Chimeric antigen receptors for binding to dysfunctional p2x7 receptor

Abstract

The present invention relates to chimeric antigen receptors for binding to dysfunctional P2X7 receptor, genetically modified cells comprising the same, and uses thereof.

Description

Chimeric antigen receptors for binding to dysfunctional P2X? receptorField of the invention

[0001] The present invention relates to chimeric antigen receptors, immune cells expressing chimeric antigen receptors and methods of using the same for the prevention and / or treatment of cancer.Related application

[0002] This application claims priority from Australian provisional application AU 2023900626, the contents of which are hereby incorporated by reference in their entirety.Background of the invention

[0003] Current T cell therapies rely on enriched or modified human T cells to target and kill cancer cells in a patient. To increase the ability of T cells to target and kill a particular cancer cell, methods have been developed to engineer T cells to express constructs which direct T cells to a particular target cancer cell. Chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs), which comprise binding domains capable of interacting with a particular tumour antigen, allow T cells to target and kill cancer cells that express the particular tumour antigen. However, some tumour types, and in particular solid tumours, are resistant to T cell immunotherapy.

[0004] Although CARs have been designed for binding to various tumour-specific antigens, the architecture and binding affinity of the CARs often requires optimisation to ensure appropriate levels of binding in the context of T cells binding to cancer cells. In other words, antigen binding domains derived directly from therapeutic antibodies, may have an inappropriate affinity for use in the context of a CAR, and may potentially result in the manufacture of a CAR T cell which leads to toxicity in patients.

[0005] There is a need for improved CARs for use in therapeutic settings and compositions comprising the same.

[0006] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in anyjurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.Summary of the invention

[0007] The present invention is based on the surprising finding by the inventors that particular antigen recognition domain sequences provide for improved functionality and cell killing in the context of chimeric antigen receptors.

[0008] In a first aspect, the present invention provides a chimeric antigen receptor (CAR) comprising: i) an antigen recognition domain that recognises dysfunctional P2X? receptor(nfP2X? receptor) or an epitope derived therefrom; ii) a transmembrane domain; and iii) an intracellular domain, wherein the antigen recognition domain comprises the complementary determining regions (CDRs) from a VH comprising a sequence as set forth in SEQ ID NO: 4 and the CDRs from a VL comprising a sequence as set forth in SEQ ID NO: 12.

[0009] In a second aspect, the present invention provides a chimeric antigen receptor (CAR) comprising: i) an antigen recognition domain that recognises dysfunctional P2X? receptor (nfP2X? receptor) or an epitope derived therefrom; ii) a transmembrane domain; and iii) an intracellular domain, wherein the antigen recognition domain comprises the complementary determining regions (CDRs) from a VH comprising a sequence as set forth in any of SEQ ID NOs: 135, 136 or 137.

[0010] In accordance with the first aspect, the antigen recognition domain of the CAR comprises:FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4, andFR1 a - CDR1 a - FR2a - CDR2a - FR3a - CDR3a - FR4a, wherein:FR1 , FR2, FR3 and FR4 are each framework regions;CDR1 , CDR2 and CDR3 are each complementarity determining regions;FR1 a, FR2a, FR3a and FR4a are each framework regions;CDR1 a, CDR2a and CDR3a are each complementarity determining regions wherein the complementarity determining regions comprise an amino acid sequence as described in Table 1 below.

[0011] In accordance with the second aspect of the invention, the antigen recognition domain of the CAR comprises:FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 wherein:FR1 , FR2, FR3 and FR4 are each framework regions;CDR1 , CDR2 and CDR3 are each complementarity determining regions; wherein the complementarity determining regions comprise an amino acid sequence as described in Table 3 below.

[0012] Preferably, and in accordance with the first aspect of the invention, there is provided a chimeric antigen receptor (CAR) comprising: an antigen recognition domain, a transmembrane domain; and an intracellular domain, wherein the antigen recognition domain comprises:(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 1 , 29, 36 or 43, a CDR2 comprisinga sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 2, 30, 37 or 44 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 3, 31 , 38 or 45;(ii) a VH comprising a sequence at least about 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in SEQ ID NO: 4;(iii) a VL comprising a CDR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 9, 50, 57 or 64, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 10, 51 , 58 or 65, and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 11 , 52, 59 or 66;(iv) a VL comprising a sequence at least about 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in SEQ ID NO: 12;(v) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 1 , 29, 36 or 43, a CDR2 comprising a sequence set forth in SEQ ID NO: 2, 30, 37 or 44, and a CDR3 comprising a sequence set forth in SEQ ID NO: 3, 31 , 38 or 45;(vi) a VH comprising a sequence set forth in SEQ ID NO: 4;(vii) a VL comprising a CDR1 comprising a sequence set SEQ ID NO: 9, 50, 57 or 64, a CDR2 comprising a sequence set forth in SEQ ID NO: 10, 51 , 58 or 65 and a CDR3 comprising a sequence set forth in SEQ ID NO: 1 1 , 52, 59 or 66;(viii) a VL comprising a sequence set forth in SEQ ID NO: 12;(ix) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 1 , 29, 36 or 43, a CDR2 comprising a sequence set forth in SEQ ID NO: 2, 30, 37 or 44 and aCDR3 comprising a sequence set forth in SEQ ID NO: 3, 31 , 38 or 45; and a VL comprising a CDR1 comprising a sequence set SEQ ID NO: 9, 50, 57 or 64, a CDR2 comprising a sequence set forth in SEQ ID NO: 10, 51 , 58 or 65 and a CDR3 comprising a sequence set forth in SEQ ID NO: 11 , 52 59 or 66; or(x) a VH comprising a sequence set forth in SEQ ID NO: 4 and a VL comprising a sequence set forth in SEQ ID NO: 12.

[0013] In any embodiment of the first aspect invention, the antigen recognition domain further comprises at least one of:(i) a VH comprising a framework region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 5, 32, 39, or 46, a FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set in SEQ ID NO: 6, 33, 40 or 47, a FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 7, 34, 41 or 48, and a FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 8, 35, 42 or 49;(ii) a VL comprising a FR1 comprising a at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 13, 53, 60 or 67, a FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 14, 54, 61 or 68, a FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 15, 55, 62 or 69, and a FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%,at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 16, 56, 63 or 70;(iii) a VH comprising a FR1 comprising a sequence set forth in SEQ ID NO: 5, 32, 39 or 46, a FR2 comprising a sequence set forth in SEQ ID NO: 6, 33, 40 or 47, a FR3 comprising a sequence set forth in SEQ ID NO: 7, 34, 41 or 48, and a FR4 comprising a sequence set forth in SEQ ID NO: 8, 35, 42 or 49;(iv) a VL comprising a FR1 comprising a sequence set forth in SEQ ID NO: 13, 53, 60 or 67, a FR2 comprising a sequence set forth in SEQ ID NO: 14, 54, 61 or 68, a FR3 comprising a sequence set forth in SEQ ID NO: 15, 55, 62 or 69, and a FR4 comprising a sequence set forth in SEQ ID NO: 16, 56, 63 or 70; or(v) a VH comprising a FR1 comprising a sequence set forth in SEQ ID NO: 5, 32, 39 or 46, a FR2 comprising a sequence set forth in SEQ ID NO: 6, 33, 40 or 47, a FR3 comprising a sequence set forth in SEQ ID NO: 7, 34, 41 or 48, and a FR4 comprising a sequence set forth in SEQ ID NO: 8, 35, 42 or 49; and a VL comprising a FR1 comprising a sequence set forth in SEQ ID NO: 13, 53, 60 or 67, a FR2 comprising a sequence set forth in SEQ ID NO: 14, 54, 61 or 68, a FR3 comprising a sequence set forth in SEQ ID NO: 15, 55, 62 or 69, and a FR4 comprising a sequence set forth in SEQ ID NO: 16, 56, 63 or 70.

[0014] In any embodiment of the first aspect, the antigen recognition domain comprises, consists essentially of or consists of the amino acid sequence of (in order of N to C terminus or C to N terminus) SEQ ID NOs: 4 and 12.

[0015] In any embodiment of the first aspect, the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 4, or a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; and a light chain variable domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 12, or sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99% identical thereto; wherein the heavy and light chain does not comprise any sequence variation in the CDRs compared to the sequence of SEQ ID NO: 4 or 12, respectively, and / or wherein the antigen binding domain retains the ability to bind to nfP2X? receptor.

[0016] In any embodiment of the first aspect, the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 4, and a light chain variable domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 12, wherein the heavy and light chain variable domain of the antigen binding domain comprises no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions or additions, compared to the amino acid sequence as set forth in SEQ ID NO: 4 or 12, respectively; preferably wherein the amino acid substitutions, deletions or additions are not in the CDRs and / or wherein the antigen binding domain retains the ability to bind to nfP2X? receptor.

[0017] Preferably the antigen recognition domain comprises, consists essentially of or consists of, in order N to C terminus, SEQ ID NO: 12 and SEQ ID NO: 4 (ie VL to VH). Optionally the antigen binding protein comprises SEQ ID NO: 12 (VL) - linker - SEQ ID NO: 4 (VH).

[0018] In any embodiment of the first aspect of the invention the antigen recognition domain competitively inhibits the binding of an antibody comprising a VH comprising a sequence as set forth in SEQ ID NO: 4 and a VL comprising a sequence as set forth in SEQ ID NO: 12.

[0019] Optionally, the variable heavy and variable light regions of the antigen recognition domain are joined via a linker. In any embodiment, the antigen recognition domain may comprise:FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 - linker - FR1a - CDR1 a - FR2a - CDR2a - FR3a - CDR3a - FR4a.

