVSS

[0174] SEQ ID No. 21 (CD33 dAb P2.A7 - 46173)

[0175] QVQLQQSGGGLVQAGGSLRLSCAASGSIFSINAMGWFRQAPGKEREFVAAISWSG GSTYYADFVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAAISGWGRSIRVGERY EYDYWGQGTQVTVSS

[0176] SEQ ID No. 22 (CD33 dAb P2.B12 - 46174)

[0177] QVQLQESGGGLVQAGGSLRLSCAASGRTSSSSTMAWFRQAPGKEREFVAAITLSG GSTHYADSAKGRFTISRESAKNTVYLQMNSLKPEDTADYYCAARRWSNNRGGYDR AGYDYWGQGTQVTVSS

[0178] SEQ I D No. 23 (CD33 dAb P2. F2 - 46176)

[0179] QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAITWS GGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAAMLLRGGLYDYTD Yl LYNYWGQGTQVTVSS

[0180] The present invention also provides: a CAR comprising such a CD33 dAb as antigen binding domain; a nucleic acid sequence encoding such a dAb or CAR.

[0181] CD 123

[0182] CD123 is the transmembrane a subunit of the interleukin-3 receptor (IL-3Ra), which together with CD131 forms a high-affinity IL-3R. Upon binding of IL-3, IL-3R promotes cell proliferation and survival. CD123 is normally expressed at high levels on plasmacytoid dendritic cells, and basophils. It is expressed at low levels on monocytes, eosinophils, and myeloid dendritic cells. The amino acid sequence of human CD123 is available from NCBI Reference Sequence: NP_002174.1. Several commercially available antibodies against CD123 are known, such as 6H6 and 5B11 (ThermoFisher).

[0183] The present invention provides a domain antibody (dAb) which binds CD123 and comprises the following complementarity determining regions:

[0184] (i) CDR1 - GRSINTYA (SEQ ID No. 24); CDR2 - INYNSRYT (SEQ ID No. 25); CDR3 - AATSYYPTDYDVASRVATWPS (SEQ ID No. 26);

[0185] (ii) CDR1 - GISLNA (SEQ ID No. 27); CDR2 - IKIGGVS (SEQ ID No. 28);

[0186] CDR3 - NTYPPYLNGMDY (SEQ ID No. 29);

[0187] (iii) CDR1 - GRSFNTDA (SEQ ID No. 30); CDR2 - ISWDGTRT (SEQ ID No.

[0188] 31); CDR3 - AAEPQKAWPIGTSAAGFRS (SEQ ID No. 32);

[0189] (iv) CDR1 - GSSISV (SEQ ID No. 33); CDR2 - ISWSDGNT (SEQ ID No. 34);

[0190] CDR3 - AVEPRGWPKGHRY (SEQ ID No. 35);

[0191] (v) CDR1 - GSSFSINV (SEQ ID No. 36); CDR2 - ISWSDGST (SEQ ID No.

[0192] 37); CDR3 - AVEPRGWPKGHRY (SEQ ID No. 38); or

[0193] (vi) CDR1 - GSIFRINA (SEQ ID No. 39); CDR2 - VNWIGGTT (SEQ ID No.

[0194] 40); CDR3 - SATDKGGSSRY (SEQ ID No. 41).

[0195] The anti-CD123 dAb may comprise one of the sequences shown as SEQ ID No: 42, 43, 44, 45, 46, 47, or 103.

[0196] SEQ ID No. 42 (CD123 dAb H11 45897)

[0197] QVQLQESGGGLVQAGGSLRLSCTASGRSINTYAMAWFRQAPGKEREFVASINYNS

[0198] RYTHYVDSVKGRFTISRDNTKNTLFLQMDSLNREDTAVYYCAATSYYPTDYDVASR

[0199] VATWPSWGQGTQVTVSS

[0200] SEQ ID No. 43 (CD123 dAb F845888)

[0201] QVQLQESGGGLVQAGESLRLTCAVSGISLNAMGWYRQAPGKQLREWVAVIKIGGV

[0202] SNYAVSVKGRFTISRDNAKNTIYLQMNSLKPEDTGVYYCNTYPPYLNGMDYWGKGT LVTVSS

[0203] SEQ ID No. 44 (CD123 dAb A7 45865)

[0204] QVQLQQSGGGLVQAGGSLRLSCAFSGRSFNTDAVAWFRQAPGKEREFVAAISWD

[0205] GTRTYYADSAKGRFTISRDNAKNTVYLQMNSLNSEDTAVYYCAAEPQKAWPIGTSA AGFRSWGQGTQVTVSS

[0206] SEQ ID No. 45 (CD123 dAb B4 45868) QVQLQESGGGSVQSGGSLRLSCAASGSSISVMGWFRQAPGKEREFVAAISWSDG

[0207] NTNYADSVNGRFSVSRDNTKNTVYLQMNSLKPEDTAIYYCAVEPRGWPKGHRYWG

[0208] QGTQVTVSS

[0209] SEQ ID No. 46 (CD123 dAb A1045866) QVQLQESGGSSVQAGGSLRLSCAASGSSFSINVMGWFRQAPGKEREFVAAISWSD GSTNYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTAIYYCAVEPRGWPKGHRYW GQGTQVTVSS

[0210] SEQ ID No. 47 (CD123 dAb C11 45874)

[0211] QVQLQESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQAPGKEREFVTAVNWIG GTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYFCSATDKGGSSRYWGQG TQVTVSS

[0212] SEQ ID No. 103 (CD123 dAb B12 45871)

[0213] QVQLQESGGGLVQAGGSLRLSCAASGLTVSRYAMGWFRQAPGKEREFVAAINWS GGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADYFEGGIMPVSA ENDFGSWGQGTQVTVSS

[0214] The present invention also provides: a CAR comprising such a CD123 dAb as antigen binding domain; a nucleic acid sequence encoding such a dAb or CAR.

[0215] FLT3

[0216] FMS-like tyrosine kinase 3 (FLT3), a receptor tyrosine kinase (RTK), is a membranebound receptor with an intrinsic tyrosine kinase domain. FLT3 is composed of a immunoglobulin-like extracellular ligand-binding domain, a transmembrane domain, a juxtamembrane dimerization domain, and highly conserved intracellular kinase domain interrupted by a kinase insert. FLT3 belongs to the class III subfamily of RTKs, which include structurally similar members such as c-FMS, c-KIT, and PDGF receptor. FLT3 is primarily expressed on committed myeloid and lymphoid progenitors with variable expression in the more mature monocytic lineage.

[0217] FLT3 expression has been described in lymphohematopoietic organs such as the liver, spleen, thymus, and placenta. In the unstimulated state, FLT3 receptor exists in a monomeric, unphosphorylated form with an inactive kinase moiety. Upon interaction of the receptor with FLT ligand (FL), the receptor undergoes a conformational change, resulting in the unfolding of the receptor and the exposure of the dimerization domain, allowing receptor-receptor dimerization to take place. This receptor dimerization is the prelude to the activation of the tyrosine kinase enzyme, leading to phosphorylation of various sites in the intracellular domain. The amino acid sequence of human FLT3 is available from NCBI Reference Sequence: NP_004110.2.

