CHIMERIC ANTIGEN RECEPTOR

FIELD OF THE INVENTION

The present invention relates to a chimeric antigen receptor (CAR). BACKGROUND TO THE INVENTION

Despite recent progress in survival rates, T-cell acute lymphocytic leukaemia (T-ALL) remains a challenging clinical problem. Long-term survival is poor for adult patients, and the outlook is particularly poor for patients of all ages with relapsed/refractory disease.

Chimeric Antigen Receptors (CARs) are synthetic signalling molecules which redirect immune effector cells, for example T-cells, to kill tumours expressing a surface target antigen. CAR T-cells against CD19 are revolutionising the treatment of children with relapsed/refractory B-acute lymphoblastic leukaemia and adults with diffuse large B-cell lymphoma. In recent clinical studies of paediatric B-ALL, remission rates close to 100% have been reported, with -60% of patients achieving sustained responses. However, some 15% of paediatric and 20-25% of adult cases of ALL are of T-lineage (T-ALL), and are therefore not suitable for treatment with anti-CD19 therapy. No other novel therapies are available, and prognosis for patients with T-ALL who have failed standard intensive chemotherapy is poor.

Development of immunotherapy for T-ALL is challenging for multiple reasons. Targeting CD19, a pan B-cell antigen, results in concomitant loss of the normal B-cell compartment.

However, this is reasonably well tolerated and is an acceptable toxicity which if needed can be mitigated by pooled immunoglobulins. In contrast, targeting a pan-T-cell antigen results in profound unacceptable immunosuppression which cannot be mitigated. In addition, CAR T-cell production would be limited by fratricide of normal T-cells expressing the target molecule. Therefore, all antigens investigated to date for treatment of T-ALL (CD36, CD47, CD58, CD79) could only be at best a bridge to bone marrow transplantation.

Accordingly, there remains a need for novel therapeutic approaches to cancers such as leukaemias, in particular T-ALL. SUMMARY OF THE INVENTION

The present inventors have developed novel binding molecules against CD21. These binding molecules have been developed for use in the context of a CAR.

Accordingly, in a first aspect the present invention provides a chimeric antigen receptor (CAR) comprising a CD21 -binding domain, a transmembrane domain and an intracellular domain.

The CD21 -binding domain may be a scFv.

The CD21 -binding domain may comprise a heavy chain variable region (VH) which comprises a complementary determining region (CDR) selected from SEQ ID NO: 4, 8, 9, 12 and 14 or a variant thereof with up to three amino add substitutions.

The CD21 -binding domain comprises a light chain variable region (VL) may comprise a CDR selected from SEQ ID NO: 17, 20, 23, 26, and 29 or a variant thereof with up to three amino add mutations.

The CD21 -binding domain may comprise six CDRs selected from (i) to (vi) as follows: (I) SEQ ID NOs: 2 to 4 and 15 to 17 or variants thereof with up to three amino add substitutions in each CDR; (ii) SEQ ID NOs: 2, 5, 8 and 18 to 20 or variants thereof with up to three amino acid substitutions in each CDR; (iii) SEQ iD NOs: 7 to 9 and 21 to 23 or variants thereof with up to three amino acid substitutions in each CDR; (iv) SEQ ID NOs: 7 to 9 and 24 to 26 or variants thereof with up to three amino acid substitutions in each CDR; (v) SEQ ID NOs: 10 to 12 and 27 to 29 or variants thereof with up to three amino acid substitutions in each CDR in each CDR; and (vi) SEQ ID NOs: 2, 13, 14, 21 , 57 and 23 or variants thereof with up to three amino acid mutations in each CDR.

The CD21 -binding domain may comprises six CDRs selected from (i) to (vi) as follows: (i) SEQ ID NOs: 2 to 4 and 15 to 17; (ii) SEQ ID NOs: 2, 5, 6 and 18 to 20; (iii) SEQ ID NOs: 7 to 9 and 21 to 23; (iv) SEQ ID NOs: 7 to 9 and 24 to 26; (v) SEQ ID NOs: 10 to 12 and 27 to 29; and (vi) SEQ ID NOs: 2, 13, 14, 21 , 57 and 23.

The six CDR sequences may be grafted onto a human antibody framework sequence. The CD21 -binding domain may comprise an amino acid sequence shown as any one of SEQ ID NO: 30-37 or 58 or a variant of any one of SEQ ID NO: 30-37 or 58 having at least 80% sequence identity thereto.

The intracellular domain may comprise a T ceil signal!ing domain. in a further aspect the present invention provides polynucleotide which encodes a CAR according to the first aspect of the invention.

In another aspect the present invention provides a vector which comprises a polynucleotide according to the present invention.

In a further aspect the present invention provides a cell expressing a CAR according to the present invention.

The cell may be an alpha-beta T cell, a NK cell, a gamma-delta T cell, or a cytokine induced killer cell.

In a further aspect the present invention relates to a pharmaceutical composition which comprises a CAR; a polynucleotide: a vector or a cell according to the present invention.

In another aspect the present invention relates to a pharmaceutical composition according to the invention for use in treating and/or preventing a disease.

In a further aspect the present invention relates to a method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to the invention to a subject in need thereof.

The method may comprise the foiiowing steps:

(i) isolation of a ceil containing sample;

(ii) transduction or transfection of the ceil with a polynucleotide or a vector according to the invention; and

(iii) administering the ceils from (II) to a subject.

The cell may be autologous or allogenic. The invention further relates to the use of a pharmaceutical composition of the invention in the manufacture of a medicament for the treatment and/or prevention of a disease.

The disease may be T-cell acute lymphoblastic leukaemia.

The disease may be a B-cell cell leukaemia or a B-cell lymphoma.

The pharmaceutical composition of the invention may be administered in combination with a NOTCH activating agent.

The NOTCH activating agent may be, for example, a PI3K inhibitor, an AKT inhibitor and/or a mTOR inhibitor.

The pharmaceutical composition of the invention and the NOTCH activating agent may be administered simultaneously, sequentially or separately.