[0020] As defined herein, the linker may be a chemical, one or more amino acids, or a disulphide bond formed between two cysteine residues.

[0021] In any embodiment of the first aspect of the invention, the antigen recognition domain may be a single-chain variable fragment (scFv). As will be understood in the art, an scFv is a fusion protein comprising two portions that may share homology with, or may be identical to, the variable-heavy (VH) and variable-light (VL) chains of an antibody, with the two portions connected, together with a linker peptide.

[0022] In some embodiments, the antigen recognition domain is a multivalent scFv. In some embodiments, the multivalent scFv is a divalent or trivalent scFv.

[0023] Preferably, and in accordance with the second aspect of the invention, there is provided a chimeric antigen receptor (CAR) comprising: an antigen recognition domain, a transmembrane domain; and an intracellular domain, wherein the antigen recognition domain comprises:(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 124, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 125, 126 or 127 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 128, 129 or 130;(ii) a VH comprising a sequence at least about 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in SEQ ID NO: 135, 136 or 137;(iii) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 124, a CDR2 comprising a sequence set forth in SEQ ID NO: 125, 126, or 127, and a CDR3 comprising a sequence set forth in SEQ ID NO: 128, 129 or 130; preferably CDR1 comprises a sequence set forth in SEQ ID NO: 124, CDR2 comprises a sequence set forth in SEQ ID NO: 125, and CDR3 comprises a sequence set forth in SEQ ID NO: 128;or preferably CDR1 comprises a sequence set forth in SEQ ID NO: 124, CDR2 comprises a sequence set forth in SEQ ID NO: 126, and CDR3 comprises a sequence set forth in SEQ ID NO: 129; or preferably CDR1 comprises a sequence set forth in SEQ ID NO: 124, CDR2 comprises a sequence set forth in SEQ ID NO: 127, and CDR3 comprises a sequence set forth in SEQ ID NO: 130; or(iv) a VH comprising a sequence set forth in SEQ ID NO: 135, 136 or 137.

[0024] In any embodiment of the second aspect of the invention, the antigen recognition domain further comprises at least one of:(i) a VH comprising a framework region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 131 , a FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set in SEQ ID NO: 132, a FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 133, and a FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 134;(ii) a VH comprising a FR1 comprising a sequence set forth in SEQ ID NO: 131 , a FR2 comprising a sequence set forth in SEQ ID NO: 132, a FR3 comprising a sequence set forth in SEQ ID NO: 133, and a FR4 comprising a sequence set forth in SEQ ID NO:134.

[0025] In any embodiment of the second aspect the antigen recognition domain comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO:135, 136 or 137.

[0026] In any embodiment, the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 135, or a sequence at least about 80%, at least 81 %, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; wherein the heavy chain does not comprise any sequence variation in the CDRs compared to the sequence of SEQ ID NO: 135, and / or wherein the antigen binding domain retains the ability to bind to nfP2X? receptor.

[0027] In any embodiment, the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 135, wherein the heavy chain variable domain of the antigen binding domain comprises no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions or additions, compared to the amino acid sequence as set forth in SEQ ID NO: 135; preferably wherein the amino acid substitutions, deletions or additions are not in the CDRs and / or wherein the antigen binding domain retains the ability to bind to nfP2X? receptor.

[0028] In any embodiment, the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 136, or a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; wherein the heavy chain does not comprise any sequence variation in the CDRs compared to the sequence of SEQ ID NO: 136, and / or wherein the antigen binding domain retains the ability to bind to nfP2X? receptor.

[0029] In any embodiment, the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 136, wherein the heavy chain variable domain of the antigen binding domain comprises no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no morethan 20 amino acid residue substitutions, deletions or additions, compared to the amino acid sequence as set forth in SEQ ID NO: 136; preferably wherein the amino acid substitutions, deletions or additions are not in the CDRs and / or wherein the antigen binding domain retains the ability to bind to nfP2X? receptor.

[0030] In any embodiment, the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 137, or a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; wherein the heavy chain does not comprise any sequence variation in the CDRs compared to the sequence of SEQ ID NO: 137, and / or wherein the antigen binding domain retains the ability to bind to nfP2X? receptor.

[0031] In any embodiment, the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 137, wherein the heavy chain variable domain of the antigen binding domain comprises no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions or additions, compared to the amino acid sequence as set forth in SEQ ID NO: 137; preferably wherein the amino acid substitutions, deletions or additions are not in the CDRs and / or wherein the antigen binding domain retains the ability to bind to nfP2X? receptor.

[0032] In any embodiment of any aspect, the CAR further comprises a hinge region (also referred to herein as a spacer region). The hinge region may be derived from CD28, CD8a or from lgG4. Preferably the hinge region is derived from CD8a. Typically the hinge is C-terminal to the antigen recognition domain. In one embodiment, the hinge region is located between the antigen recognition domain and the transmembrane domain. In preferred embodiments, the hinge region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 88.

[0033] In any embodiment of any aspect, the transmembrane domain of the CAR comprises a portion from CD8a, CD28 or ICOS. Optionally, the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 89.

[0034] In any embodiment of any aspect, the signalling domain of the CAR comprises a portion derived from an activation receptor. In some embodiments, the activation receptor is a member of the CD3 co-receptor complex. In some embodiments, the portion derived from the CD3 co-receptor complex is CD3- (CD3-zeta) and optionally comprises an amino acid sequence as set forth in SEQ ID NO: 92.

[0035] In any embodiment of any aspect, the signalling domain of the CAR comprises a portion derived from a co-stimulatory receptor. In some embodiments, the costimulatory receptor is CD28, ICOS, CD27, 0X40 and / or 4-1 BB (CD137) and optionally comprises an amino acid sequence as set forth in SEQ ID NO: 90 and / or 91 .

[0036] The signalling domain may comprise a portion derived from an activation receptor and a portion derived from a co-stimulatory receptor. In some embodiments, the activation receptor is a member of the CD3 co-receptor complex and the co-stimulatory receptor is selected from CD28, ICOS, CD27, 0X40 and / or 4-1 BB. In some embodiments, the activation receptor is a member of the CD3 co-receptor complex and the co-stimulatory receptor is CD28 and 4-1 BB (CD137). Preferably, the portion derived from the CD3 co-receptor complex is CD3- (CD3-zeta). In certain embodiments, the signalling domain comprises an amino acid sequence comprising the sequences set forth in SEQ ID NOs: 90, 91 and / or 92 and combinations thereof.

[0037] In an embodiment of any aspect, the CAR comprises (from N to C terminus) an antigen binding domain as described herein, a linker, CD8a hinge, a CD28 transmembrane domain, a CD28 signalling domain, a 4-1 BB signalling domain, and a CD3 (zeta) signalling domain. Optionally, the CAR may further comprise an epitope or affinity tag.

[0038] In a preferred embodiment of the first aspect, the chimeric antigen receptor comprises an amino acid sequence as set forth in SEQ ID NO: 94, or a functional variant thereof comprising a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% identical thereto; wherein the functional variant comprises an antigen recognition domain as herein defined, preferably comprising a VH comprising the amino acid sequence of SEQ ID NO: 4; and a VL comprising the amino acid sequence of SEQ ID NO: 12.

[0039] In particularly preferred embodiment of the first aspect, the chimeric antigen receptor comprises an amino acid sequence as set forth in SEQ ID NO: 94.

[0040] In a preferred embodiment of the second aspect, the chimeric antigen receptor comprises an amino acid sequence as set forth in SEQ ID NO: 138, or a functional variant thereof comprising a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; wherein the functional variant comprises an antigen recognition domain as herein defined, preferably comprising a VH comprising the amino acid sequence of SEQ ID NO: 135.

[0041] In particularly preferred embodiment of the second aspect, the chimeric antigen receptor comprises an amino acid sequence as set forth in SEQ ID NO: 138.

[0042] In a preferred embodiment of the second aspect, the chimeric antigen receptor comprises an amino acid sequence as set forth in SEQ ID NO: 139, or a functional variant thereof comprising a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; wherein the functional variant comprises an antigen recognition domain as herein defined, preferably comprising a VH comprising the amino acid sequence of SEQ ID NO: 136.

[0043] In particularly preferred embodiment of the second aspect, the chimeric antigen receptor comprises an amino acid sequence as set forth in SEQ ID NO: 139.

[0044] In a preferred embodiment of the second aspect, the chimeric antigen receptor comprises an amino acid sequence as set forth in SEQ ID NO: 140, or a functional variant thereof comprising a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% identical thereto; wherein the functional variant comprises an antigen recognition domain as herein defined, preferably comprising a VH comprising the amino acid sequence of SEQ ID NO: 137.

[0045] In particularly preferred embodiment of the second aspect, the chimeric antigen receptor comprises an amino acid sequence as set forth in SEQ ID NO: 140.

[0046] In one aspect, any variation described herein, for example % identity with an amino acid sequence or substitutions, deletions or additions to an amino acid sequence, is variation in the framework region of a variable domain or a constant region.

[0047] In a further aspect, there is provided a nucleic acid encoding a chimeric antigen receptor according to the first or second aspects of the invention.

[0048] In another aspect, the present invention provides a nucleic acid construct that includes a nucleic acid molecule according to the invention described herein. In some embodiments, expression of the nucleic acid molecule is under the control of a transcriptional control sequence. In some embodiments, the transcriptional control sequence may be a constitutive promoter or an inducible promoter.

[0049] In some embodiments, the nucleic acid construct further includes an internal ribosome entry site (IRES) that allows for translation initiation within the mRNA once expressed from the nucleic acid construct.

[0050] In some embodiments, the nucleic acid construct is a vector such as a viral vector, which can be used to transform an immune cell, for example a T cell, to induce expression of the CAR.