[0218] FLT3 is important to the biology of some cases of AML with mutations in FLT3 being among the most commonly found mutations in this disease. These mutations typically result in constitutive activation. Some cases of AML respond to small-molecule inhibition of FLT3.

[0219] Several commercially available antibodies against FLT3 are known, such as A2F10 and BV10A4H2 (ThermoFisher).

[0220] The present invention provides a domain antibody (dAb) which binds FLT3 and comprises the following complementarity determining regions:

[0221] (i) CDR1 - GIFKTNY (SEQ ID No. 72); CDR2 - FTNDGST (SEQ ID No. 73); CDR3 - YGLGH (SEQ ID No. 74);

[0222] (ii) CDR1 - GTISSIRY (SEQ ID No. 75); CDR2 - ITSSGNT (SEQ ID No. 76); CDR3 - YTMGY (SEQ ID No. 77);

[0223] (iii) CDR1 - GIFSTNY (SEQ ID No. 78); CDR2 - FTNDGGT (SEQ ID No. 79); CDR3 - CGLGH (SEQ ID No. 80);

[0224] (iv) CDR1 - GSISSIRY (SEQ ID No. 81); CDR2 - ITSSGST (SEQ ID No. 82); CDR3 - YTMGY (SEQ ID No. 83); or

[0225] (v) CDR1 - GIFSTNH (SEQ ID No. 84); CDR2 - FTNDGST (SEQ ID No. 85); CDR3 - YGLGH (SEQ ID No. 86).

[0226] The anti-FLT3 dAb may comprise one of the sequences shown as SEQ ID No. 87, 88, 89, 90 or 91.

[0227] SEQ ID No. 87 (FLT3 dAb B5)

[0228] QVQLQQSGGGLVQAGGSLRLSCAASGIFKTNYMAWYRQAPGKQRELVAAFTNDG STLYGDSVKGRFTISRDDAKYTVSLQMNSLKPEDTAVYYCYGLGHWGQGTQVIVSS EPKTPKPQPAAADDDDKEQKLISEEDLNGAAHHHHHHGAA

[0229] SEQ ID No. 88 (FLT3 dAb G3) QVQLQESGGGLVQAGGSLRLSCAASGTISSIRYMNWYRQAPGKQREWAYITSSG

[0230] NTNYADSVKGRFTISRDNAKNTVYLQMDNLKPEDTAAYYCYTMGYWGQGTQVTVS

[0231] SEPKIPQPQPAAADDDDKEQKLISEEDLNGAAHHHHHHGAA

[0232] SEQ ID No. 89 (FLT3 dAb H5) QVQLQESGGGLVQAGGSLRLSCAASGIFSTNYMVWCRQAPGKQRELVAAFTNDG GTLYADSLKGRFSISQDNAKNTVLLLMNSLKPEDTAVYYCCGLGHWGRGTKVTVSS EPKIPQPQPAAADDDDKEQKLISEEDLNGAAHHHHHHGAA

[0233] SEQ ID No. 90 (FLT3 dAb D12)

[0234] QVQLQESGGGLVQAGGSLRLSCAASGSISSIRYMNWYRQAPGKQRESVAWITSSG STNYADSVQGRFTISRDNAKNTVYLQMDNLKPEDTAVYYCYTMGYWGQGTQVTVS SEPKIPQPQPAAADDDDKEQKLISEEDLNGAAHHHHHHGAA

[0235] SEQ ID No. 91 (FLT3 dAb F10)

[0236] QAQVQLQESGGGLVQAGGSLRLSCAASGIFSTNHMAWYRQAPGKQRELVAAFTND GSTLYGDSVKGRFVISRDNAKYTVFLQMNSLKPEDTAVYYCYGLGHWGQGTQVTV SSEPKTPKPQPAAADDDDKEQKLISEEDLNGAAHHHHHHGAA

[0237] The present invention also provides: a CAR comprising such a FLT3 dAb as antigen binding domain; a nucleic acid sequence encoding such a dAb or CAR.

[0238] CLL1

[0239] Human C-type lectin-like molecule-1 (CLL-1, MICL or CLEC12A), is a type II transmembrane glycoprotein and member of the large family of C-type lectin-like receptors involved in immune regulation. The intracellular domain of CLL-1 contains an ITIM motif as well as a binding site for PI-3 kinase. The pattern of expression of CLL-1 in hematopoietic cells is restricted; it is found in particular in myeloid cells derived from peripheral blood and bone marrow. The amino acid sequence of human CLL1 is available from Uniprot accession No. Q5QGZ9.

[0240] Several antibodies have been described against CLL-1 , for example in WQ2009051974, WO2013169625, WQ2016205200 and WQ2016040868.

[0241] The present invention provides a domain antibody (dAb) which binds CLL1 and comprises the following complementarity determining regions: (i) CDR1 - GFTFGNHD (SEQ ID No. 48); CDR2 - IDSGGNVI (SEQ ID No. 49); CDR3 - ATDLDSGAESLESVY (SEQ ID No. 50);

[0242] (ii) CDR1 - GFAFGSAD (SEQ ID No. 51); CDR2 - IDSGGNTQ (SEQ ID No. 52); CDR3 - TDLDPTTDSLENVY (SEQ ID No. 53);

[0243] (iii) CDR1 - GRTFSAYF (SEQ ID No. 54); CDR2 - INWNGDSS (SEQ ID No. 55); CDR3 - AADTHGAVGLGSERLYDY (SEQ ID No. 56);

[0244] (iv) CDR1 - GIGVSSTG (SEQ ID No. 57); CDR2 - IDRDGTT (SEQ ID No. 58); CDR3 - TVVGDYY (SEQ ID No. 59);

[0245] (v) CDR1 - GFIFGNYD (SEQ ID No. 60); CDR2 - ISSGGNDI (SEQ ID No.

[0246] 61); CDR3 - AADLDPGTDSLDNIH (SEQ ID No. 62); or

[0247] (vi) CDR1 - GFTLDYYA (SEQ ID No. 63); CDR2 - ISSSDGST (SEQ ID No. 64); CDR3 - AEAVYYAGVCVAMYDS (SEQ ID No. 65)

[0248] The anti-CLL-1 dAb may comprise one of the sequences shown as SEQ ID No. 66, 67, 68, 69, 70 or 71.

[0249] SEQ ID No. 66 (CLL-1 dAb 44548)

[0250] QVQLQQSGGGLVQPGGSLRLSCVGSGFTFGNHDMSWVRQAPGKEVEFVAGIDSG GNVIVYEEVVKGRFTISRDNAKNTLYLQMDGLKPEDAGMYFCATDLDSGAESLESV

[0251] YHGQGTQVTVSS

[0252] SEQ ID No. 67 (CLL-1 dAb 44544)

[0253] QVQLQESGGGLVESGGSLRISCTGFGFAFGSADMSWVRQAPGKEVEFVAGIDSGG NTQTYEDTVKGRFTISRDNAKNTLYLQMNSLQSEDAGVYFCATDLDPTTDSLENVY

[0254] HGQGTQVIVSS

[0255] SEQ ID No. 68 (CLL-1 dAb 44538)

[0256] QVQLQESGGGLVQTGDSLRLSCVASGRTFSAYFMGWFRQAPGKEREFVSAINWN GDSSWYRDSVKGRFTVSRDNAKNTVYLQMNSLEPEDTAVYYCAADTHGAVGLGSE RLYDYWGQGTQVTVSS