In a further aspect the present invention provides a kit comprising (i) a CAR, a polynucleotide; a vector; a cell or a pharmaceutical composition according to the invention; and (ii) a NOTCH activating agent.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 - a) Schematic diagram illustrating a classical CAR. (b) to (d): Different generations and permutations of CAR endodomains: (b) initial designs transmitted ITAM signals alone through FcsRI-y or CD3z endodomain, while later designs transmitted additional (c) one or (d) two co-stimulatory signals in the same compound endodomain.

Figure 2 - CD21 is expressed on T-ALL cell lines and primary samples (a) Proportion of T- ALL cell lines which were CD21+ or CD21- (b) Guantification of surface copies of CD21 on T-ALL cell lines by flow cytometry. Grey = Raji, B-lymphoma line (c) CD21 expression by microarray in normal/ T-ALL blood/ marrow, data MILE 2004 database (d) CD21 expression in 6 patients with T-ALL. 4/6 samples = CD21+ (highlighted in red, panels 3-6). CD7 = pan-T marker (e) Primary T-ALL samples were engrafted in NSG mice by intravenous injection. Flow cytometry plots demonstrate CD21 expression in blast cells isolated from spleens harvested from injected animals.

Figure 3 - CD21 expression in normal tissues (a) RNA-seq expression data for CR2/CD21 from a compendium of normal tissues. Data from GTEx (http://www.gtexportal.org/) (b) Expression of CD21 on peripheral B- and T-cells. Data was obtained by flow cytometry of peripheral blood. (n=11 healthy donors).

Figure 4 - Identification of anti-CD21 binders by genetic vaccination and phage display (a) Schematic of protocol (b) Pre- and post-vaccination serum from 1 representative rat was incubated with untransfected 293T cells or 293T cells transfected with CD21 , then stained with anti-rat Fc. (c) Myc-tagged phage from unselected library and after each round of CD21 biopanning was incubated with CD21+ Raji cells, then stained with anti-Myc. Enrichment of CD21 phage was seen (d) Single phage clones from 3rd round of biopanning were cloned as ScFv-MuFc fusions, then incubated with non-transduced H9 cells (red, left-hand peak in each panel) or H9 cells transduced to express CD21 (blue, right-hand peak in each panel) (e) Binding kinetics of selected ScFv-Fc fusions to CD21 were evaluated by surface plasmon resonance. Variable heavy and light chain usage detailed.

Figure 5 - Anti-CD21 CAR targeting the SupT1 T-cell leukaemia cell line (a) A histogram of flow-cytometric analysis for SupT1 (a T-cell leukaemia cell line which expresses CD21) and SupT1 KO (a control cell line which was genomically edited at the CD21 locus to generate SupT1 derivative which was CD21 negative) (b) Normal donor human T-cells were transduced with anti-CD21 CAR derived from scFv C43 with a CD8 stalk spacer or an lgG1 hinge spacer, or with anti-CD21 CAR derived from scFv C48 with either a CD8 stalk spacer or an lgG1 hinge spacer. Control non-transduced T-cells and T-cells transduced with a CD19 CAR were also tested. These effectors were co-cultures with SupT1 targets or SupT1 cells which don’t express CD21 target cells. Proportion of remaining targets is shown.

Figure 6 - Inhibition of PI3K-mTOR pathway in T-ALL cell lines leads to upregulation of CD21 expression (a) Schematic of Pi3K / Akt/mTOR pathway. Solid blue lines with arrowheads - positive regulation, dotted blue lines (long dots) = negative regulation, solid red lines with block ends = drug inhibition. Dotted box = proposed mechanism for CD21 upregulation by drugs acting on PI3K/ AKT/ mTOR pathway (b) CD21 expression in RPMI- 8402 cell line after 48hr co-culture with indicated drugs, targets in brackets IC = isotype control (c) Fold change in CD21 protein surface copy number v no-drug condition on RPMI- 8402 48hrs after incubation with drugs acting on PI3K/AKT/mTOR pathway and for 48hrs after drug has been removed.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention relates to a CAR comprising a CD21-binding domain, a transmembrane domain and an intracellular domain. CD21

CD21/Complement receptor type 2 (CR2) (also known as complement C3d receptor and Epstein-Barr virus receptor), is a protein that in humans is encoded by the CR2 gene.

CD21 is involved in the complement system. It binds to iC3b (inactive derivative of C3b), C3dg, or C3d. B cells have CD21 on their surfaces, allowing the complement system to play a role in B-cell activation and maturation.

The present inventors have determined that CD21 may be expressed in T-cell acute lymphomablastic leukaemia (T-ALL) and that CD21 is not expressed on normal non lymphoid tissue (see present Example 2). Tatsumi et al. have also reported CD21 expression in T-ALL (Am J Hematol 45, 150-155 (1994)).

Thus, without wishing to be bound by theory, a CAR targeting CD21 may result in B cell depletion (as seen in CD19-targeted CAR therapy) but no other significant haematological toxicity.

An illustrative human CD21 protein is provided by UniProt Accession Number P20023, the amino acid sequence of which is shown as SEQ ID NO: 1.