[0051] In another aspect, the present invention provides a genetically modified cell that comprises a CAR according to the invention described herein.

[0052] In another aspect, the present invention provides a genetically modified cell that comprises a nucleic acid molecule according to the invention described herein, or a nucleic acid construct of the invention described herein, or a genomically integrated form of the construct.

[0053] In another aspect, the present invention provides a method of generating a genetically modified cell, said method comprising transducing the cell, preferably animmune cell, with a nucleic acid construct encoding a CAR of the invention as described herein, so that the transduced cell expresses the CAR, thereby generating a genetically modified cell.

[0054] In some embodiments, the cell is an immune cell such as a leukocyte. In some embodiments, the cell is a Peripheral Blood Mononuclear Cell (PBMC), a lymphocyte, a T cell (including a CD4+ T cell or a CD8+ T cell), a natural killer (NK) cell, a natural killer T cell or a tumour infiltrating lymphocyte (TIL).

[0055] In a preferred embodiment, the immune cell that expresses the CAR is a T cell. Illustrative examples of suitable T cells include helper T cells (HTL; CD4+T cell), a cytotoxic T cell (CTL; CD8+T cell), CD4+CD8+T cell, CD4 CD8’ T cell, or any other subset of T cells. Other illustrative examples of suitable T cells include T cells expressing one or more of the following markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, CD197, and HLA-DR.

[0056] In another aspect, the present invention provides a method of generating a CAR-T cell, the method comprising transducing a T cell with a nucleic acid construct encoding a CAR of the invention as described herein so that the transduced T cell expresses the CAR, thereby generating a CAR-T cell.

[0057] In another aspect, the present invention provides a method of killing a cell expressing nfP2X? receptor, the method comprising exposing the cell expressing nfP2X? receptor to a genetically modified cell of the invention described herein, thereby killing a cell expressing nfP2X? receptor.

[0058] In another aspect, the present invention provides a method of killing a cancer cell, the method comprising exposing the cancer cell to a genetically modified cell of the invention, thereby killing the cancer cell.

[0059] In some embodiments, the cell expressing nfP2X? receptor (ie the cell for being bound by the CAR of the invention) is a cancer cell. In some embodiments the cancer is selected from the group consisting of; brain cancer, oesophageal cancer, mouth cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cervical cancer, epithelial cell cancers, skin cancer, leukaemia, lymphoma, myeloma, breast cancer, ovarian cancer, endometrial cancer and testicular cancer. In some embodiments thecancer is selected from the group consisting of; lung cancer, oesophageal cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer, cervical cancer, vaginal cancers, epithelial cell cancers, skin cancer, blood-related cancers, breast cancer, endometrial cancer, uterine cancer and testicular cancer.

[0060] In some embodiments, the cancer is metastatic. In some embodiments, the cancer is stage III cancer or is stage IV cancer.

[0061] In another aspect, the present invention provides a method of expanding in vitro the genetically modified cell of the invention described herein, the method comprising the step of exposing the cell to an antigen for the CAR. In some embodiments, the method includes the further step of exposing the cell to a cytokine.

[0062] In another aspect, the present invention provides a method of expanding in vitro the genetically modified cell of the invention described herein, the method comprising the step of exposing the cell to an antigen for the CAR, e.g. nfP2X? receptor, and simultaneously exposing the cell to a cytokine.

[0063] In some embodiments, the cytokine is a member of the IL-2 subfamily, the interferon subfamily, the IL-10 subfamily, the IL-1 subfamily, the IL-17 subfamily or the TGF-[3 subfamily.

[0064] In some embodiments, the cytokine is selected from the group consisting of IFN- y, IL-2, IL-5, IL- 7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, TNF-a, TGF-[31 , TGF-[32, TGF-[33 and GM-CSF, or a combination thereof.

[0065] In another aspect, the present invention provides a method of expanding in vitro the genetically modified cell of the invention described herein, the method comprising- exposing the cell to immobilised CD3 and CD28 agonists; and- contacting the cells with a media under conditions that allow proliferation of the cells, preferably T cells, preferably human T cells.

[0066] In some embodiments, the agonists are immobilised on a beaded substrate. In one embodiment, the agonists may be immobilised on “Human Activator” Dynabeads™. In another preferred example, the agonists are immobilised on a colloidal polymericnanomatrix beaded substrate conjugated to recombinant humanized CD3 and CD28 agonist (for example on a “MACS GMP” TransAct™ beaded substrate).

[0067] In some embodiments the CD3 and CD28 agonists are anti-CD3 and anti-CD28 antibodies.

[0068] In some embodiments the media is TexMACS™ GMP media. In some embodiments the media supplemented with interleukins, for example IL-7 and IL-15.

[0069] In some embodiments, the antibodies are immobilised on a surface of a tissue culture vessel such as a surface of a culture flask, plate or bioreactor.

[0070] In another aspect, the present invention provides a pharmaceutical composition including a genetically modified cell of the invention described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises suitable adjuvants which may consist of cytokines. In some embodiments, the pharmaceutical composition may also comprise an intermediate as described herein.

[0071] In another aspect, the present invention provides a method of:• treating, preventing or minimising progression of cancer in a subject,• minimising, reducing or preventing growth of a tumour in a subject,• minimising, reducing or preventing cancer metastasis in a subject, or• increasing survival of a subject suffering from cancer, optionally wherein the cancer or tumour is characterised by the expression of nfP2X? receptor; the method comprising, administering to the subject a CAR of the invention described herein, a nucleic acid construct of the invention as described herein, a genetically modified cell of the invention as described herein, or a pharmaceutical composition of the invention as described herein, thereby:• treating, preventing or minimising progression of cancer in the subject,• minimising, reducing or preventing growth of a tumour in the subject,• minimising, reducing or preventing metastasis in the subject, or• increasing survival of the subject suffering from cancer.

[0072] In another aspect, the present invention provides use of a CAR of the invention described herein, a nucleic acid construct of the invention as described herein, a genetically modified cell of the invention as described herein, or a pharmaceutical composition of the invention as described herein, in the manufacture of a medicament for:• treating, preventing or minimising progression of cancer in a subject,• minimising, reducing or preventing growth of a tumour in a subject,• minimising, reducing or preventing cancer metastasis in a subject, or• increasing survival of a subject suffering from cancer, optionally wherein the cancer or tumour is characterised by the expression of nfP2X? receptor.

[0073] In another aspect, the present invention provides a CAR of the invention described herein, a nucleic acid construct of the invention as described herein, a genetically modified cell of the invention as described herein, or a pharmaceutical composition of the invention as described herein for use in:• treating, preventing or minimising progression of cancer in a subject,• minimising, reducing or preventing growth of a tumour in a subject,• minimising, reducing or preventing cancer metastasis in a subject, or• increasing survival of a subject suffering from cancer, optionally wherein the cancer or tumour is characterised by the expression of nfP2X? receptor.

[0074] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

[0075] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.Brief description of the drawings

[0076] Figure 1 : MOLM-13 cell count following contact with untransduced T cells (UTD), or T cells transduced with CAR12AV1 or CAR10A. NB = no bridging molecule (indicative of direct cell killing). “With BRiDGE” = co-incubation with 200 ng / mL anti-CD33 BRIDGE molecule comprising E200 sequence for binding by CAR antigen recognition domain.

[0077] Figure 2: JeKo-1 cell viability following contact with untransduced T cells (UTD), or T cells transduced with CAR10A, CAR12A, 3a-B9-CAR, or 4A7-CAR, supplemented with anti-CD19 BRiDGE. BRiDGE concentrations are from Ong / mL to 100ng / mL. “BRiDGE” = fusion protein comprising E200 sequence for binding by CAR antigen recognition domain and anti-CD19 antigen binding domain. Effector target cell ratio = 2.77 / 1 . Cell viability was read 21 hours post-co-incubation.

[0078] Figure 3: MOLM-13 cell viability following contact with untransduced T cells (UTD), or T cells transduced with 3a-B9-CAR, or 4A7-CAR. CAR positive cell and cancer cell ratio of 10:1. “BRiDGE” = co-incubation with 100 ng / mL BRiDGE molecule comprising E200 sequence for binding by CAR antigen recognition domain and an anti-CD33 binding domain or an anti-CD19 binding domain. Cell viability was read 24 hours post-co-incubation.

[0079] Figure 4: JeKo-1 cell viability following contact with untransduced T cells (UTD), or T cells transduced with 3a-B9-CAR, or 4A7-CAR. CAR positive cell and cancer cell ratio of 10:1 in (A) and 10:1 , 5:1 and 2.5:1 in (B). “BRiDGE” = co-incubation with 100 ng / mL BRiDGE molecule comprising E200 sequence for binding by CAR antigen recognition domain and either an anti-CD19 antigen binding domain or anti- CD33 binding domain. Cell viability was read 24 hours post-co-incubation.Sequence information

[0080] Table 1 : sequence informationTable 2: further sequence informationTable 3: sequence information for sdAb-based CARs of the inventionDetailed description of the embodiments

[0081] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

[0082] One skilled in the art will recognise many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0083] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0084] All of the patents and publications referred to herein are incorporated by reference in their entirety.

[0085] The present invention seeks to address one or more deficiencies of the prior art and is based on the recognition of the inventors that a specific antigen recognition domain provides for an improved anti-nfP2X? receptor CAR function. More specifically, preferred CARs of the invention are understood to provide higher affinity for binding to cancer cells expressing nfP2X? receptor and therefore have a stronger cell-killing capacity (potency). The inventors have found that particular single domain (sdAb-based) CARs and a particular scFv-based CAR provide for superior efficacy compared with sdAb-based CARs of the prior art.Definitions - general

[0086] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0087] For purposes of interpreting this specification, the following definitions will generally apply and whenever appropriate, terms used in the singular will also include the plural and vice versa.