[0257] SEQ ID No. 69 (CLL-1 dAb 44546)

[0258] QVQLQESGGGVVQAGGSLRLSCAVSGIGVSSTGMGWSRQTPGKQVELVALIDRDG TTNYADTVKGRFTISKDNSKNMVYLQMNSLKPEDTALYHCTVVGDYYWGQGTQVT VSS

[0259] SEQ ID No. 70 (CLL-1 dAb 44545) QVQLQQSGGGLVQPGGSLRLSCVGSGFIFGNYDMSWVRQAPGKEVEFVAGISSG

[0260] GNDIVYEDAVKGRFSISRDNARNTVYLDMASVKPEDAGVYYCAADLDPGTDSLDNIH

[0261] HGQGTQVFVSS

[0262] SEQ ID No. 71 (CLL-1 dAb 44536)

[0263] QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSD GSTAYADSVKGRFTISRDNAKNSVYLQMNSLKPEDTAVYYCAEAVYYAGVCVAMYD SWGQGTQVTVSS

[0264] The present invention also provides: a CAR comprising such a CLL-1 dAb as antigen binding domain; a nucleic acid sequence encoding such a dAb or CAR.

[0265] SPACER

[0266] Classical CARs comprise a spacer sequence to connect the antigen-binding domain with the transmembrane domain and spatially separate the antigen-binding domain from the endodomain. A flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding.

[0267] The spacer may cause two CAR-forming polypeptide chains to dimerise. Two of the polypeptide chains may, for example, comprise one or more suitable cysteine residues to form di-sulphide bridge(s). Commonly used spacers include the lgG1 Fc region, the lgG1 hinge, CD28 stalk or a human CD8 stalk. A hinge spacer may comprise the sequence shown as SEQ ID No. 92

[0268] SEQ ID No. 92 (hinge spacer) EPKSCDKTHTCPPCP

[0269] A CD8-derived spacer may comprise the sequence shown as SEQ ID No. 97

[0270] SEQ ID No. 97 (CD8-derived spacer)

[0271] PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0272] The spacer may be chosen to suit the target antigen, i.e. the location and orientation of the epitope on the target antigen and the distance of the target epitope from the target cell membrane. In an OR gate, different spacers may be used to suit the different relative locations of the target epitopes and also to prevent cross-pairing between the two CARs.

[0273] The tri-specific CAR of the present invention utilises a CD8-derived spacer domain. The present inventors have found that the use of this spacer in the tri-specific format leads to lower basal activation. Without wishing to be bound by any one particular theory, the present inventors propose that lower basal activation will reduce T cell exhaustion and enhance in vivo performance.

[0274] The use of a CD28-derived spacer is also contemplated herein, which may comprise the sequence shown in SEQ ID No. 101

[0275] SEQ ID No. 101 (CD28-derived spacer) PAKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPKDPKACDI

[0276] In the case of a compound CAR system, the different spacers may be chosen to optimise the formation of an appropriate immune synapse, based on considerations such as the size of the relevant antigens and the lengths of the spacers. In the compound CAR of the present invention, the binder / spacer combination may be aCD33 / HNG, aCD123 / CD8, and aCLL1 / CD28 as shown in figure 3. Another preferred binder / spacer combination is aCD33 / CD8, aCD123 / CD28, and aCLL1 / HNG.

[0277] TRANSMEMBRANE DOMAIN

[0278] The transmembrane domain is the portion of a CAR which spans the membrane. The transmembrane domain may be any protein structure which is thermodynamically stable in a membrane. This is typically an alpha helix comprising of several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to supply the transmembrane portion of the chimeric receptor. The presence and span of a transmembrane domain of a protein can be determined by those skilled in the art using the TMHMM algorithm (http: / / www.cbs. dtu.dk / services / TMHMM-2.0 / ). Alternatively, an artificially designed TM domain may be used. A CD8 transmembrane domain may comprise the sequence shown as SEQ ID No. 98.

[0279] SEQ ID No. 98 (CD8 transmembrane domain)

[0280] IYIWAPLAGTCGVLLLSLVITLYC ENDODOMAIN

[0281] The endodomain is the signal-transmission portion of a CAR. It may be part of or associate with the intracellular domain of the CAR. After antigen recognition, receptors cluster, native CD45 and CD148 are excluded from the synapse and a signal is transmitted to the cell. The most commonly used endodomain component is that of CD3-zeta which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signalling may be needed. Co-stimulatory signals promote T-cell proliferation and survival. There are two main types of co-stimulatory signals: those that belong the Ig family (CD28, ICOS) and the TNF family (0X40, 41 BB, CD27, GITR etc). For example, chimeric CD28 and 0X40 can be used with CD3-Zeta to transmit a proliferative I survival signal, or all three can be used together.

[0282] The endodomain may comprise:

[0283] (i) an ITAM-containing endodomain, such as the endodomain from CD3 zeta; and / or

[0284] (ii) a co-stimulatory domain, such as the endodomain from CD28 or ICOS; and / or

[0285] (iii) a domain which transmits a survival signal, for example a TNF receptor family endodomain such as OX-40, 4-1 BB, CD27 or GITR.

[0286] A CD3 zeta based endodomain may comprise the sequence shown in SEQ ID No. 99.

[0287] SEQ ID No. 99 (CD3 zeta endodomain) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR

[0288] A 41 BB based endodomain may comprise the sequence shown in SEQ ID No. 100

[0289] SEQ ID No. 100 (41 BB endodomain)

[0290] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL A number of systems have been described in which the antigen recognition portion is on a separate molecule from the signal transmission portion, such as those described in WQ015 / 150771; WO2016 / 124930 and WO2016 / 030691. The cell of the present invention may therefore express a CAR system which comprises an antigen-binding component comprising an antigen-binding domain(s) and a transmembrane domain; which is capable of interacting with a separate intracellular signalling component comprising a signalling domain.

[0291] The CAR may comprise a signal peptide so that when it is expressed inside a cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed. The signal peptide may be at the amino terminus of the molecule.

[0292] Examples of signal peptides include the following sequences:

[0293] SEQ ID NO: 107 (Signal peptide) MGTSLLCWMALCLLGADHADA

[0294] SEQ ID NO: 108 (MuIG Hsignal peptide)

[0295] M ETDTLLLWVLLLWVPGSTG

[0296] SUICIDE GENE

[0297] The cell of the present invention may also express a suicide gene.

[0298] A suicide-gene is a genetically encoded mechanism which allows selective destruction of adoptively transferred cells, such as T-cells, in the face of unacceptable toxicity, such as on-target off-tumour toxicity, cytokine release syndrome (CRS) or neurotoxicity.

[0299] When the cells of the present invention are used to treat acute myeloid leukemia (AML), the treatment may cause myeloid aplasia in the patient which may be long- lasting or permanent. It is possible to rescue the patient from this state using an allogeneic transplant, such as an allogeneic hematopoietic stem cell transplantation (alloHSCT). The incorporation of a suicide gene in the CAR-expressing cells also enables the CAR-expressing cells to be deleted if a transplant is used, to prevent CAR mediated graft rejection. The cells of the present invention may comprise one of the suicide-genes previously tested in clinical studies, such as Herpes Simplex Virus thymidine kinase (HSV-TK) or inducible caspase 9 (iCasp9).