SEQ ID NO: 1

MGAAGLLGVFLALVAPGVLGISCGSPPPILNGRI SYYSTPIAVGTVIRYSCSGTFRLIGEKSLLCITK DKVDGTWDKPAPKCEYFNKYSSCPEPIVPGGYKIRGSTPYRHGDSVTFACKTNFSMNGNK SVWCQANN MWGPTRLPTCVSVFPLECPALPMIHNGHHTSENVGS IAPGLSVTYSCESGYLLVGEKI INCLSSGKWS AVPPTCEEARCKSLGRFPNGKVKEPPILRVGVTANFFCDEGYRLQGPPSSRCVIAGQGVA WTKMPVCE EIFCPSPPPILNGRHIGNSLANVSYGSIVTYTCDPDPEEGVNFILIGESTLRCTVDSQKT GTWSGPAP RCELSTSAVQCPHPQILRGRMVSGQKDRYTYNDTVIFACMFGFTLKGSKQIRCNAQGTWE PSAPVCEK ECQAPPNILNGQKEDRHMVRFDPGTS IKYSCNPGYVLVGEES IQCTSEGVWTPPVPQCKVAACEATGR QLLTKPQHQFVRPDVNSSCGEGYKLSGSVYQECQGTIPWFMEIRLCKEITCPPPPVIYNG AHTGSSLE DFPYGTTVTYTCNPGPERGVEFSLIGESTIRCTSNDQERGTWSGPAPLCKLSLLAVQCSH VHIANGYK ISGKEAPYFYNDTVTFKCYSGFTLKGSSQIRCKADNTWDPEIPVCEKETCQHVRQSLQEL PAGSRVEL VNTSCQDGYQLTGHAYQMCQDAENGIWFKKIPLCKVIHCHPPPVIVNGKHTGMMAENFLY NEVSYECD QGFYLLGEKKLQCRSDSKGHGSWSGPSPQCLRSPPVTRCPNPEVKHGYKLNKTHSAYSHN DIVYVDCN PGFIMNGSRVIRCHTDNTWVPGVPTCIKKAFIGCPPPPKTPNGNHTGGNIARFSPGMS ILYSCDQGYL LVGEALLLCTHEGTWSQPAPHCKEVNCSSPADMDGIQKGLEPRKMYQYGAWTLECEDGYM LEGSPQS QCQSDHQWNPPLAVCRSRSLAPVLCGIAAGLILLTFLIVITLYVISKHRARNYYTDTSQK EAFHLEAR EVYSVDPYNPAS

CHIMERIC ANTIGEN RECEPTOR (CAR)

Classical CARs, which are shown schematically in Figure 1 , are chimeric type I trans membrane proteins which connect an extracellular antigen-binding 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 or on a ligand for the target antigen. A spacer domain may be necessary to isolate the binder from the membrane and to allow it a suitable orientation. A common spacer domain used is the Fc of lgG1. 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.

Early CAR designs had endodomains derived from the intracellular parts of either the g chain of the FcsR1 or CD3z. 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 CD3z 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 4-1 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.

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

ANTIGEN BINDING DOMAIN

The antigen-binding domain is the portion of a classical CAR which recognizes antigen.

Numerous antigen-binding domains are known in the art, including those based on the antigen binding site of an antibody, antibody mimetics, and T-cell receptors. For example, the antigen-binding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a natural ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain binder such as a camelid; an artificial binder single as a Darpin; or a single-chain derived from a T-cell receptor. The antigen-binding domain may be a polypeptide having an antigen binding site which comprises at least one complementarity determining region (CDR). The antigen-binding domain may comprise 3 CDRs and have an antigen binding site which is equivalent to that of a domain antibody (dAb). The antigen-binding domain may comprise 6 CDRs and have an antigen binding site which is equivalent to that of a classical antibody molecule. The remainder of the polypeptide may be any sequence which provides a suitable scaffold for the antigen binding site and displays it in an appropriate manner for it to bind the antigen. The antigen-binding domain may be part of an immunoglobulin molecule such as a Fab, F(ab)’ ₂ , Fv, single chain Fv (ScFv) fragment, Nanobody or single chain variable domain (which may be a VH or VL chain, having 3 CDRs). The antigen-binding domain may be non-human, chimeric, humanised or fully human.

The antigen-binding domain may comprise a binding domain which is not derived from or based on an immunoglobulin. A number of "antibody mimetic" designed repeat proteins (DRPs) have been developed to exploit the binding abilities of non-antibody polypeptides. Such molecules include ankyrin or leucine-rich repeat proteins e.g. DARPins (Designed Ankyrin Repeat Proteins), Anticalins, Avimers and Versabodies.

The binding domain may“specifically bind” to the antigen as defined herein. As used herein, “specifically bind” means that the binding domain binds to the antigen but does not bind to other peptides, or binds at a lower affinity to other peptides.

The binding affinity between two molecules, e.g. an antigen binding domain and an antigen, may be quantified, for example, by determination of the dissociation constant (KD). The KD can be determined by measurement of the kinetics of complex formation and dissociation between the antigen-binding domain and antigen, e.g. by a surface plasmon resonance (SPR) method (e.g. Biacore™). The rate constants corresponding to the association and the dissociation of a complex are referred to as the association rate constants ka (or kon) and dissociation rate constant kd. (or koff), respectively. KD is related to ka and kd through the equation KD = kd / ka.

Binding affinities associated with different molecular interactions, e.g. comparison of the binding affinity of different antigen-binding domains and an antigen, may be compared by comparison of the KD values for the individual antigen-binding domain and antigen.

The present invention provides a CAR comprising a CD21-binding domain.

The CD21-binding domain may be based on a CD21 binder as shown in Tables 1 and 2. Table i - Variable Heavy Chain Usage * C-ORs

Table 2 - Variable U¾h† Chain Usage ~ CD Rs

The CD21 -binding domain may comprise a VH which comprises a CDR selected from SEQ ID NO: 4, 6, 9, 12, or 14 or a variant thereof with up to three amino acid mutations (e.g. 1 , 2 or 3 mutations).

The CD21 -binding domain may comprise a VL which comprises a CDR selected from SEQ ID NO: 17, 20, 23, 26 or 29 or a variant thereof with up to three amino acid mutations (e.g. 1 , 2 or 3 mutations).

It may be possible to introduce one or more mutations (substitutions, additions or deletions) into a CDR without negatively affecting CD21 -binding activity. The CDR may, for example, haveone, two or three amino acid mutations, for example one, two or three amino acid substitutions. Preferably, the amino acid substitutions are conservative substitutions. Conservative amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobscity, hydrophilicity, and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to Table 3 below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other: Table 3

The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc.

Unless otherwise explicitly stated herein by way of reference to a specific, individual amino acid, amino acids may be substituted using conservative substitutions as recited below.

An aliphatic, non-polar amino acid may be a glycine, alanine, proline, isoleucine, leucine or valine residue.

An aliphatic, polar uncharged amino may be a cysteine, serine, threonine, methionine, asparagine or glutamine residue.