[0088] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms “a”, “an” and “the” include plural aspects, and vice versa, unless the context clearly dictates otherwise. For example, reference to “a” includes a single as well as two or more; reference to “an” includes a single as well as two or more; reference to “the” includes a single as well as two or more and so forth.

[0089] Those skilled in the art will appreciate that the present invention is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0090] Any example or embodiment of the present invention herein shall be taken to apply mutatis mutandis to any other example or embodiment of the invention unless specifically stated otherwise.

[0091] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0092] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991 ), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1 -4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0093] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0094] As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.

[0095] When comparing amino acid sequences, the sequences should be compared over a comparison window which is determined by the length of the polypeptide. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms such as the BLAST family of programs as, for example, disclosed by Altschul etal., 1997, Nucl. Acids Res. 25: 3389-3402. Global alignment programs may also be used to align similar sequences of roughly equal size. Examples of global alignment programs include NEEDLE (available at www.ebi.ac.uk / Tools / psa / emboss_needle / ) which is part of the EMBOSS package (Rice P et al., 2000, Trends Genet., 16: 276-277), and the GGSEARCH program (available at fasta.bioch.virginia.edu / fasta_www2 / fasta_www.cgi?rm=compare&pgm=gnw) which is part of the FASTA package (Pearson W and Lipman D, 1988, Proc. Natl. Acad. Sci. USA,85: 2444-2448). Both of these programs are based on the Needleman-Wunsch algorithm which is used to find the optimum alignment (including gaps) of two sequences along their entire length. A detailed discussion of sequence analysis can also be found in Unit 19.3 of Ausubel et al ("Current Protocols in Molecular Biology" John Wiley & Sons Inc, 1994- 1998, Chapter 15, 1998).

[0096] "Purinergic receptor" generally refers to a receptor that uses a purine (such as ATP) as a ligand.

[0097] "P2X? receptor" generally refers to a purinergic receptor formed from three protein subunits or monomers, with at least one of the monomers having an amino acid sequence substantially as shown in SEQ ID NO: 141 below:

[0098] SEQ ID NO: 141MPACCSCSDVFQYETNKVTRIQSMNYGTIKWFFHVIIFSYVCFALVSDKLYQRKEPVIS SVHTKVKGIAEVKEEIVENGVKKLVHSVFDTADYTFPLQGNSFFVMTNFLKTEGQEQRL CPEYPTRRTLCSSDRGCKKGWMDPQSKGIQTGRCVVYEGNQKTCEVSAWCPIEAVE EAPRPALLNSAENFTVLIKNNIDFPGHNYTTRNILPGLNITCTFHKTQNPQCPIFRLGDIF RETGDNFSDVAIQGGIMGIEIYWDCNLDRWFHHCRPKYSFRRLDDKTTNVSLYPGYNF RYAKYYKENNVEKRTLIKVFGIRFDILVFGTGGKFDIIQLVVYIGSTLSYFGLAAVFIDFLID TYSSNCCRSHIYPWCKCCQPCVVNEYYYRKKCESIVEPKPTLKYVSFVDESHIRMVNQ QLLGRSLQDVKGQEVPRPAMDFTDLSRLPLALHDTPPIPGQPEEIQLLRKEATPRSRD SPVWCQCGSCLPSQLPESHRCLEELCCRKKPGACITTSELFRKLVLSRHVLQFLLLYQEPLLALDVDSTNSRLRHCAYRCYATWRFGSQDMADFAILPSCCRWRIRKEFPKSEGQ YSGFKSPY

[0099] To the extent that P2X? receptor is formed from three monomers, it is a "trimer" or "trimeric". "P2X? receptor" encompasses naturally occurring variants of P2X? receptor, e.g., wherein the P2X? monomers are splice variants, allelic variants, SNPs and isoforms including naturally-occurring truncated or secreted forms of the monomers forming the P2X? receptor (e.g., a form consisting of the extracellular domain sequence or truncated form of it), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants. In certain embodiments of the invention, the native sequence P2X7 monomeric polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acids sequence shown in SEQID NO: 141 . In certain embodiments the P2X? receptor may have an amino acid sequence that is modified, for example various of the amino acids in the sequence shown in SEQ ID NO: 141 may be substituted, deleted, or a residue may be inserted.

[0100] "Functional P2X? receptor" generally refers to a form of the P2X? receptor having three intact binding sites or clefts for binding to ATP. When bound to ATP, the functional receptor forms a non-selective sodium / calcium channel that converts to a porelike structure that enables the ingress of calcium ions and molecules of up to 900 Da into the cytosol, one consequence of which may be induction of programmed cell death. In normal homeostasis, expression of functional P2X? receptors is generally limited to cells that undergo programmed cell death such as thymocytes, dendritic cells, lymphocytes, macrophages and monocytes. There may also be some expression of functional P2X? receptors on erythrocytes and other cell types.

[0101] "Dysfunctional P2X? receptor" (also called “non-functional” or (nf) P2X?) is a P2X? receptor that has an impaired response to ATP such that it is unable to form an apoptotic pore under normal physiological conditions. A dysfunctional P2X? receptor (or nfP2X? receptor) generally refers to a form of a P2X7 receptor having a conformation, distinct from functional P2X7, whereby the receptor is unable to form an apoptotic pore, but which is still able to operate as a non-selective channel through the maintenance of a single functional ATP binding site located between adjacent monomers. One example arises where one or more of the monomers has a cis isomerisation at Pro210 (according to the sequence set forth in SEQ ID NO: 141 ). The isomerisation may arise from any molecular event that leads to misfolding of the monomer, including for example, mutation of monomer primary sequence or abnormal post translational processing. One consequence of the isomerisation is that the receptor is unable to bind to ATP at one, or more particularly two, ATP binding sites on the trimer and as a consequence not be able to extend the opening of the channel. In the circumstances, the receptor cannot form a pore and this limits the extent to which calcium ions may enter the cytosol. Dysfunctional P2X7 receptors are expressed on a wide range of epithelial and haematopoietic cancers. As used herein, the term “dysfunctional P2X7 receptors” may be used interchangeably with the term “non-functional P2X7 receptors” or “nfP2X7 receptors”.

[0102] "Cancer associated-P2X7 receptors" are generally P2X7 receptors that are found on cancer cells (including, pre-neoplastic, neoplastic, malignant, benign or metastatic cells), but not on non-cancer or normal cells.

[0103] "E200 epitope" generally refers to an epitope having the sequence GHNYTTNILPGLNITC (SEQ ID NO: 95) and variants thereof (e.g. SEQ ID NOs: 96 to 1 19). The antigen recognition domains of the CARs of the invention are ones that preferably are capable of binding to E200 or a variant thereof.

[0104] "E300 epitope" generally refers to an epitope having the sequence KYYKENNVEKRTLIK and variants thereof (eg SEQ ID NOs: 120 and 121 ).

[0105] A "composite epitope" generally refers to an epitope that is formed from the juxtaposition of the E200 and E300 epitopes or parts of these epitopes. An example of a composite epitope comprising E200 and E300 epitopes is GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 122).

[0106] As used herein, the term “chimeric antigen receptor” or CAR, refers to an artificially constructed protein for expression on the surface of an immune cell, the protein comprising an extracellular domain (extracellular part) comprising an antigen binding domain (also called an antigen recognition domain), a transmembrane domain and an intracellular signaling domain. The extracellular domain may be linked to the transmembrane domain by a linker. The extracellular domain may also comprise a signal peptide.

[0107] The terms "binds to", “specifically binds to” or "specific for" with respect to the antigen-binding domain of a CAR, refers to a domain that recognises and binds to a specific antigen and does not substantially recognise or bind to other molecules in a sample. An antigen-binding domain that binds specifically to an antigen from one species also may bind to that antigen from another species. This cross-species reactivity is typical of many antibodies and therefore not contrary to the definition that the antigen-binding domain is specific. An antigen-binding domain that specifically binds to an antigen may bind also to different allelic forms of the antigen (allelic variants, splice variants, isoforms etc.) or homologous variants of this antigen from the same gene family. This cross reactivity is typical of many antibodies and therefore not contrary to the definition that the antigen-binding domain is specific.

[0108] The terms "engineered cell" and "genetically modified cell" as used herein can be used interchangeably. The terms mean containing and / or expressing a foreign gene or nucleic acid sequence that in turn modifies the genotype or phenotype of the cell or itsprogeny. Especially, the terms refer to the fact that cells, preferentially immune cells, can be manipulated by recombinant methods well known in the art to express stably or transiently peptides or proteins that are not expressed in these cells in the natural state. For example, immune cells are engineered to express an artificial construct such as a chimeric antigen receptor on their cell surface. For example, nucleic acid sequences encoding a CAR may be delivered into cells using an adenoviral, adeno-associated viral (AAV)-based, retroviral or lentiviral vector or any other pseudotyped variations thereof or any other gene delivery mechanism such as electroporation or lipofection with CRISPR / Cas9, transposons (e.g. sleeping-beauty) or variations thereof. The gene delivery may be in the form of mRNA (transient) or DNA (transient or permanent).