[0300] WO201 3 / 153391 describes a compact sort-suicide gene comprising a CD20 epitope which enables cells expressing the polypeptide to be selectively killed using Rituximab. The cells of the present invention may express a suicide gene having the sequence shown as SEQ ID No. 93

[0301] SEQ ID No. 93

[0302] CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSP APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLS LVITLYCNHRNRRRVCKCPRPW

[0303] WO2016 / 135470 describes a suicide gene which dimerizes in the presence of a chemical inducer of dimerization (CID) such as rapamycin or a rapamycin analogue causing caspase-mediated apoptosis of the cell.

[0304] The suicide gene may have the structure:

[0305] Ht1-HT2-Casp in which:

[0306] Ht1 and Ht2 are heterodimerisation domains, one of which comprises an FK506- binding protein (FKBP) and the other of which comprises an FRB domain of mTOR; and

[0307] Casp is a caspase 9 domain.

[0308] The suicide gene may have the sequence shown as SEQ ID No. 94 or a variant thereof having 90, 95, or 99% sequence identity.

[0309] SEQ ID No. 94 (FRB-FKBP12-L3-dCasp9)

[0310] MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFN QAYGR - FRB - ><L1-><— FKBP12 -

[0311] DLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKLEYSGGGSLEGVQVETIS

[0312] PGDGR

[0313] - FKBP12 -

[0314] TFPKRGQTCWHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMS VGQRAK

[0315] SLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS

[0316] NUCLEIC ACID CONSTRUCT

[0317] A nucleic acid sequence encoding a CAR may have the following structure:

[0318] AgB-spacer-TM-endo in which:

[0319] AgB is a nucleic acid sequence encoding an antigen binding domain of the CAR; spacer is a nucleic acid sequence encoding a spacer of the CAR;

[0320] TM is a nucleic acid sequence encoding a transmembrane domain of the CAR; endo is a nucleic acid sequence encoding an endodomain of the CAR.

[0321] A nucleic acid sequence encoding a tri-specific CAR may have the following structure:

[0322] AgB1-linker1-AgB2-linker2-AgB3-spacer-TM-endo in which

[0323] AgB1 is a nucleic acid sequence encoding a first antigen-binding domain of the tri- specific CAR; linked is a nucleic acid sequence encoding a linker of the tri-specific;

[0324] AgB2 is a nucleic acid sequence encoding the second antigen binding domain of the tri-specific CAR;

[0325] Linker2 is a nucleic acid sequence encoding a linker of the tri-specific;

[0326] AgB3 is a nucleic acid sequence encoding a first antigen-binding domain of the tri- specific CAR; spacer is a nucleic acid sequence encoding a spacer of the tri-specific CAR;

[0327] TM is a nucleic acid sequence encoding a transmembrane domain of the tri-specific CAR; endo is a nucleic acid sequence encoding an endodomain of the tri-specific CAR.

[0328] When expressed in a cell, the nucleotide sequence encodes a polypeptide expressing the first, second, and third antigen binding domains in series at the cell surface.

[0329] The linkers may be or comprise a Gly-Ser flexible linker.

[0330] The antigen binding domain(s) for the CAR or tri-specific CAR may, for example, be scFv(s) or dAb(s). In particular, all three antigen binding domains in the tri-specific CAR may be dAbs. The present invention also provides a nucleic acid construct encoding an scFv / dAb CAR and a tri-specific CAR. In this embodiment, the nucleic acid construct may have the structure:

[0331] AgB1-linker-AgB2-linker-AgB3-spacer1-TM1-endo1-coexpr-AgB4-spacer2-TM2- endo2 in which:

[0332] AgB1 is a nucleic acid sequence encoding the first antigen-binding domain of the tri- specific CAR; linker is a nucleic acid sequence encoding a linker of the tri-specific CAR;

[0333] AgB2 is a nucleic acid sequence encoding the second antigen binding domain of the tri-specific CAR;

[0334] AgB3 is a nucleic acid sequence encoding the third antigen binding domain of the tri- specific CAR; spaced is a nucleic acid sequence encoding a spacer of the tri-specific CAR;

[0335] TM1 is a nucleic acid sequence encoding a transmembrane domain of the tri-specific CAR; endol is a nucleic acid sequence encoding an endodomain of the tri-specific CAR; coexpr is a nucleic acid sequence enabling co-expression the CAR and the tri-specific CAR;

[0336] AgB4 is a nucleic acid sequence encoding the antigen-binding domain of the CAR spacer2 is a nucleic acid sequence encoding a spacer of the CAR;

[0337] TM2 is a nucleic acid sequence encoding a transmembrane domain of the CAR; endo2 is a nucleic acid sequence encoding an endodomain of the CAR; or the structure:

[0338] AgB1-spacer1-TM1-endo1-coexpr- AgB2-linker-AgB3-linker-AgB4-spacer2-TM2- endo2 in which:

[0339] AgB1 is a nucleic acid sequence encoding the antigen-binding domain of the CAR spaced is a nucleic acid sequence encoding a spacer of the CAR;

[0340] TM1 is a nucleic acid sequence encoding a transmembrane domain of the CAR; endol is a nucleic acid sequence encoding an endodomain of the CAR; coexpr is a nucleic acid sequence enabling co-expression the CAR and the tri-specific CAR;

[0341] AgB2 is a nucleic acid sequence encoding the first antigen-binding domain of the tri- specific CAR; linker is a nucleic acid sequence encoding a linker of the tri-specific CAR;

[0342] AgB3 is a nucleic acid sequence encoding the second antigen binding domain of the tri-specific CAR;

[0343] AgB4 is a nucleic acid sequence encoding the third antigen binding domain of the tri- specific CAR; spacer2 is a nucleic acid sequence encoding a spacer of the tri-specific CAR;

[0344] TM2 is a nucleic acid sequence encoding a transmembrane domain of the tri-specific CAR; endo2 is a nucleic acid sequence encoding an endodomain of the tri-specific CAR.

[0345] The present invention also provides a nucleic acid construct encoding two tri-specific CARs. In this embodiment, the nucleic acid construct may have the structure:

[0346] AgB1-linker1-AgB2-AgB3-linker1-spacer1-TM1-endo1-coexpr-AgB4-linker2-AgB5-

[0347] Hnker2AgB6-spacer2-TM2-endo2 in which:

[0348] AgB1 is a nucleic acid sequence encoding the first antigen-binding domain of the first tri-specific CAR; linked is a nucleic acid sequence encoding a linker of the first tri-specific CAR;

[0349] AgB2 is a nucleic acid sequence encoding the second antigen binding domain of the first tri-specific CAR;

[0350] AgB3 is a nucleic acid sequence encoding the third antigen binding domain of the first tri-specific CAR; spaced is a nucleic acid sequence encoding a spacer of the first AgB2 is a nucleic acid sequence encoding the second antigen binding domain of the first tri-specific CAR;

[0351] TM1 is a nucleic acid sequence encoding a transmembrane domain of the first tri- specific CAR; endol is a nucleic acid sequence encoding an endodomain of the first tri-specific CAR; coexpr is a nucleic acid sequence enabling co-expression the first and second tri- specific CARs; AgB4 is a nucleic acid sequence encoding the first antigen-binding domain of the second tri-specific CAR;

[0352] Iinker2 is a nucleic acid sequence encoding a linker of the second tri-specific CAR;

[0353] AgB5 is a nucleic acid sequence encoding the second antigen binding domain of the second tri-specific CAR;

[0354] AgB6 is a nucleic acid sequence encoding the third antigen binding domain of the second tri-specific CAR; spacer2 is a nucleic acid sequence encoding a spacer of the second tri-specific CAR;

[0355] TM2 is a nucleic acid sequence encoding a transmembrane domain of the second tri- specific CAR; endo2 is a nucleic acid sequence encoding an endodomain of the second tri-specific CAR.