An aliphatic, polar charged amino acid may be an aspartic acid, glutamic acid, lysine or arginine residue. An aromatic amino acid may be a histidine, phenylalanine, tryptophan or tyrosine residue. Suitably, a conservative substitution may be made between amino acids in the same line in Table 3.

The CD21 -binding domain may comprise six CDRs selected from (I) to (vi) as follows: (i) SEQ ID NOs: 2 to 4 and 15 to 17 or variants thereof with up to three amino acid substitutions in each CDR; (ii) SEQ ID NOs: 2, 5, 6 and 18 to 20 or variants thereof with up to three amino acid substitutions in each CDR; (iii) SEQ ID NOs: 7 to 9 and 21 to 23 or variants thereof with up to three amino acid substitutions in each CDR; (iv) SEQ ID NOs: 7 to 9 and 24 to 26 or variants thereof with up to three amino acid substitutions in each CDR; (v) SEQ ID NOs: 10 to 12 and 27 to 29 or variants thereof with up to three amino acid substitutions in each CDR in each CDR; and (vi) SEQ ID NOs: 2, 13, 14, 21 , 57 and 23 or variants thereof with up to three amino acid mutations in each CDR.

The CD21-binding domain may comprise six CDRs of a CD21-binder as shown in Tables 2 and 3.

The CD21 -binding domain may comprise six CDRs selected from (i) to (vi) as follows: (i) SEQ ID NOs: 2 to 4 and 15 to 17; (ii) SEQ ID NOs: 2, 5, 6 and 18 to 20; (iii) SEQ ID NOs: 7 to 9 and 21 to 23; (iv) SEQ ID NOs: 7 to 9 and 24 to 28; (v) SEQ ID NOs: 10 to 12 and 27 to 29; and (vi) SEQ ID NOs: 2, 13, 14, 21 , 57 and 23.

The CD21 -binding domain may comprise six CDRs shown as SEQ ID NOs: 2 to 4 and 15 to 17 or variants thereof with up to three amino acid mutations, preferably substitutions, in each CDR. The CD21 -binding domain may comprise six CDRs shown as SEQ ID NOs: 2 to 4 and 15 to 17.

The CD21 -binding domain may comprise six CDRs shown as SEQ ID NOs: 2, 5, 6 and 18 to

20 or variants thereof with up to three amino acid mutations, preferably substitutions, in each CDR. The CD21-binding domain may comprise six CDRs shown as SEQ ID NOs: 2, 5, 6 and 18 to 20.

The CD21 -binding domain may comprise six CDRs shown as SEQ ID NOs: 7 to 9 and 21 to

23 or variants thereof with up to three amino acid mutations, preferably substitutions, in each CDR. The CD21 -binding domain may comprise six CDRs shown as SEQ ID NOs: 7 to 9 and

21 to 23.

The CD21-binding domain may comprise six CDRs shown as SEQ ID NOs: 7 to 9 and 24 to 26 or variants thereof with up to three amino acid mutations, preferably substitutions, in each CDR. The CD21 -binding domain may comprise six CDRs shown as SEQ ID NOs: 7 to 9 and

24 to 26. The CD21 -binding domain may comprise six CDRs shown as SEQ ID NOs: 10 to 12 and 27 to 29 or variants thereof with up to three amino acid mutations, preferably substitutions, in each CDR. The CD21 -binding domain may comprise six CDRs shown as SEQ ID NOs: 10 to 12 and 27 to 29. The CD21-binding domain may comprise six CDRs shown as SEQ ID NOs: 13. 14, 21 , 57 and 23 or variants thereof with up to three amino acid mutations, preferably substitutions, in each CDR. The CD21 -binding domain may comprise six CDRs shown as SEQ ID NOs: 2, 13, 14, 21 57 and 23.

The CD21 -binding domain may comprise a variable heavy chain shown as any one of SEQ !D NO: 38-46 or a variant thereof having at least 80% sequence identity thereto.

The CD21 -binding domain may comprise a variable light chain shown as any one of SEQ ID NO: 47-55 or a variant thereof having at least 80% sequence identity thereto.

The CD21 -binding domain may comprise a variable heavy chain and a variable light chain pair as shown in the table below, or a variant of the corresponding sequence shown as SEQ ID NO: 38-55 having at least 80% sequence identity thereto, such that the variable heavy and light chain pair retains the ability to bind to CD21. Suitably, the variant variable heavy and iight chain pair may bind to CD21 at least as weli as the corresponding variable heavy and iight chain pair in the table. In other words, the variant may specifically bind to CD21 with a binding affinity which is at ieast equivalent to the binding affinity between the corresponding variable heavy and iight chain pair shown below and CD21.

The variant may have at Ieast 80, 85, 90, 95, 98 or 99% sequence identity to any one of SEQ ID NO: 38-55.

The CDRs may be grafted onto the framework of a human antibody or scFv.

The CD21 -binding domain may be a scFv. A scFv is a fusion protein of the heavy variable region (VH) and Sight chain variable region (VL) of an antibody, connected with a short linker peptide of ten to about 25 amino acids. The scFv may be in the orientation VH-VL, i.e. the VH is at the amino-terminus of the CAR molecule and the VL domain is linked to the spacer and, in turn the transmembrane domain and endodomain.

The scFv may comprise a sequence shown as SEQ ID NO: 30-37 or 58 or a variant of any one of SEQ ID NO: 30-37 or 58 having at least 80% sequence identity thereto and retaining the ability to bind to CD21. Suitably, the variant may bind to CD21 at least as well as the corresponding scFv shown as SEQ ID NO: 30-37 or 58. In other words, the variant may specifically bind to CD21 with a binding affinity which is at least equivalent to the binding affinity between the corresponding scFv shown as SEQ ID NO: 30-37 or 58 and CD21.

Binding affinities may be determined using SPR methodologies, for example as described herein.

The scFv may comprise a sequence shown as SEQ ID NO: 58 or a variant having at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 58 and retaining the ability to bind to CD21 The scFv may comprise a sequence shown as SEQ ID NO: 30 or a variant having at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 30 and retaining the ability to bind to CD21.