[0109] The terms "immune cell" or "immune effector cell" refer to a cell that may be part of the immune system, either the adaptive (i.e. cellular or humoral) or innate immune system, and executes a particular effector function such as alpha-beta T cells, NK cells, NKT cells, B cells, Breg cells, Treg cells, innate lymphoid cells (ILC), cytokine induced killer (CIK) cells, lymphokine activated killer (LAK) cells, gamma-delta T cells, mesenchymal stem cells or mesenchymal stromal cells (MSC), monocytes or macrophages or any hematopoietic progenitor cells such as pluripotent stem cells and early progenitor subsets that may mature or differentiate into somatic cells. The cells may be naturally occurring or generated by cytokine exposure, artif icial / genetically modified cells (such as iPSCs and other artificial cell types). The immune cell may be an artificial cell subset including induced pluripotent stem cells and cells maturated therefrom. Preferred immune cells are cells with cytotoxic effector function such as alpha-beta T cells, NK cells, NKT cells, ILC, CIK cells, LAK cells or gamma-delta T cells. "Effector function" means a specialised function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper cell activity including the secretion of cytokines.

[0110] The term "treat" (treatment of) a disorder as used herein means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0111] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter in a cell.

[0112] Antibodies" or "immunoglobulins" or "Igs" are gamma globulin proteins that are found in blood, or other bodily fluids of vertebrates that function in the immune system to bind antigen, hence identifying and / or neutralising foreign objects.

[0113] Antibodies are generally a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. Each L chain is linked to a H chain by one covalent disulfide bond. The two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges.

[0114] H and L chains define specific Ig domains. More particularly, each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and y chains and four CH domains for p and £ isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1 ).

[0115] Antibodies can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, Ig D, Ig E, IgG, and IgM, having heavy chains designated a, 5, £, y, and p, respectively. The y and a classes are further divided into subclasses on the basis of relatively minor differences in % sequence and function, e.g., humans express the following subclasses: lgG1 , lgG2, lgG3, lgG4, IgAI, and lgA2. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0116] The constant domain includes the Fc portion that comprises the carboxyterminal portions of both H chains held together by disulfides. The effector functions of antibodies such as ADCC are determined by sequences in the Fc region, which region is also the part recognised by Fc receptors (FcR) found on certain types of cells.

[0117] The pairing of a VH and VL together forms a "variable region" or "variable domain" including the amino -terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH." The variable domain of the light chain may be referred to as "VL." The V domain contains an "antigen binding site" that affects antigen binding and defines specificity of a particular antibody for its particular antigen. V regions span about 1 10 amino acid residues and consist of relativelyinvariant stretches called framework regions (FRs) (generally about 4) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" (generally about 3) that are each generally 9-12 amino acids long. The FRs largely adopt a [3-sheet configuration and the hypervariable regions form loops connecting, and in some cases forming part of, the [3-sheet structure.

[0118] "Hypervariable region" refers to the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six hypervariable regions (also called complementarity determining regions, or CDRs); three in the VH (H1 , H2, H3), and three in the VL (L1 , L2, L3).

[0119] The complementarity determining region sequences (CDRs) of an antigen binding protein may be defined according to any one of a number of different numbering systems, including the IMGT numbering system, Kabat or Chothia systems. It is well within the purview of the skilled person to be able to identify and determine the CDRs once provided with the full variable heavy and light sequences.

[0120] The description and definitions of variable regions and parts thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by the discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md„ 1987 and 1991 , Bork et al., J Mol. Biol. 242, 309-320, 1994, Chothia and Lesk J. Mol Biol. 196:901 -917, 1987, Chothia et al. Nature 342, 877-883, 1989, Martin (“enhanced Chothia”; Mol Immunol. (2008) 45:3832-9; and / or or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

[0121] As used herein, the term “complementarity determining regions” (syn. CDRs; i.e., CDRi, CDR2, and CDR3) refers to the amino acid residues of an antibody variable region the presence of which are major contributors to specific antigen binding. Each variable region domain (VH or VL) typically has three CDRs identified as CDR1, CDR2 and CDR3. The CDRs of VH are also referred to herein as CDR Hi, CDR H2 and CDR H3, respectively, wherein CDR Hi corresponds to CDR 1 of VH, CDR H2 corresponds to CDR 2 of VH and CDR H3 corresponds to CDR 3 of VH. Likewise, the CDRs of VL are referred to herein as CDR Li , CDR L2 and CDR L3, respectively, wherein CDR Li corresponds to CDR 1 of VL, CDR L2 corresponds to CDR 2 of VL and CDR L3 corresponds to CDR 3 of VL. In one example, the amino acid positions assigned to CDRs and FRs are defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutesof Health, Bethesda, Md., 1987 and 1991 (also referred to herein as “the Kabat numbering system”). In another example, the amino acid positions assigned to CDRs and FRs are defined according to the Enhanced Chothia Numbering Scheme (http: / / www.bioinfo.org.uk / mdex.html). The present invention is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including the canonical numbering system or of Chothia and Lesk J. Mol. Biol. 196: 901 - 917, 1987; Chothia et al., Nature 342: 877-883, 1989; and / or Al-Lazikani et al., J. Mol. Biol. 273: 927-948, 1997; the numbering system of Honnegher and Plukthun J. Mol. Biol. 309: 657-670, 2001 ; or the IMGT system discussed in Giudicelli et al., Nucleic Acids Res. 25: 206-21 1 1997.

[0122] "Framework regions" (FRs) are those variable region residues other than the CDR residues. The FRs of VH are also referred to herein as FR Hi, FR H2, FR H3 and FR H4, respectively, wherein FR Hi corresponds to FR 1 of VH, FR H2 corresponds to FR 2 of VH, FR H3 corresponds to FR 3 of VH and FR H4 corresponds to FR 4 of VH. Likewise, the FRs of VL are referred to herein as FR Li, FR L2, FR L3 and FR L4, respectively, wherein FR Li corresponds to FR 1 of VL, FR L2 corresponds to FR 2 of VL, FR L3 corresponds to FR 3 of Vr and FR L4 corresponds to FR 4 of VL.

[0123] An "antigen binding site" generally refers to a molecule that includes at least the hypervariable and framework regions that are required for imparting antigen binding function to a V domain. An antigen binding site may be in the form of an antibody or an antibody fragment, (such as a mAb, single domain (SD)-mAb, dAb, Fab, SD-Fab, Fd, SD- Fv, Fv, F(ab')2 or scFv) in a method described herein.

[0124] An "intact" or "whole" antibody is one that comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CH1 , CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof.

[0125] "Whole antibody fragments including a variable domain" include SD-mAb, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies, single-chain antibody molecules; and multi-specific antibodies formed from antibody fragments.

[0126] The "Fab fragment" consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CH1 ). EachFab fragment is monovalent with respect to antigen binding, i.e., it has a single antigenbinding site.

[0127] A "Fab' fragment" differs from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab'- SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.

[0128] A "F(ab')2 fragment" roughly corresponds to two disulphide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen.

[0129] An "Fv" is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This fragment consists of a dimer of one heavy and one light chain variable region domain in tight, non-covalent association.

[0130] In a single-chain Fv (scFv) species, one heavy and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody.

[0131] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the VH and VL antibody domains connected to form a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0132] A "single variable domain" is half of an Fv (comprising only three CDRs specific for an antigen) that has the ability to recognise and bind antigen, although generally at a lower affinity than the entire binding site.

[0133] "Diabodies" refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). The small antibody fragments are prepared by constructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the VH and VL domains such that interchain but not intrachain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites.

[0134] Diabodies may be bivalent or bispecific. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Triabodies and tetrabodies are also generally known in the art.

[0135] An "isolated antibody" is one that has been identified and separated and / or recovered from a component of its pre-existing environment. Contaminant components are materials that would interfere with therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0136] A "human antibody" refers to an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a human. Human antibodies can be produced using various techniques known in the art, including phage - display libraries. Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled.

[0137] "Humanised' forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanised antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanised antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanised antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. Thehumanised antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0138] "Monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site or determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesised uncontaminated by other antibodies. Monoclonal antibodies may be prepared by the hybridoma methodology. The "monoclonal antibodies" may also be isolated from phage antibody libraries using molecular engineering techniques.

[0139] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high- affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention.

[0140] As used herein, the term "antigen" is intended to include substances that bind to or evoke the production of one or more antibodies and may comprise, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates, and combinations thereof, for example a glycosylated protein or a glycolipid. The term "antigen" as used herein refers to a molecular entity that may be expressed on a target cell and that can be recognised by means of the adaptive immune system including but not restricted to antibodies or TCRs, or engineered molecules including but not restricted to transgenic TCRs, CARs, scFvs or multimers thereof, Fab-fragments or multimers thereof, antibodies or multimers thereof, single chain antibodies or multimers thereof, or any other molecule that can execute binding to a structure with high affinity.

[0141] "Epitope" generally refers to that part of an antigen that is bound by the antigen binding site of an antibody. An epitope may be "linear" in the sense that the hypervariable loops of the antibody CDRs that form the antigen binding site bind to a sequence of amino acids as in a primary protein structure. In certain embodiments, the epitope is a "conformational epitope" i.e. one in which the hypervariable loops of the CDRs bind to residues as they are presented in the tertiary or quaternary protein structure.

[0142] The term "target cell" as used herein refers to a cell that expresses nfP2X? receptor. The target cell may be a cancer cell or any other diseased cell.

[0143] The term "disorder" or “condition” means a functional abnormality or disturbance in a subject such as a cancer, an autoimmune disorder, or an infection by virus, bacteria, parasite, or others.

[0144] For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated". An isolated nucleic acid or protein can also exist in a non-native environment such as, for example, in a host cell.

[0145] The term "autologous" as used herein refers to any material derived from the same subject to whom it is later re-introduced.

[0146] The term "allogeneic" as used herein refers to any material derived from a different subject of the same species as the subject to whom the material is re-introduced.