[0356] The nucleic acid construct of the present invention may also comprise a nucleic acid sequence encoding a suicide gene.

[0357] As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other.

[0358] It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described here to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed.

[0359] Nucleic acids according to the invention may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest. The terms “variant”, “homologue” or “derivative” in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence.

[0360] In the structure above, “coexpr” is a nucleic acid sequence enabling co-expression of two polypeptides as separate entities. It may be a sequence encoding a cleavage site, such that the nucleic acid construct produces both polypeptides, joined by a cleavage site(s). The cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into individual peptides without the need for any external cleavage activity.

[0361] The cleavage site may be any sequence which enables the two polypeptides to become separated.

[0362] The term “cleavage” is used herein for convenience, but the cleavage site may cause the peptides to separate into individual entities by a mechanism other than classical cleavage. For example, for the Foot-and-Mouth disease virus (FMDV) 2A selfcleaving peptide (see below), various models have been proposed for to account for the “cleavage” activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect (Donnelly et al (2001) J. Gen. Virol. 82:1027-1041). The exact mechanism of such “cleavage” is not important for the purposes of the present invention, as long as the cleavage site, when positioned between nucleic acid sequences which encode proteins, causes the proteins to be expressed as separate entities.

[0363] The cleavage site may, for example be a furin cleavage site, a Tobacco Etch Virus (TEV) cleavage site or encode a self-cleaving peptide.

[0364] A ‘self-cleaving peptide’ refers to a peptide which functions such that when the polypeptide comprising the proteins and the self-cleaving peptide is produced, it is immediately “cleaved” or separated into distinct and discrete first and second polypeptides without the need for any external cleavage activity.

[0365] The self-cleaving peptide may be a 2A self-cleaving peptide from an aphtho- or a cardiovirus. The primary 2A / 2B cleavage of the aptho- and cardioviruses is mediated by 2A “cleaving” at its own C-terminus. In apthoviruses, such as foot-and-mouth disease viruses (FMDV) and equine rhinitis A virus, the 2A region is a short section of about 18 amino acids, which, together with the N-terminal residue of protein 2B (a conserved proline residue) represents an autonomous element capable of mediating “cleavage” at its own C-terminus (Donelly et al (2001) as above).

[0366] “2A-like” sequences have been found in picornaviruses other than aptho- or cardioviruses, ‘picornavirus-like’ insect viruses, type C rotaviruses and repeated sequences within Trypanosoma spp and a bacterial sequence (Donnelly et al (2001) as above).

[0367] The cleavage site may comprise the 2A-like sequence shown as SEQ ID Nos.95, 109, 110, and 111:

[0368] SEQ ID No. 95:

[0369] RAEGRGSLLTCGDVEENPGP

[0370] SEQ ID No. 109 (T2A):

[0371] EGRGSLLTCGDVEENPGP

[0372] SEQ ID No. 110 (E2A):

[0373] QCTNYALLKLAGDVESNPGP

[0374] SEQ ID No. 111 (P2A):

[0375] ATNFSLLKQAGDVEENPGP

[0376] SEQUENCE VARIANTS

[0377] The present invention also provides sequence variants of the amino acid and nucleic acid sequences provided herein. 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. Thus, a CAR, compound CAR, or TanCAR of the invention may comprise a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a sequence described herein. As used herein, “variant” is synonymous with “mutant” and refers to an amino acid sequence which differs in comparison to the corresponding wild-type sequence. The term “wild-type” is used to mean a protein having an amino acid sequence respectively, which is identical with the native protein respectively. It may be possible to introduce one or more mutations (substitutions, additions or deletions) into each sequence without negatively affecting activity. The term may similarly be applied to nucleic acid sequences.

[0378] Methods of sequence alignment are well known in the art and are accomplished using suitable alignment programs. The % sequence identity refers to the percentage of amino acid or nucleotide residues that are identical in the two sequences when they are optimally aligned. Nucleotide and protein sequence homology or identity may be determined using standard algorithms such as a BLAST program (Basic Local Alignment Search Tool at the National Center for Biotechnology Information) using default parameters, which is publicly available at http: / / blast.ncbi.nlm.nih.gov. Other algorithms for determining sequence identity or homology include: LALIGN and http: / / www.ebi.ac.uk / Tools / psa / lalign / nucleotide.html), AMAS (Analysis of Multiply Aligned Sequences, at http: / / www.compbio.dundee.ac.uk / Software / Amas / amas.html), FASTA (http: / / www.ebi.ac.uk / Tools / sss / fasta / ) , Clustal Omega 10 (http: / / www.ebi.ac.uk / Tools / msa / clustalo / ), SIM (http: / / web.expasy.org / sim / ), and EMBOSS Needle (http: / / www.ebi.ac.uk / Tools / psa / emboss needle / nucleotide.html).

[0379] VECTOR

[0380] The present invention also provides a vector, or kit of vectors, which comprises one or more nucleic acid sequence(s) encoding one or more chimeric antigen receptor(s) of the cell of the invention. Such a vector may be used to introduce the nucleic acid sequence(s) into a host cell so that it expresses the or each CAR(s).

[0381] The vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon based vector or synthetic mRNA.

[0382] The vector may be capable of transfecting or transducing a cell such as a T cell or a NK cell.

[0383] CELL

[0384] The present invention provides a cell which comprises a tri-specific chimeric antigen receptor. A cell comprising a triple compound CAR is also provided.

[0385] The cell may be a cytolytic immune cell such as a T cell or an NK cell. T cells or T lymphocytes are a type of lymphocyte that play a central role in cell- mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. There are various types of T cell, as summarised below.

[0386] Helper T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells become activated when they are presented with peptide antigens by MHC class II molecules on the surface of antigen presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1 , TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines to facilitate different types of immune responses.

[0387] Cytolytic T cells (TC cells, or CTLs) destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. CTLs express the CD8 at their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of all nucleated cells. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevent autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0388] Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with "memory" against past infections. Memory T cells comprise three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells may be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0389] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell- mediated immunity toward the end of an immune reaction and to suppress auto- reactive T cells that escaped the process of negative selection in the thymus. Two major classes of CD4+ Treg cells have been described — naturally occurring Treg cells and adaptive Treg cells.

[0390] Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and have been linked to interactions between developing T cells with both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells that have been activated with TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations of the FOXP3 gene can prevent regulatory T cell development, causing the fatal autoimmune disease IPEX.

[0391] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originate during a normal immune response.

[0392] The cell may be a Natural Killer cell (or NK cell). NK cells form part of the innate immune system. NK cells provide rapid responses to innate signals from virally infected cells in an MHC independent manner

[0393] NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph node, spleen, tonsils and thymus where they then enter into the circulation.