The scFv may comprise a sequence shown as SEQ ID NO: 31 or a variant having at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 31 and retaining the ability to bind to CD21. The scFv may comprise a sequence shown as SEQ ID NO: 32 or a variant having at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 32 and retaining the ability to bind to CD21.

The scFv may comprise a sequence shown as SEQ ID NO: 33 or a variant having at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 33 and retaining the ability to bind to CD21.

The scFv may comprise a sequence shown as SEQ ID NO: 34 or a variant having at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 34 and retaining the ability to bind to CD21.

The scFv may comprise a sequence shown as SEQ ID NO: 35 or a variant having at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 35 and retaining the ability to bind to CD21.

The scFv may comprise a sequence shown as SEQ ID NO: 36 or a variant having at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 36 and retaining the ability to bind to CD21.

The scFv may comprise a sequence shown as SEQ ID NO: 37 or a variant having at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 37 and retaining the ability to bind to CD21.

The“GGGGSGGGGSGGGGS” (SEQ ID NO: 56) linker sequence of any one of the scFVs shown as SEQ ID NO: 29-37 may be replaced with a suitable, alternative linker sequence.

TRANSMEMBRANE DOMAIN

The transmembrane domain is the sequence of a classical CAR that 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 a transmembrane portion. 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/). Further, given that the transmembrane domain of a protein is a relatively simple structure, i.e a polypeptide sequence predicted to form a hydrophobic alpha helix of sufficient length to span the membrane, an artificially designed TM domain may also be used (US7052906 B1 describes transmembrane components). The transmembrane domain of the CAR may comprise a hydrophobic aipha helix. The transmembrane domain may be derived from CD28, which gives good receptor stability. The transmembrane domain may comprise the sequence shown as SEQ ID NO: 24 or a variant thereof having at least 80% sequence identity.

SEQ ID NO; St

FWVLVVVGGVLACYSLLVTVAFIIFWV

The variant may have at least 80, 85, 90, 95, 98 or 99% sequence identify with SEQ ID NO: 59, provided that the variant sequence retains the capacity to traverse the membrane.

SIGNAL PEPTIDE

The CAR may comprise a signal peptide so that when it is expressed in a ceil, such as a T- cei!, the nascent protein is directed to the endoplasmic reticulum and subsequently to the ceil surface, where it is expressed.

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

SPACER DOMAIN

The CAR may comprise a spacer sequence to connect the antigen-binding domain with the transmembrane domain. A flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding.

The spacer sequence may, for example, comprise an !gG1 Fc region, an !gG1 hinge or a human CD8 stalk or the mouse CD8 stalk. The spacer may alternatively comprise an alternative linker sequence which has similar length and/or domain spacing properties as an lgG1 Fc region, an lgG1 hinge or a GD8 stalk. A human lgG1 spacer may be altered to remove Fc binding motifs.

INTRACELLULAR DOMAIN

The intracellular domain is the signal-transmission portion of a classical CAR. The most commonly used signalling domain component is that of CD3-zeta endodomain, which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co stimulatory signalling may be needed. For example, chimeric CD28 and 0X40 can be used with CD3-Zeta to transmit a proliferative / survival signal, or all three can be used together (illustrated in Figure 1 B).

The intracellular signalling domain may be or comprise a T cell signalling domain.

The intracellular signalling domain may comprise one or more immunoreceptor tyrosine- based activation motifs (ITAMs). An ITAM is a conserved sequence of four amino acids that is repeated twice in the cytoplasmic tails of certain cell surface proteins of the immune system. The motif contains a tyrosine separated from a leucine or isoleucine by any two other amino acids, giving the signature YxxL/I. Two of these signatures are typically separated by between 6 and 8 amino acids in the tail of the molecule (YxxL/Ix _(6-8) YxxL/I).

ITAMs are important for signal transduction in immune cells. Hence, they are found in the tails of important cell signalling molecules such as the CDS and z-chains of the T cell receptor complex, the CD79 alpha and beta chains of the B ceil receptor complex, and certain Fc receptors. The tyrosine residues within these motifs become phosphorylated following interaction of the receptor molecules with their ligands and form docking sites for other proteins involved in the signalling pathways of the ceil.

The intracellular signalling domain component may comprise, consist essentially of, or consist of the CD3-z endodomain, which contains three ITAMs. Classically, the CD3-z endodomain transmits an activation signal to the T cell after antigen is bound. However, in the context of the present invention, the CD3-z endodomain transmits an activation signal to the T cell after the MHC/peptide complex comprising the engineered B2M binds to a TCR on a different T cell.

The intracellular signalling domain may comprise additional co-stimulatory signalling. For example, 4-1 BB (also known as CD137) can be used with CD3-z, or CD28 and 0X40 can be used with CD3-z to transmit a proliferative / survival signal.

Accordingly, intracellular signalling domain may comprise the CD3-z endodomain alone, the CD3-z endodomain in combination with one or more co-stimulatory domains selected from 4- 1 BB, CD28 or 0X40 endodomain, and/or a combination of some or all of 4-1 BB, CD28 or QX40

The endodomain may comprise one or more of the following: an ICOS endodomain, a CD2 endodomain, a CD27 endodomain, or a CD40 endodomain.

The endomain may comprise the sequence shown as SEQ ID NO: 60 to 63 or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence retains the capacity to transmit an activating signal to the cell.

As used throughout this disclosure, the percentage identity between two polypeptide sequences may be readily determined by programs such as BLAST, which is freely available at http://blast.ncbi.nlm.nih.gov. Suitably, the percentage identity is determined across the entirety of the reference and/or the query sequence.