[0147] The terms "therapeutically effective amount" or "therapeutically effective population" mean an amount of, for example, a cell population that provides a therapeutic benefit in a subject.Chimeric antigen receptors

[0148] Generally, an "antigen binding domain" or “antigen recognition domain” refers to the region of the CAR that specifically binds to an antigen (and thereby is able to target a cell containing the antigen). The CARs of the invention may comprise one or more antigen binding domains, wherein at least one of the antigen binding domains is for binding to nfP2X? receptor. The antigen binding domain may comprise an antibody or an antibody binding fragment thereof. The antigen binding domain may comprise, for example, full length heavy chain, Fab fragments, single chain Fv (scFv) fragments,divalent single chain antibodies or diabodies. Any molecule that binds specifically to a given antigen such as affibodies or ligand binding domains from naturally occurring receptors may be used as an antigen binding domain. Often the antigen binding domain is a scFv. Normally, in a scFv the variable regions of an immunoglobulin heavy chain and light chain are fused by a flexible linker to form a scFv. Such a linker may be for example the "(G4 / Si)3-linker" and variations thereof but the skilled person will appreciate that various linker sequences and formats may be used.

[0149] In some instances, it is beneficial for the antigen binding domain to be derived from the same species in which the CAR will be used in. For example, when it is planned to use it therapeutically in humans, it may be beneficial for the antigen binding domain of the CAR to comprise a human or humanised antibody or antigen binding fragment thereof. Human or humanised antibodies or antigen binding fragments thereof can be made by a variety of methods well known in the art.

[0150] A "signal peptide" refers to a peptide sequence that directs the transport and localisation of the protein within a cell, e.g. to a certain cell organelle (such as the endoplasmic reticulum) and / or the cell surface.

[0151] "Spacer" or "hinge" as used herein refers to the hydrophilic region that is between the antigen binding domain and the transmembrane domain. The CARs of the invention may comprise an extracellular spacer domain but it is also possible to leave out such a spacer. The spacer may include e.g. Fc fragments of antibodies or fragments thereof, hinge regions of antibodies or fragments thereof, CH2 or CH3 regions of antibodies, accessory proteins, artificial spacer sequences or combinations thereof. A prominent example of a spacer is the CD8alpha hinge.

[0152] The transmembrane domain of the CAR may be derived from any desired natural or synthetic source for such a domain. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane domain may be derived for example from CD8alpha or CD28. When the key signalling and antigen recognition modules (domains) are on two (or even more) polypeptides, then the CAR may have two (or more) transmembrane domains. The splitting of key signalling and antigen recognition modules enables small moleculedependent, titratable and reversible control over CAR cell expression (Wu et al, 2015,Science 350: 293-303) due to small molecule-dependent heterodimerising domains in each polypeptide of the CAR.

[0153] The cytoplasmic domain (or the intracellular signaling domain) of the CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed. "Effector function" means a specialised function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper cell activity including the secretion of cytokines. The intracellular signalling domain refers to the part of a protein that transduces the effector function signal and directs the cell expressing the CAR to perform a specialised function. The intracellular signalling domain may include any complete, mutated or truncated part of the intracellular signalling domain of a given protein sufficient to transduce a signal that initiates or blocks immune cell effector functions.

[0154] The signalling domain of the CAR can be any suitable domain that is capable of inducing, or participating in the induction of, an intracellular signalling cascade upon activation of the CAR as a result of recognition of an antigen by the antigen-recognition domain of the CAR. The signalling domain of a CAR will be specifically chosen depending on the cellular outcome desired following activation of the CAR. Whilst there are many possible signalling domains, when used in immunotherapy and cancer therapy the signalling domains can be grouped into two general categories based on the receptor from which they are derived, namely activation receptors and co-stimulatory receptors. Therefore, in some embodiments, the signalling domain of the CAR includes a portion derived from an activation receptor. In some embodiments, the signalling domain includes a portion derived from a co-stimulatory receptor.

[0155] As used throughout the specification the term “activation receptor” relates to receptors, or co-receptors that form a component of, or are involved in the formation of, the T cell receptor (TCR) complex, or receptors involved in the specific activation of immune cells as a result of recognition of an antigenic or other immunogenic stimuli.

[0156] The function of the intracellular domains may be pro- or anti-inflammatory and / or immunomodulatory, or a combination thereof.

[0157] Some examples of intracellular signalling domains for use in the CARs include the cytoplasmic signaling sequences of the T cell receptor (TCR) and co-receptors that initiate signal transduction following antigen receptor engagement.

[0158] Generally, T cell activation can be mediated by two distinct classes of cytoplasmic signalling sequences, firstly those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signalling sequences) and secondly those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signalling sequences, co-stimulatory signalling domain). Therefore, an intracellular signalling domain of a CAR may comprise one or more primary cytoplasmic signalling domains and / or one or more secondary cytoplasmic signalling domains.

[0159] Primary cytoplasmic signalling sequences that act in a stimulatory manner may contain ITAMs (immunoreceptor tyrosine-based activation motifs) signalling motifs.

[0160] Examples of ITAM containing primary cytoplasmic signalling sequences often used in CARs are those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b and CD66d. Most prominent is the sequence derived from CD3 zeta.

[0161] The cytoplasmic domain of the CAR may be designed to comprise the CD3-zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s). The cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion and a co- stimulatory signalling region. The co-stimulatory signalling region refers to a part of the CAR comprising the intracellular domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples for a co- stimulatory molecule are CD27, CD28, 4-1 BB (CD137), 0X40, CD30, CD40, PD-1 , ICOS, lymphocyte function-associated antigen- 1 (LFA-1 ), CD2, CD7, LIGHT, NKG2C and B7- H3.

[0162] The cytoplasmic signalling sequences within the cytoplasmic signalling part of the CAR may be linked to each other with or without a linker in a random or specified order. A short oligo-or polypeptide linker, which is preferably between 2 and 10 amino acids in length, may form the linkage. A prominent linker is the glycine-serine doublet.

[0163] As an example, the cytoplasmic domain may comprise the signalling domain of CD3-zeta and the signalling domain of CD28. In another example the cytoplasmic domain may comprise the signalling domain of CD3-zeta and the signalling domain of CD27. In a further example, the cytoplasmic domain may comprise the signalling domain of CD3- zeta, the signalling domain of CD28, and the signalling domain of CD27.

[0164] Either the extracellular part or the transmembrane domain or the cytoplasmic domain of a CAR may also comprise a heterodimerising domain for the aim of splitting key signalling and antigen recognition modules of the CAR.

[0165] A CAR for use in accordance with the present invention, i.e. a CAR comprising an nfP2X? E200 binding domain, may be designed to comprise any portion or part of the above-mentioned domains as described herein in any order and / or combination resulting in a functional CAR.

[0166] The CARs as disclosed herein, or polypeptide(s) derived therefrom, nucleic acid molecule(s) or recombinant expression vectors cells encoding said CARs, or populations of cells expressing said CARs, may be isolated and / or purified. The term "isolated" means altered or removed from the natural state. For example, an isolated population of cells means an enrichment of such cells and separation from other cells that are normally associated in their naturally occurring state with said isolated cells. An isolated population of cells means a population of substantially purified cells that are a more homogenous population of cells than found in nature. Preferably, the enriched cell population comprises at least about 90% of the selected cell type. In particular aspects, the cell population comprises at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% of the selected cell type.

[0167] The affinity at which the antigen recognition domain of the CAR binds to the nfP2X? recognition site E200 on a cell (or the E200 sequence comprised in a “bridging molecule as described elsewhere herein) can vary, but generally the binding affinity may be in the range of 100 pM, 1 nM, 10 nM, or 100 nM, preferably at least about 1 pM or 10 pM, even more preferably at least about 100 pM.Linkers

[0168] A linker may be a peptide having a length of up to 20 amino acids. The term “linked to” or “fused to” refers to a covalent bond, e.g., a peptide bond, formed betweentwo moieties. Accordingly, in the context of the present invention the linker may have a length of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acids. For example, the herein provided CAR may comprise a linker between the VH and VL of the antigen binding domain and / or the antigen binding domain and the hinge. Such linkers have the advantage that they can make it more likely that the different polypeptides of the fusion protein fold independently and behave as expected.

[0169] The skilled person will be familiar with the design and use of various peptide linkers comprised of various amino acids, and of various lengths, which would be suitable for use as linkers in accordance with the present invention. The linker may comprise various combinations of repeated amino acid sequences. The linker may be a flexible linker (such as those comprising repeats of glycine and serine residues), a rigid linker (such as those comprising glutamic acid and lysine residues, flanking alanine repeats) and / or a cleavable linker (such as sequences that are susceptible by protease cleavage).

[0170] The peptide linker may be any one or more repeats of Gly-Ser (GS), Gly-Gly- Ser (GGS), Gly-Gly-Gly-Ser (GGGS) (SEQ ID NO: 17) or Gly-Gly-Gly-Gly-Ser (GGGGS) (SEQ ID NO: 18) or variations thereof. In any embodiment, the linker may comprise or consist of the sequence GGGGSGGGGSGGGGS, i.e. (G4S)3 (SEQ ID NO: 93).

[0171] In any embodiment, the peptide linker can include the amino acid sequence GGGGGS (a linker of 6 amino acids in length, SEQ ID NO: 19) or even longer. The linker may be a series of repeating glycine and serine residues (GS) of different lengths, i.e., (GS)n where n is any number from 1 to 15 or more. For example, the linker may be (GS)3 (i.e., GSGSGS) (SEQ ID NO: 20) or longer (GS)n (SEQ ID NO: 142) or longer. It will be appreciated that n can be any number including 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or more.Nucleic acids

[0172] In another aspect, the present invention provides a nucleic acid molecule, such as a recombinant nucleic acid molecule, including a nucleotide sequence encoding a chimeric antigen receptor according to the invention.