[0394] The cells of the invention may be any of the cell types mentioned above.

[0395] T or NK cells according to the first aspect of the invention may either be created ex vivo either from a patient’s own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2nd party), or peripheral blood from an unconnected donor (3rd party).

[0396] Alternatively, T or NK cells according to the first aspect of the invention may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to T or NK cells. Alternatively, an immortalized T-cell line which retains its lytic function and could act as a therapeutic may be used. In all these embodiments, chimeric polypeptide-expressing cells are generated by introducing DNA or RNA coding for the chimeric polypeptide by one of many means including transduction with a viral vector, transfection with DNA or RNA.

[0397] The cell of the invention may be an ex vivo T or NK cell from a subject. The T or NK cell may be from a peripheral blood mononuclear cell (PBMC) sample. T or NK cells may be activated and / or expanded prior to being transduced with nucleic acid encoding the molecules providing the chimeric polypeptide according to the first aspect of the invention, for example by treatment with an anti-CD3 monoclonal antibody.

[0398] The T or NK cell of the invention may be made by:

[0399] (i) isolation of a T or NK cell-containing sample from a subject or other sources listed above; and

[0400] (ii) transduction or transfection of the T or NK cells with a nucleic acid construct, vector or kit of vectors of the invention.

[0401] The T or NK cells may then by purified, for example, selected on the basis of expression of the antigen-binding domain of the antigen-binding polypeptide.

[0402] PHARMACEUTICAL COMPOSITION

[0403] The present invention also relates to a pharmaceutical composition containing a plurality of cells according to the invention.

[0404] The pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and / or compounds. Such a formulation may, for example, be in a form suitable for intravenous infusion.

[0405] METHOD OF TREATMENT

[0406] The present invention provides a method for treating a disease which comprises the step of administering the cells of the present invention (for example in a pharmaceutical composition as described above) to a subject. A method for treating a disease relates to the therapeutic use of the cells of the present invention. Herein the cells may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and / or to slow down, reduce or block the progression of the disease.

[0407] The method may involve the steps of:

[0408] (i) isolating a T or NK cell-containing sample;

[0409] (ii) transducing or transfecting such cells with a nucleic acid sequence or vector provided by the present invention;

[0410] (iii) administering the cells from (ii) to a subject.

[0411] The T or NK cell-containing sample may be isolated from a subject or from other sources, for example as described above. The T or NK cells may be isolated from a subject’s own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2nd party), or peripheral blood from an unconnected donor (3rd party).

[0412] The method may involve the following steps:

[0413] (i) administering cells of the first aspect of the invention to a subject;

[0414] (ii) administering an allogeneic hematopoietic stem cell transplant (alloHSCT) to the subject.

[0415] For example, the method may involve the following steps:

[0416] (i) administering cells of the first aspect of the invention to a subject;

[0417] (ii) monitoring the subject for myeloid aplasia

[0418] (ii) administering an allogeneic hematopoietic stem cell transplant (alloHSCT) to the patient if myeloid aplasia is detected.

[0419] Myeloid metaplasia is a clinical and pathologic syndrome which is characterized by the constant occurrence of extramedullary hematopoiesis in the spleen and almost always in the liver, splenomegaly and usually hepatomegaly, and an anemia with immature red and white cells in the peripheral blood.

[0420] The present invention provides a cell of the present invention for use in treating and / or preventing a disease. The invention also relates to the use of a cell of the present invention in the manufacture of a medicament for the treatment and / or prevention of a disease.

[0421] The disease to be treated by the m Acute Myeloid Leukemia (AML) ethods of the present invention may be a cancerous disease. In particular, the disease may be Acute Myeloid Leukemia (AML).

[0422] Acute myeloid leukemia (AML) is a cancer of the myeloid line of blood cells, characterized by the rapid growth of abnormal cells that build up in the bone marrow and blood and interfere with normal blood cells. Symptoms may include feeling tired, shortness of breath, easy bruising and bleeding, and increased risk of infection. Diagnosis is usually based on bone marrow aspiration and blood tests As an acute leukemia, AML progresses rapidly and is typically fatal within weeks or months if left untreated.

[0423] The cells of the present invention may be capable of killing target cells, such as cancer cells. The target cell may be characterised by the expression of one or more target antigens, such as one, two, three or all four of the following: CD33, CD123, CLL-1 and FLT3.

[0424] The cells and pharmaceutical compositions of present invention may be for use in the treatment and / or prevention of the diseases described above.

[0425] 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.

[0426] EXAMPLES

[0427] Example 1 - Evidence for auto-activation of multi-specific CAR with HNG spacer

[0428] In order to determine if basal activation was caused by the HNG stalk, experiments were conducted with both CD8 and CD28 stalks.

[0429] A panel of CAR-encoding nucleic acid constructs are generated as follows:

[0430] FMC63CAR - control CAR expressing the aCD19 FMC63 binder • V5 GD2 CAR - second control CAR expressing aGD2 binder

[0431] • aCLL1-G4S-aCD33-G4S-aCD123(F8)-HNG-CD8TM-41 BB-CD3zeta - trispecific CAR construct expressing CLL1, CD33, and CD123 binders. The F8 CD123 binder was used, together with a HNG spacer;

[0432] • aCLL1-G4S-aCD33-G4S-aCD123(F8)-CD8Stk-CD8TM-41BB-CD3zeta - trispecific CAR construct expressing CLL1, CD33, and CD123 binders. The F8 CD123 binder was used, together with a CD8Stk spacer

[0433] • aCLL1-G4S-aCD33-G4S-aCD123(F8)-CD28Stk-CD8TM-41 BB-CD3zeta - trispecific CAR construct expressing CLL1 , CD33, and CD123 binders. The F8 CD123 binder was used, together with a CD28Stk spacer

[0434] • aCLL1-G4S-aCD33-G4S-aCD123(C11)-CD8Stk-CD8TM-41 BB-CD3zeta - trispecific CAR construct expressing CLL1, CD33, and CD123 binders. The C11 CD123 binder was used, together with a CD8Stk spacer;

[0435] • aCLL1-G4S-aCD33-G4S-aCD123(B12)-CD8Stk-CD8TM-41BB-CD3zeta - trispecific CAR construct expressing CLL1, CD33, and CD123 binders. The B12 CD123 binder was used, together with a CD8Stk spacer

[0436] • aCLL1-G4S-aCD33-G4S-aCD123(B12)-CD28Stk-CD8TM-41BB-CD3zeta - tri-specific CAR construct expressing CLL1 , CD33, and CD123 binders. The B12 CD123 binder was used, together with a CD28Stk spacer

[0437] All constructs co-express the sort-suicide gene RQR8, which is described in WO2013 / 153391.

[0438] A variety of CD123 binders were used in order to determine if this binder, in the membrane proximal position, contributed to basal activation. With the exception of the FMC63 CAR and CD2 CAR, all CARs are dAb CARs having a second generation endodomain comprising CD3 and a 4-1 BB co-stimulatory domain.

[0439] Peripheral blood-derived CD4+ and CD8+ T cells were transduced with the constructs.