SEQ ID NO: 60 - CD3-z endodomain

RVKFSRSADAPAYQQGQNQLYNELNLGRRESYDVLDKRRGRDPEMGGKPRRKNPQEGLYN EL QKDKMAEAYSE I GMKGERRRGKGHDGLYQGLS TATKDT YDALHMQAL PPR

SEQ ID HO: 61 - 4-1 BB and CD3-z end odomams

MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQR TC

DXCRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKD CCFGT

FNDQKRGICRPWTNCSLDGKSVLVNGTKERDWCGPS PADLS PGAS SVTPPAPAREFGHS PQ

I IS FFLALT8TALLFLLFFLTLRFSWKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEE

EEGGCELRVKFSRSADAPAYQQGQNQLYKELNLGRREEYDVLDKRRGRDPEMGGKPQ RRKNP

QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R

SEQ ID NO: 62 - CD28 and CD3-z endodomains

SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQL YN

ELNLGRREEYDVLDKRRGRBPEMGGB1PRRKMPQEGLYMELQKDKMAEAY3EIGMKG ERRRGK

GHDGLYQGL3TATKDTYDALHMQALPPR

SEQ ID NO: 63 - CD28, 0X40 and CD3-z endodomains

SKRSRLLH3DYMKMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPFGGGS FRT PI

QEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGK

PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALP

PR

Suitably, the CAR may have the general format: antigen-binding domain - TCR element. As used herein“TCR element” means a domain or portion thereof of a component of the TCR receptor complex. The TCR element may comprise (e.g. have) an extracellular domain and/or a transmembrane domain and/or an intracellular domain e.g. intracellular signalling domain of a TCR element.

The TCR element may selected from TCR alpha chain, TCR beta chain, a CD3 epsilon chain, a CD3 gamma chain, a CD3 delta chain, CD3 epsilon chain.

Suitably, the TCR element may comprise the extracellular domain of the TCR alpha chain, TCR beta chain, a CD3 epsilon chain, a CD3 gamma chain, a CD3 delta chain, or CD3 epsilon chain.

Suitably, the TCR element may comprise the transmembrane domain of the TCR alpha chain, TCR beta chain, a CD3 epsilon chain, a CD3 gamma chain, a CD3 delta chain, or CD3 epsilon chain.

Suitably, the TCR element may comprise the intracellular domain of the TCR alpha chain, TCR beta chain, a CD3 epsilon chain, a CD3 gamma chain, a CD3 delta chain, or CD3 epsilon chain.

Suitably, the TCR element may comprise the TCR alpha chain, TCR beta chain, a CD3 epsilon chain, a CD3 gamma chain, a CD3 delta chain, or CD3 epsilon chain.

POLYNUCLEOTIDE

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

It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed. Suitably, the polynucleotides of the present invention are codon optimised to enable expression in a mammalian cell, in particular an immune effector cell as described herein. 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.

VECTOR

The present invention also provides a vector, which comprises one or more nucleic acid sequence(s) of the invention. Such a vector may be used to introduce the nucleic acid sequence(s) or construct(s) into a host cell so that it expresses a CAR of the present invention.

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.

The vector may be capable of transfecting or transducing a cell.

CELL

The cell of the present invention may be an immune effector cell, such as a T-cell, a natural killer (NK) cell or a cytokine induced killer cell.

The T cell may be an alpha-beta T cell or a gamma-delta T cell.

The cell may be derived 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). T or NK cells, for example, may be activated and/or expanded prior to being transduced with nucleic acid molecule(s) encoding the polypeptides of the invention, for example by treatment with an anti-CD3 monoclonal antibody. Alternatively, the cell may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to T cells. Alternatively, an immortalized T-cell line which retains its lytic function may be used.

The cell may be a haematopoietic stem cell (HSC). HSCs can be obtained for transplant from the bone marrow of a suitably matched donor, by leukopheresis of peripheral blood after mobilization by administration of pharmacological doses of cytokines such as G-CSF [peripheral blood stem cells (PBSCs)], or from the umbilical cord blood (UCB) collected from the placenta after delivery. The marrow, PBSCs, or UCB may be transplanted without processing, or the HSCs may be enriched by immune selection with a monoclonal antibody to the CD34 surface antigen.

PHARMACEUTICAL COMPOSITION

The present invention also relates to a pharmaceutical composition containing a cell, a nucleic acid construct, a first nucleic acid sequence and a second nucleic acid sequence; a vector or a first and a second vector of the present invention. In particular, the invention relates to a pharmaceutical composition comprising a cell according to the present invention.

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

METHOD OF TREATMENT

The present invention provides a method for treating and/or preventing a disease which comprises the step of administering a cell, a polynucleotide; or a vector of the present invention (for example in a pharmaceutical composition as described above) to a subject.

Suitably, the present methods for treating and/or preventing a disease may comprise administering a cell of the 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. In this respect, the cells may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease. The method for preventing a disease relates to the prophylactic use of the cells of the present invention. In this respect, the cells may be administered to a subject who has not yet contracted the disease and/or who is not showing any symptoms of the disease to prevent or impair the cause of the disease or to reduce or prevent development of at least one symptom associated with the disease. The subject may have a predisposition for, or be thought to be at risk of developing, the disease.

The method may involve the steps of:

(i) isolating a cell-containing sample;

(ii) transducing or transfecting such cells with a polynucleotide or vector provided by the present invention;

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

The present invention provides a cell, a nucleic acid construct, a first nucleic acid sequence and a second nucleic acid sequence, a vector, or a first and a second vector of the present invention for use in treating and/or preventing a disease. In particular 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, a polynucleotide or a vectorof the present invention in the manufacture of a medicament for the treatment and/or prevention of a disease. In particular, the invention relates to the use of a cell in the manufacture of a medicament for the treatment and/or prevention of a disease

The disease to be treated and/or prevented by the method of the present invention may be cancer. In particular, the disease may be a cancer comprising tumour cells which express CD21.

Suitably, the disease may be T-cell acute lymphoblastic leukaemia or a B-cell leukaemia or lymphoma.

Suitably, the disease may be T-cell acute lymphoblastic leukaemia.