[0173] In some embodiments of the invention, the nucleic acid molecule includes a nucleotide sequence which encodes the amino acid sequence set forth in SEQ ID NO: 4 and / or 12, such as SEQ ID NO: 94, or encodes a functional variant thereof, wherein the functional variant retains the ability to bind to nfP2X? receptor. In some embodiments, thenucleic acid molecule encodes a functional variant that includes an amino acid sequence which is at least 80% identical to SEQ ID NO: 94 and retains the ability to bind to nfP2X? receptor. In some embodiments, the nucleic acid molecule encodes a functional variant that includes an amino acid sequence which is at least 80% identical to SEQ ID NO: 94, retains the ability to bind to nfP2X? and comprises an antigen recognition domain comprising the amino acid sequences of SEQ ID NOs 4 and 12.

[0174] The nucleic acid molecule may comprise any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified, or modified, RNA or DNA. For example, the nucleic acid molecule may include single- and / or double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, doublestranded or a mixture of single- and double-stranded regions. In addition, the nucleic acid molecule may comprise triple-stranded regions comprising RNA or DNA or both RNA and DNA. The nucleic acid molecule may also comprise one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. A variety of modifications can be made to DNA and RNA; thus the term "nucleic acid molecule" embraces chemically, enzymatically, or metabolically modified forms.

[0175] In some embodiments of the invention, the nucleic acid molecule includes the nucleotide sequence set forth in SEQ ID NO: 74 and / or SEQ ID NO: 82.

[0176] It would be understood by a person skilled in the art that any nucleotide sequence which encodes a chimeric antigen receptor comprising the amino acid sequence set forth in in SEQ ID NOs: 4 and / or 12, for example SEQ ID NO: 94, or a functional variant thereof, is contemplated by the present invention. For example, variants of SEQ ID NO: 94, are contemplated which comprise one or more different nucleic acids to SEQ ID NOs: 74 and / or 82, but which still encode identical amino acid sequences. Because of the degeneracy of the genetic code, a large number of nucleic acids can encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Accordingly, every nucleotide sequence herein which encodes a chimeric antigen receptor having the amino acid sequence set forth in SEQ ID NO: 94, or a functional variant thereof also describes every possible silent variation of the nucleotide sequence. One of skill will recognise that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily theonly codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleotide sequence that encodes a polypeptide is implicit in each described sequence.

[0177] In another aspect, the present invention provides a nucleic acid construct including a nucleic acid molecule according to the invention. The nucleic acid construct may further comprise one or more of: an origin of replication for one or more hosts; a selectable marker gene which is active in one or more hosts; and / or one or more transcriptional control sequences.

[0178] As used herein, the term “selectable marker gene” includes any gene that confers a phenotype on a cell in which it is expressed, to facilitate the identification and / or selection of cells which are transfected or transformed with the construct.

[0179] “Selectable marker genes” include any nucleotide sequences which, when expressed by a cell transformed with the construct, confer a phenotype on the cell that facilitates the identification and / or selection of these transformed cells. A range of nucleotide sequences encoding suitable selectable markers are known in the art (for example Mortesen, RM. and Kingston RE. Curr Protoc Mol Biol, 2009; Unit 9.5). Exemplary nucleotide sequences that encode selectable markers include: Adenosine deaminase (ADA) gene; Cytosine deaminase (CDA) gene; Dihydrofolate reductase (DHFR) gene; Histidinol dehydrogenase (hisD) gene; Puromycin-N-acetyl transferase (PAC) gene; Thymidine kinase (TK) gene; Xanthine-guanine phosphoribosyltransferase (XGPRT) gene or antibiotic resistance genes such as ampicillin-resistance genes, puromycin-resistance genes, Bleomycin-resistance genes, hygromycin-resistance genes, kanamycin-resistance genes and ampicillin-resistance gene; fluorescent reporter genes such as the green, red, yellow or blue fluorescent protein-encoding genes; and luminescence-based reporter genes such as the luciferase gene, amongst others which permit optical selection of cells using techniques such as Fluorescence-Activated Cell Sorting (FACS).

[0180] Furthermore, it should be noted that the selectable marker gene may be a distinct open reading frame in the construct or may be expressed as a fusion protein with another polypeptide (e.g. the CAR).

[0181] As set out above, the nucleic acid construct may also comprise one or more transcriptional control sequences. The term “transcriptional control sequence” should be understood to include any nucleic acid sequence which effects the transcription of an operably connected nucleic acid. A transcriptional control sequence may include, for example, a leader, polyadenylation sequence, promoter, enhancer or upstream activating sequence, and transcription terminator. Typically, a transcriptional control sequence at least includes a promoter. The term “promoter” as used herein, describes any nucleic acid which confers, activates or enhances expression of a nucleic acid in a cell.

[0182] In some embodiments, at least one transcriptional control sequence is operably connected to the nucleic acid molecule of the invention. For the purposes of the present specification, a transcriptional control sequence is regarded as “operably connected” to a given nucleic acid molecule when the transcriptional control sequence is able to promote, inhibit or otherwise modulate the transcription of the nucleic acid molecule. Therefore, in some embodiments, the nucleic acid molecule is under the control of a transcription control sequence, such as a constitutive promoter or an inducible promoter.

[0183] The "nucleic acid construct" may be in any suitable form, such as in the form of a plasmid, phage, transposon, cosmid, chromosome, vector, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences, contained within the construct, between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. In some embodiments, the nucleic acid construct is a vector. In some embodiments the vector is a viral vector.

[0184] A promoter may regulate the expression of an operably connected nucleic acid molecule constitutively, or differentially, with respect to the cell, tissue, or organ at which expression occurs. As such, the promoter may include, for example, a constitutive promoter, or an inducible promoter. A “constitutive promoter” is a promoter that is active under most environmental and physiological conditions. An “inducible promoter” is a promoter that is active under specific environmental or physiological conditions. The present invention contemplates the use of any promoter which is active in a cell of interest. As such, a wide array of promoters would be readily ascertained by one of ordinary skill in the art.

[0185] Mammalian constitutive promoters may include, but are not limited to, Simian virus 40 (SV40), cytomegalovirus CMV), P-actin, Ubiquitin C (UBC), elongation factor-1alpha (EF1 A), phosphoglycerate kinase (PGK) and CMV early enhancer / chicken [3 actin (CAGG).

[0186] Inducible promoters may include, but are not limited to, chemically inducible promoters and physically inducible promoters. Chemically inducible promoters include promoters which have activity that is regulated by chemical compounds such as alcohols, antibiotics, steroids, metal ions or other compounds. Examples of chemically inducible promoters include: tetracycline regulated promoters (e.g. see US Patent 5,851 ,796 and US Patent 5,464,758); steroid responsive promoters such as glucocorticoid receptor promoters (e.g. see US Patent 5,512,483), ecdysone receptor promoters (e.g. see US Patent 6,379,945) and the like; and metal-responsive promoters such as metallothionein promoters (e.g. see US Patent 4,940,661 , US Patent 4,579,821 and US 4,601 ,978) amongst others.

[0187] As mentioned above, the control sequences may also include a terminator. The term “terminator” refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3'-non-translated DNA sequences generally containing a polyadenylation signal, which facilitate the addition of polyadenylate sequences to the 3'-end of a primary transcript. As with promoter sequences, the terminator may be any terminator sequence which is operable in the cells, tissues or organs in which it is intended to be used. Suitable terminators would be known to a person skilled in the art.

[0188] As will be understood, the nucleic acid construct of the invention can further include additional sequences, for example sequences that permit enhanced expression, cytoplasmic or membrane transportation, and location signals. Specific non-limiting examples include an Internal Ribosome Entry Site (IRES).

[0189] The present invention extends to all genetic constructs essentially as described herein. These constructs may further include nucleotide sequences intended for the maintenance and / or replication of the genetic construct in eukaryotes and / or the integration of the genetic construct or a part thereof into the genome of a eukaryotic cell.

[0190] Methods are known in the art for the deliberate introduction (transfection / transduction) of exogenous genetic material, such as the nucleic acid construct of the present invention, into eukaryotic cells. As will be understood the methodbest suited for introducing the nucleic acid construct into the desired host cell is dependent on many factors, such as the size of the nucleic acid construct, the type of host cell the desired rate of efficiency of the transfection / transduction and the final desired, or required, viability of the transfected / transduced cells. Non-limiting examples of such methods include; chemical transfection with chemicals such as cationic polymers, calcium phosphate, or structures such as liposomes and dendrimers; non-chemical methods such as electroporation, sonoporations, heat-shock or optical transfection; particle-based methods such as ‘gene gun’ delivery, magnetofection, or impalefection or viral transduction.

[0191] The nucleic acid construct will be selected depending on the desired method of transfection / transduction. In some embodiments of the invention, the nucleic acid construct is a viral vector, and the method for introducing the nucleic acid construct into a host cell is viral transduction. Methods are known in the art for utilising viral transduction to elicit expression of a CAR in a PBMC (Parker, LL. et al. Hum Gene Ther. 2000;1 1 : 2377-87) and more generally utilising retroviral or lentiviral systems for transduction of mammalian cells (Barde et al, “Production and Titration of Lentiviral Vectors,” Current Protocols in Neuroscience, Volume 53, Issue 1 (2010) (also cited as Current Protocols in Neuroscience 4.21.1 -4.21.23 (October 2010) and Cepko, C. and Pear, W. Curr Protoc Mol Biol. 2001 , unit 9.9). In other embodiments, the nucleic acid construct is a plasmid, a cosmid, an artificial chromosome or the like, and can be transfected into the cell by any suitable method known in the art.Genetically modified cells

[0192] As described herein, in certain embodiments the invention includes methods of treatment involving the use of a cell expressing a chimeric antigen receptor (CAR) of the invention.