[0440] We noted dilution of cell trace violet, particularly with the HNG stalk (Figure 4). This was also associated with basal elaboration of IFNg over 7 days of culturing the CAR T cells on their own (Figure 5B). The level was similar to the GD2 CAR, which is known to cause basal activation in association with T cell exhaustion (Long et al., Nat Med. 2015 Jun; 21(6): 581-590). Encouragingly, there was no evidence of basal activation when a CD8 stalk was used to replace the HNG stalk with the same binder.

[0441] Example 2 - Comparison of tri-specific CAR and triple compound CAR

[0442] A comparison of the CD8 stalk and HNG stalk based tri-specific CARs was carried out. Furthermore, these constructs were compared to the triple compound CAR described in W02020 / 035676 (Figure 3).

[0443] A panel of CAR-encoding nucleic acid constructs are generated as follows:

[0444] • FMC63CAR - control CAR expressing the aCD19 FMC63 binder;

[0445] • CD123CAR-2A-CD33CAR-2A-CLL1CAR - triple CAR construct expressing CD123 CAR; CD33 CAR; and CLL1 CAR;

[0446] • aCLL1-G4S-aCD33-G4S-aCD123-HNG-CD8TM-41 BB-CD3zeta - tri-specific CAR construct expressing CLL1 , CD33, and CD123 binders;

[0447] • aCLL1-G4S-aCD33-G4S-aCD123-CD8Stk-CD8TM-41BB-CD3zeta - tri- specific CAR construct expressing CLL1, CD33, and CD123 binders

[0448] All constructs co-express the sort-suicide gene RQR8, which is described in WO2013 / 153391.

[0449] Figure 6 shows that the cytotoxicity is equivalent for all 3 constructs against either SupT1-NT cells (which are CD33, CD123, and CLL1 negative) and MOLM14. Figure 7 also shows that antigen-specific IL-2 production is also equivalent. There is no evidence of non-specific cytokine elaboration with the CD8-based tri-specific format.

[0450] Cytotoxicity experiments comparing triple compound CAR and tri-specific CAR formats in co-cultures with MOLM14 cell lines manipulated to knockdown one of the AML antigens each were also conducted (Figure 8)

[0451] There was no evidence that any of the formats was disadvantaged against another in killing Ag negative MOLM14 variants

[0452] Example 3 - In vivo model The tri-specific CAR format described herein was also tested in an in vivo model. Specifically, luciferase expressing MOLM14 AML cells were transferred into NSG mice. Mice were irradiated and a million MOLM14 cells injected iv with CAR T cells being transferred 7 days later. The mice underwent weekly BLI to assess tumour burden.

[0453] CAR T cell dose was determined by initial experiments using a range from 0.2 to 5x10A6 CAR T cells. All treatment groups cleared the tumour, so a dose of 2x10A5 CAR T cells per mouse was chosen as the lowest dose capable of controlling tumour over a time interval of 33 days.

[0454] The single CARs and the tri-specific CAR (labelled TanCAR) were able to control the tumour burden in this model (Figure 9).

[0455] At the end of the experiment, there were 4 / 5 mice in the tri-specific CAR (labelled TanCAR) and CD123 CAR groups alive, 3 / 5 in the CD33 CAR group and 1 / 5 of the triple compound group alive, with all of the mice receiving NT cells or CLL1CAR T cells only surviving to 22 and 25 days respectively (Figure 10).

[0456] Example 4 - Comparison of triple compound CAR and single CARs

[0457] A comparison of a triple compound CAR using a specific spacer configuration and corresponding single CARs was carried out.

[0458] A panel of CAR-encoding nucleic acid constructs are generated as follows:

[0459] • FMC63CAR - control CAR expressing the aCD19 FMC63 binder;

[0460] • RQR8-T2A-aCD33-VHH_CD8_CD8TMw1_41 BBzwl ;

[0461] • RQR8-T2A-aCD123-VHH_CD28_CD8TMw2_41 BBzw2;

[0462] • RQR8-T2A-aCLL1-VHH_HNG_CD8TMw3;

[0463] • RQR8-T2A-aCLL1-VHH_HNG_CD8TMw3_41BBzw3-E2A-aCD33- VHH_CD8_CD8TMw1_41BBzw1-P2A-aCD123- VHH_CD28_CD8TMw2_41 BBzw2;

[0464] All constructs co-express the sort-suicide gene RQR8, which is described in WO2013 / 153391. Cells expressing these constructs were challenged with either SupT1-NT cells (which are CD33, CD123, and CLL1 negative), M0LM14 cells, or SupTI cells engineered to express CD33, CD123, and CLL1 at a high level. Figure 11 shows that triple compound CAR T-cells show potent in vitro cytotoxic responses which are equivalent or greater than those of single CAR-expressing T-cells. Figure 12 also shows that antigen-specific IFN-gamma and IL-2 production is also equivalent or improved.

[0465] An in vivo experiment was also conducted comparing cells expressing the above constructs. To model sub-clonal heterogeneity and emergence of antigen-loss AML variants, CD33, CD123 and CLL1 gene knock outs of parental MOLM14 was carried out, creating 3 KO cell lines each lacking one antigen. These were mixed 1 :1 :1 and injected at a total of 1x10A6 cells into mice. Recipient groups received either NT cells, single CAR T-cells or triple CAR T-cells. Improved in vivo anti-tumour efficacy of triple compound CAR T-cells compared to single CAR T-cells was observed (Figure 13). Median OS was 29-31 days (recipients of NT cells and single CARs) but recipients of triple CAR T cells remained alive to end of experiment (D40, p=0.003 compared to NT).

[0466] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS1. A cell comprising a tri-specific CAR, the tri-specific CAR comprising:(i) an anti-CD33 antigen binding domain;(ii) an anti-CLL1 antigen binding domain;(iii) an anti-CD123 antigen binding domain;(iv) a spacer derived from CD8;(v) a transmembrane domain; and(vi) an intracellular signalling domain.

2. A cell according to claim 1 , wherein one or more antigen binding domains comprise a domain antibody (dAb) antigen binding domain.

3. A cell according to any of claims 1 to 2, wherein each antigen binding domain comprises a domain antibody (dAb) antigen binding domain.

4. A cell according to claim 2 wherein the anti-CD33 domain antibody (dAb) antigen binding domain comprises the following complementarity determining regions:(i) CDR1 - GRTFSMHS (SEQ ID No. 1); CDR2 - VTWSGDTF (SEQ ID No. 2); CDR3 - KDDPYRPAYDY (SEQ ID No. 3);(ii) CDR1 - GRTFSSYV (SEQ ID No. 4); CDR2 - ISWSGGST (SEQ ID No. 5); CDR3 - AAMELRGGSYNYASSRQYDY (SEQ ID No. 6);(iii) CDR1 - EIAFSNFN (SEQ ID No. 7); CDR2 - ISSHGDTNY (SEQ ID No. 8); CDR3 - NANDPFLSVSDF (SEQ ID No. 9);(iv) CDR1 - GSIFSINA (SEQ ID No. 10); CDR2 - ISWSGGST (SEQ ID No. 5); CDR3 - AAISGWGRSIRVGERYEYDY (SEQ ID No. 11);(v) CDR1 - GRTSSSST (SEQ ID No. 12); CDR2 - ITLSGGST (SEQ ID No. 13); CDR3 - AARRWSNNRGGYDRAGYDY (SEQ ID No. 14); or(vi) CDR1 - GRTFSSYA (SEQ ID No. 15); CDR2 - ITWSGGST (SEQ ID No. 16); CDR3 - AMLLRGGLYDYTDYILYNY (SEQ ID No. 17).