Suitably, the disease may be a B-cell leukaemia or lymphoma. For example, the disease may be B-cell chronic lymphocytic leukaemia/small lymphocytic lymphoma, Acute lymphoblastic leukaemia, mature B-cell type, B-cell prolymphocytic leukaemia, Precursor B lymphoblastic leukaemia, Hairy cell leukaemia, Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, Marginal zone B-cell lymphoma (MZL) or Mucosa-Associated Lymphatic Tissue lymphoma (MALT), Small lymphocytic lymphoma, Mantle cell lymphoma (MCL), Burkitt's lymphoma, Lymphoplasmacytic lymphoma, Nodal marginal zone B cell lymphoma (NMZL), Splenic marginal zone lymphoma (SMZL), Intravascular large B-cell lymphoma, Primary effusion lymphoma, Lymphomatoid granulomatosis, Primary central nervous system lymphoma, ALK-positive large B-cell lymphoma or Plasmablastic lymphoma.

COMBINATION THERAPY

The present invention further provides a method of treating and/or preventing a disease which comprises the step of administering a cell, a polynucleotide; or a vector of the present invention (for example in a pharmaceutical composition as described above) in combination with a NOTCH activating agent to a subject.

The disease may be any disease as described herein.

The Notch protein spans the cell membrane, with part of it inside and part outside. Ligand proteins binding to the extracellular domain induce proteolytic cleavage and release of the intracellular domain, which enters the cell nucleus to modify gene expression. Once the notch extracellular domain interacts with a ligand, an ADAM-family metalloprotease called ADAM 10, cleaves the notch protein just outside the membrane. This releases the extracellular portion of notch (NECD), which continues to interact with the ligand. The ligand plus the notch extracellular domain is then endocytosed by the ligand-expressing cell. There may be signalling effects in the ligand-expressing cell after endocytosis. After this first cleavage, an enzyme called g-secretase (which is implicated in Alzheimer's disease) cleaves the remaining part of the notch protein just inside the inner leaflet of the cell membrane of the notch-expressing cell. This releases the intracellular domain of the notch protein (NICD), which then moves to the nucleus, where it can regulate gene expression by activating the transcription factor CSL.

The NOTCH pathway is activated by translocation or mutation (of Notchl and/or FBXW7) in -80% of T-ALL cases (see Van Vlierberghe, P. & Ferrando, A.; J. Clin. Invest. 122, 3398- 3406 (2012)).

NOTCH activation was associated with upregulation of CD21 , suggesting that CD21 is a downstream target of NOTCH. Furthermore, incubation of NOTCH-mutated T-ALL cell lines with the dual PI3K/mTOR kinase inhibitor P1103 led to feedback activation of NOTCH signalling and consequently CD21 expression, in a manner that could be blocked by concomitant NOTCH inhibition (Shepherd, C. et al. Leukemia 27, 650-660 (2013)). The present inventors have determined that NOTCH mutated and unmutated cell lines incubated with multiple agents which block components of the PI3K/ AKT/ mTOR pathway results in significant CD21 upregulation in NOTCH mutated T-ALL cell lines only, and loss of CD21 protein expression upon incubation with a gamma-secretase inhibitor, which inhibits NOTCH.

Suitably, the disease may be T-cell acute lymphoblastic leukaemia or a B-cell leukaemia or lymphoma. In particular, the disease may be T-cell acute lymphoblastic leukaemia.

Suitably, the disease may be a cancer in which the tumour cells comprise a mutation which results in activation of the NOTCH pathway. In other words, the cancer may be a NOTCH+ tumour. For example, the tumour cells may comprise a mutation, such as a translocation, substitution, deletion or insertion which results in activation or increased expression of e.g. Notchl and/or inactivation of FBXW7. The mutation may be a t(7;9)(q34;q34.3) translocation.

Methods for identifying such mutations are well known in the art and include, for example, next-generation sequencing technology such as Solexa and SOLiD platforms. Mutations which result in the activation of NOTCH may be determined by identifying a translocation using metaphase spread and FISH techniques, sequencing Notch gene and determining for upregulation of genes controlled by NOTCH. An illustrative human Notchl amino acid sequence is provided by UniProt Accession Number P46531. An illustrative human FBXW7 amino acid sequence is provided by UniProt Accession Number Q969H0. A summary of previously identified NOTCH activating mutations and suitable methods for identifying such mutations is provided in Van Vlierberghe, P. & Fernando, A (as above).

Suitably, the cancer may be a T-cell acute lymphoblastic leukaemia in which tumour cells comprise a mutation which results in activation of the NOTCH pathway.

Accordingly, and without wishing to be bound by theory, the present inventors consider that co-administration of a CD21 CAR-based therapy with an agent that increases NOTCH signalling will improve the efficacy of the CD21 CAR by increasing the level and/or density of CD21 expression on target tumour cells.

“A NOTCH activating agent” is intended to encompass any agent which induces and/or upregulates NOTCH signalling and is suitable for use in a pharmaceutical composition. For example, suitable NOTCH activating agents may be determined using a reporter assay in a cell line. Suitable reporter assays are known in the art, e.g. as described by Pinchot et al. (Cancer. 2011 Apr 1 ; 117(7): 1386-1398; hereby incorporated by reference) or by determining an upregulation of NOTCH regulated gene expression following treatment with the agent. Upregulation of NOTCH regulated gene expression may be determined using commercially available kits and assays (e.g. as provided by Giagen - RT ² Profiler™ PCR Array Human Notch Signalling Pathway).

Suitably, NOTCH activating agent increases CD21 expression in a target cell. Increased CD21 expression may be determined using methods which are known in the art, for example northern blotting, serial analysis of gene expression (SAGE) or quantitative polymerase chain reaction (qPCR). Protein levels in a population of cells may be measured by techniques such as flow cytometry, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), immunohistochemistry, Western blotting or enzyme-linked immunosorbent assay (ELISA).

For example, the agent may be capable of inhibiting the PI3K/AKT/mTOR pathway.

Suitably, the agent may be a PI3K inhibitor, an AKT inhibitor, a mTOR inhibitor or a combination inhibitor (e.g. an agent which inhibits at least two of PI3K, AKT and mTOR).

The agent may be an allosteric, kinase or dual inhibitor of Pi3K, AKT and/or mTOR. The inhibitor may be a dual inhibitor of both PI3K and mTOR.

Suitably, the agent may be a small molecule.

An illustrative list of PI3K inhibitors includes, but is not limited to, pictilisib, idelalisib, buparlisib, ZSTK474, alpelisib, AZD6482, duvelisib, copanlisib, taselisib, serabelisib, GDC0326, umbralisib, AZD8835, nemiralisib, AZD8186, pilaralisib, PX866, MLN1117 and IPI549.

An illustrative list of dual PI3K/mTOR inhibitor includes, but is not limited to, dactolisib (BEZ235), P1103, voxtalisib, omipalisib, PF04691502, apitolisib, GSK1059615, gedatolisib, GDC0084, voxtalisib, SF1126, and PQR309.

An illustrative list of allosteric mTOR inhibitors includes, but is not limited to, sirolimus/rapamycin, everolimus, temsirolimus, and ridaforolimus. An illustrative list of mTOR kinase inhibitors includes, but is not limited to, AZD8055, KU0063794, Torkinib, sapanisertib, Torinl , Torin2, OSI-027, WYE-354, WYE-132, WYE- 687, vistusertib, WAY-600, GDC0349, XL388, MHY1485, CZ415, CC-223, and PP242.

An illustrative list of allosteric/kinase dual TOR inhibitors includes, but is not limited to, Rapalink-1 , Rapalink-2, and Rapalink-3.

An illustrative list of AKT inhibitors includes but is not limited to MK2206, Perifosine, GCK690693, ipatasertib, AZD5363, AT7867, CCT128930, A-674563, PHT-427, afurusertib, AT13148, uprosertib, miransertib, ARQ751 , BAY1125976, GSK2141795, LY2780301 , and tricribine.

As used herein,‘in combination’ means that the cell, polynucleotide; or vector of the present invention (for example in a pharmaceutical composition as described above) may be used simultaneously, sequentially or separately with an agent which increases NOTCH signalling.

METHOD OF MAKING A CELL

CAR- expressing cells of the present invention may be generated by introducing DNA or RNA coding for the CAR of the present invention.

The cell of the invention may be made by:

(i) isolation of a cell-containing sample from a subject or one of the other sources listed above; and

(ii) transduction or transfection of the cells with one or more a polynucleotide(s) or nucleic acid construct as defined above in vitro or ex vivo.

The cells may then by purified, for example, selected on the basis of expression of the antigen-binding domain of the antigen-binding polypeptide.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

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

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

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

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.

EXAMPLES

Example 1 - CD21 is a potential target for T-ALL

Flow cytometry data demonstrated that CD21 is expressed in -70% (11/16) of T-ALL cell lines with a median surface copy number of 2545/cell in positive lines. In addition, interrogation of publicly available gene-expression data show that CD21 is upregulated in primary T-ALL. Analysis of CD21 expression by flow cytometry in diagnostic specimens from 22 primary T-ALL cases and showed that 15 (68%) were CD21-positive (Figure 2D).

Example 2 - CD21 is not expressed on normal non-lymphoid tissues

CD21 is primarily described on the surface of B cells and follicular dendritic cells (FDCs) where its role as a member of the B cell co-receptor complex binding C3d and as the receptor for EBV is well established. In addition to B-cells, CD21 has also historically been reported to be expressed on about 10% of normal thymocytes, where its function is unknown. RNA expression data across a panel of normal tissues showed a restricted pattern of CD21 expression confined to B-lymphocytes, spleen and terminal ileum (Figure 3A).

Peripheral blood subset analysis of 11 healthy donors has shown restricted expression of CD21 in cells other than B-lymphocytes, with expression only on <10% of ab T-cells (Figure 3B), which was not affected by T-cell stimulation. No expression on erythroid or myeloid compartments was seen in blood or in bone marrow from patients with T-ALL.

Example 3 - Binder discovery for CD21

Binders against human CD21 were isolated. Briefly, 3 rats were genetically vaccinated with human CD21. After confirmation of seroconversion against CD21 (Figure 4B), lymphoid tissues were harvested, and B-cell RNA extracted. Immunoglobulin heavy and light chains were cloned into an immune phage-display library by PCR. This library was panned against beads coated with immobilised CD21 (Figure 4C). 6 specific, high-affinity binders against CD21 were identified (Figure 4D, Table 2 and Table 3).

Example 4 - Illustrative CD21 CAR functionality

SupT1 is a T-cell leukaemia cell line which expresses CD21. To generate a control cell line which doesn’t express CD21 , SupT1 cells were genomically edited at the CD21 locus using CRISPR/Cas9 and a CD21 guide RNA which recognized the CD21 genomic. Wild-type and edited supT1 cells were stained with either an isotype antibody or an anti-CD21 antibody and analysed by flow-cytometry (see Figure 5A).

Normal donor human T-cells were transduced (using retroviral constructs) with anti-CD21 CAR derived from scFv C43 with a CD8 stalk spacer or an lgG1 hinge spacer, or with anti- CD21 CAR derived from scFv C48 with either a CD8 stalk spacer or an lgG1 hinge spacer. Control non-transduced T-cells and T-cells transduced with a CD19 CAR were also tested.

Normal donor T-cells were isolated by ficoll, stimulated with soluble anti-CD3/anti-CD28 antibodies and retrovirally transduced with the above constructs. After transduction was confirmed by flow-cytometry, these effectors were co-cultures with SupT 1 targets or SupT 1 cells which don’t express CD21 target cells. The co-culture was analyzed by flow cytometry calibrated by counting beads and remaining SupT1 cells were quantified (see Figure 5B).

Example 5 - CD21 expression in T-ALL is regulated in a NOTCH-dependent manner

NOTCH mutated and unmutated cell lines incubated with multiple agents which block components of the PI3K/ AKT/ mTOR pathway confirms significant CD21 upregulation in NOTCH mutated T-ALL cell lines only, and loss of CD21 protein expression upon incubation with a gamma-secretase inhibitor, which inhibits NOTCH. This effect was seen to persist up to at least 24hrs after the drug has been removed (see Figure 6).

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.