5. A cell according to claim 4, wherein the antigen binding domain comprises one of the sequences shown as SEQ ID No. 18, 19, 20, 21 , 22 or 23.

6. A cell according to claim 2 wherein the anti-CLL1 domain antibody (dAb) antigen binding domain comprising the following complementarity determining regions:(i) CDR1 - GFTFGNHD (SEQ ID No. 48); CDR2 - IDSGGNVI (SEQ ID No. 49); CDR3 - ATDLDSGAESLESVY (SEQ ID No. 50);(ii) CDR1 - GFAFGSAD (SEQ ID No. 51); CDR2 - IDSGGNTQ (SEQ ID No. 52); CDR3 - TDLDPTTDSLENVY (SEQ ID No. 53);(iii) CDR1 - GRTFSAYF (SEQ ID No. 54); CDR2 - INWNGDSS (SEQ ID No. 55); CDR3 - AADTHGAVGLGSERLYDY (SEQ ID No. 56);(iv) CDR1 - GIGVSSTG (SEQ ID No. 57); CDR2 - IDRDGTT (SEQ ID No. 58); CDR3 - TVVGDYY (SEQ ID No. 59);(v) CDR1 - GFIFGNYD (SEQ ID No. 60); CDR2 - ISSGGNDI (SEQ ID No. 61); CDR3 - AADLDPGTDSLDNIH (SEQ ID No. 62); or(vi) CDR1 - GFTLDYYA (SEQ ID No. 63); CDR2 - ISSSDGST (SEQ ID No. 64); CDR3 - AEAVYYAGVCVAMYDS (SEQ ID No. 65).

7. A cell according to claim 6, wherein the antigen binding domain comprises one of the sequences shown as SEQ ID No. 66, 67, 68, 69, 70 or 71.

8. A cell according to claim 2 wherein the anti-CD123 domain antibody (dAb) antigen binding domain comprises the following complementarity determining regions:(i) CDR1 - GRSINTYA (SEQ ID No. 24); CDR2 - INYNSRYT (SEQ ID No. 25); CDR3 - AATSYYPTDYDVASRVATWPS (SEQ ID No. 26);(ii) CDR1 - GISLNA (SEQ ID No. 27); CDR2 - IKIGGVS (SEQ ID No. 28); CDR3 - NTYPPYLNGMDY (SEQ ID No. 29);(iii) CDR1 - GRSFNTDA (SEQ ID No. 30); CDR2 - ISWDGTRT (SEQ ID No. 31); CDR3 - AAEPQKAWPIGTSAAGFRS (SEQ ID No. 32);(iv) CDR1 - GSSISV (SEQ ID No. 33); CDR2 - ISWSDGNT (SEQ ID No. 34); CDR3 - AVEPRGWPKGHRY (SEQ ID No. 35);(v) CDR1 - GSSFSINV (SEQ ID No. 36); CDR2 - ISWSDGST (SEQ ID No. 37); CDR3 - AVEPRGWPKGHRY (SEQ ID No. 38); or(vi) CDR1 - GSIFRINA (SEQ ID No. 39); CDR2 - VNWIGGTT (SEQ ID No. 40); CDR3 - SATDKGGSSRY (SEQ ID No. 41).

9. A cell according to claim 8, wherein the antigen binding domain comprises one of the sequences shown as SEQ ID No. 42, 43, 44, 45, 46, 47, or 103.

10. A cell according to any one of claims 1 to 9, wherein the endodomain comprises 4-1BB and CD3 .

11. A cell according to claim 1, wherein the tri-specific CAR comprises the sequence shown in SEQ ID No. 96.

12. A nucleic acid construct encoding: a tri-specific CAR as defined in claim 1.

13. A method for making a cell according to claim 1 which comprises the step of transducing or transfecting a cell with a nucleic acid construct according to claim 12.

14. A vector comprising a nucleic acid construct according to claim 13.

15. A pharmaceutical composition which comprises a plurality of cells according any of claims 1 to 11, together with a pharmaceutically acceptable carrier, diluent or excipient.

16. A method for treating cancer which comprises the step of administering a pharmaceutical composition according to claim 15 to a subject.

17. A method according to claim 16, wherein the cancer is acute myeloid leukemia (AML).

18. A method according to claim 17, which also involves the step of subsequently administering an allogeneic transplant to the subject.

19. A pharmaceutical composition according to claim 15 for use in treating cancer.

20. The use of a cell according to any of claims 1 to 11 in the manufacture of a pharmaceutical composition for treating cancer.

21. A cell comprising: an anti-CD33 chimeric antigen receptor (CAR) having a CD8 spacer; an anti-CLL1 CAR having a hinge spacer; and an anti-CD123 CAR having a CD28 spacer.

22. A cell according to claim 21 , wherein one or more CAR(s) comprise(s) a domain antibody (dAb) antigen binding domain.

23. A cell according to any of claims 21 and 22, wherein each CAR comprises a domain antibody (dAb) antigen binding domain.

24. A cell according to any of claims 21 to 23, wherein the anti-CD33 CAR has the amino acid sequence shown in SEQ ID NO: 104.

25. A cell according to any of claims 21 to 23, wherein the anti-CLL1 CAR has the amino acid sequence shown in SEQ ID NO: 105.

26. A cell according to any of claims 21 to 23, wherein the anti-CD123 CAR has the amino acid sequence shown in SEQ ID NO: 106.

27. A cell according to claim 21 , wherein the anti-CD33 CAR has the amino acid sequence shown in SEQ ID NO: 104, the anti-CLL1 CAR has the amino acid sequence shown in SEQ ID NO: 105, and the anti-CD123 CAR has the amino acid sequence shown in SEQ ID NO: 106.

28. A nucleic acid construct encoding: an anti-CD33 chimeric antigen receptor (CAR) having a CD8 spacer; an anti-CLL1 CAR having a hinge spacer; and an anti- CD123 CAR having a CD28 spacer.

29. A method for making a cell according to claim 21 which comprises the step of transducing or transfecting a cell with a nucleic acid construct according to claim 28.

30. A vector comprising a nucleic acid construct according to claim 28.

31. A kit of vectors, which comprises:(i) a first vector which comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR) which binds CD33 and has a CD8 spacer;(ii) a second vector which comprises a nucleic acid sequence encoding a CAR which binds CLL1 and has a hinge spacer; and(iii) a third vector which comprises a nucleic acid sequence encoding a CAR which binds CD123 and has a CD28 spacer.

32. A pharmaceutical composition which comprises a plurality of cells according to any of claims 21 to 27, together with a pharmaceutically acceptable carrier, diluent or excipient.

33. A method for treating cancer which comprises the step of administering a pharmaceutical composition according to claim 32 to a subject.

34. A method according to claim 33, wherein the cancer is acute myeloid leukemia (AML).

35. A method according to claim 34, which also involves the step of subsequently administering an allogeneic transplant to the subject.

36. A pharmaceutical composition according to claim 32 for use in treating cancer.

37. The use of a cell according to any of claims 21 to 27, in the manufacture of a pharmaceutical composition for treating cancer.

38. The cell of any of claims 1 to 11 or 21 to 26, wherein the cell is a T cell or a natural killer (NK) cell.

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