[0193] The cell may be an "engineered cell", "genetically modified cell", “immune cell” or “immune effector cell” as described herein. Further, the cell may be capable of differentiating into an immune cell. A cell that is capable of differentiating into an immune cell (e.g. T cell that will express the dysfunctional P2X? CAR) may be a stem cell, multilineage progenitor cell or induced pluripotent stem.

[0194] In any embodiment, the cell may be a T cell, wherein optionally said T cell does not express TcRap, PD1 , CD3 or CD96 (e.g. by way of knocking down or knocking out one of these genes on a genetic level or functional level).

[0195] In any embodiment, the cell may be an immune cell, wherein optionally said cell does not express accessory molecules that can be checkpoint, exhaustion or apoptosis- associated signalling receptors as well as ligands such as PD-1 , LAG-3, TIGIT, CTLA-4, FAS-L and FAS-R, (e.g. by way of knocking out one of these genes on a genetic level or functional level).

[0196] In some embodiments, the genetically modified cell includes two or more different CARs. For example, the genetically modified cell may include CARs of differing architectures (eg with differing signalling domains) but which comprise the same antigen recognition sequence. Alternatively, the genetically modified cell may include CARs which have differing antigen recognition domains, but which bind to nfP2X? receptor. The CARs may bind to the same or differing epitopes of the nfP2X? receptor. In alternative embodiments, the genetically modified cell may comprise CARs for binding to different antigens (eg nfP2X? receptor and to a different antigen on a cancer cell).

[0197] In some embodiments of the invention, the genetically modified cell includes a nucleic acid molecule, or a nucleic acid construct, that encodes for two or more different CARs. In some embodiments of the invention, the genetically modified cell includes two or more nucleic acid molecules, or two or more nucleic acid constructs, each of which encodes for a different CAR.

[0198] As referred to herein, a "genetically modified cell" includes any cell comprising a non-naturally occurring and / or introduced nucleic acid molecule or nucleic acid construct encompassed by the present invention. The introduced nucleic acid molecule or nucleic acid construct may be maintained in the cell as a discreet DNA molecule, or it may be integrated into the genomic DNA of the cell.

[0199] Genomic DNA of a cell should be understood in its broadest context to include any and all endogenous DNA that makes up the genetic complement of a cell. As such, the genomic DNA of a cell should be understood to include chromosomes, mitochondrial DNA and the like. As such, the term "genomically integrated" contemplates chromosomal integration, mitochondrial DNA integration, and the like. The "genomically integratedform" of the construct may be all or part of the construct. However, in some embodiments the genomically integrated form of the construct at least includes the nucleic acid molecule of the second aspect of the invention.

[0200] As used herein, the term “different CARs” or “different chimeric antigen receptors” refers to any two or more CARs that have either non-identical antigenrecognition and / or non-identical signalling domains. In one example, “different CARs” includes two CARs with the same antigen-recognition domains (e.g. both CARs may recognise a dysfunctional P2X? receptor), but have different signalling domains, such as one CAR having a signalling domain with a portion of an activation receptor and the other CAR having a signalling domain with a portion of an co-stimulatory receptor. As will be understood, at least one of the two or more CARs within this embodiment will have an antigen-recognition domain that recognises the dysfunctional P2X? receptor and the other CAR(s) may take any suitable form and may be directed against any suitable antigen.

[0201] Accordingly, in some embodiments of the invention the two or more different CARs have different signalling domains, and may have identical, or different, antigenrecognition domains. Specifically, the genetically modified cell of the invention may include a first chimeric antigen receptor with a signalling domain that includes a portion derived from an activation receptor and a second chimeric antigen receptor with a signalling domain including a portion derived from a co-stimulatory receptor.

[0202] In some embodiments, the activation receptor (from which a portion of signalling domain is derived) is the CD3 co-receptor complex or is an Fc receptor.

[0203] In some embodiments, the co-stimulatory receptor (from which a portion of signalling domain is derived) is selected from the group consisting of CD27, CD28, CD- 30, CD40, DAP10, 0X40, 4-1 BB (CD137) and ICOS.

[0204] In some embodiments, the co-stimulatory receptor (from which a portion of signalling domain is derived) is selected from the group consisting of CD28, 0X40 or 4- 1 BB.

[0205] In some embodiments, the genetically modified cell is further modified to constitutively express co-stimulatory receptors.

[0206] As described above, a cellular immune response is typically only induced when an activation signal (typically in response to an antigen) and a co-stimulation signal are simultaneously experienced. Therefore, by having a genetically modified cell in accordance with some of the above embodiments, which includes two or more CARs that in combination provide both an intracellular activation signal and an intracellular costimulation signal, ensures that a sufficient immune response can be induce in response to the recognition by the CAR(s) of their cognate antigen. Alternatively, the genetically modified cell may include only one CAR, which has an antigen-recognition domain that recognises a dysfunctional P2X? receptor, and may constitutively express co-stimulatory receptors, thereby increasing the likelihood of co-stimulation being provided simultaneously when the CAR is activated. Alternatively, the genetically modified cell may be further modified to constitutively express both co-stimulatory receptor(s) and its / their ligand(s). In this way the cell is continuously experiencing co-stimulation and only needs the activation of a CAR, with a signalling domain including a portion from an activation receptor, for immune activation of the cell.

[0207] Therefore, in some embodiments, the genetically modified cell expressing the CAR is further modified so as to constitutively express co-stimulatory receptors. In further embodiments, the genetically modified cell is further modified so as to express ligands for the co-stimulatory receptors, thereby facilitating auto-stimulation of the cell. Examples of CAR-expressing T cells that also express both co-stimulatory receptors and their cognate ligands (so as to induce auto-stimulation) are known in the art and include, inter alia, those disclosed in Stephen MT. et al. Nat Med, 2007; 13: 1440-9.

[0208] The potency of a genetically modified cell including a CAR can be enhanced by further modifying the cell so as to secrete cytokines, preferably pro-inflammatory or pro- proliferative cytokines. This secretion of cytokines provides both autocrine support for the cell expressing the CAR and alters the local environment surrounding the CAR- expressing cell such that other cells of the immune system are recruited and activated. Consequently, in some embodiments of the fourth or fifth aspects of the invention the genetically modified cell is further modified to secret cytokines. This secretion may be constitutive, or may be inducible upon recognition of a CAR of its cognate antigen of ligand.

[0209] While any one or more cytokines can be selected depending on the desired immune response, preferable cytokines and / or chemokines include IL-2, IL-7, IL-12, IL- 15, IL-17, IL-18 and IL-21 , CCL19, CCL21 or a combination thereof.

[0210] The immune cell can be any suitable immune cell, or progenitor cell thereof, or can be a homogeneous or a heterogeneous cell population. In some embodiments, the cell is a leukocyte, a Peripheral Blood Mononuclear Cell (PBMC), a lymphocyte, a T cell, a CD4+ T cell, a CD8+ T cell, a natural killer cell, a natural killer T cell, or a yb T cell.

[0211] The immune cell may be a T cell, wherein optionally said T cell does not express TcRap, PD1 , CD3 or CD96 (e.g. by way of knocking down or knocking out one of these genes on a genetic level or functional level).

[0212] The immune cell may not express accessory molecules that can be checkpoint, exhaustion or apoptosis-associated signalling receptors as well as ligands such as PD-1 , LAG-3, TIGIT, CTLA-4, FAS-L and FAS-R, (e.g. by way of knocking out, or knocking down, one of these genes on a genetic level or functional level).Methods of treatment and administration

[0213] As discussed further in this document, the present invention finds application in the treatment of a variety of conditions, although preferably in the treatment of cancers.

[0214] The present invention also contemplates various scenarios for the use of the CAR of the invention, including the administration of genetically modified immune cells expressing a nucleic acid encoding a chimeric antigen receptor of the invention, such that the cells present the CAR on their cell surface. Preferably the genetically modified immune cells are for killing a target cell.

[0215] In preferred embodiments, the target cell is a cancer cell and the cell surface molecule of the cancer cell is an antigen that is associated with the cancer. The antigen may be a tumour-specific antigen or a tumour-associated antigen. The antigen may be one which is associated with a particular type of cancer. For example, overexpression of the antigen may be associated with a specific cancer or specific class of cancer. For example, where the cancer is breast cancer, the antigen may be associated with breast cancer but not with another form of cancer. Alternatively, the antigen may be associated with a class of cancers such as solid tumours, but not associated with haematological (ie“liquid”) tumours, or vice versa. The antigen may be associated with cancers of a particular lineage but not with others. For example the antigen may be associated with sarcomas but not lymphomas or carcinoma. As used herein the term “associated with” in relation to cancer, will be understood to mean that the antigen’s expression (whether increased or decreased) is considered a marker of the cancer. It will be appreciated that there may be low levels of expression of an antigen but this does not equate to the antigen being “associated” with a given cancer.

[0216] In preferred embodiments, the present invention provides a method of killing a target cell expressing nfP2X? receptor, the method including exposing the cell expressing a nfP2X? receptor to a genetically modified cell of the invention, thereby killing the target cell.

[0217] In accordance with preferred embodiments of the invention, the antigen recognition domain of the CAR of the invention is for directly recognising nfP2X? receptor. As used herein the term “directly recognises” includes direct binding of the antigenrecognition domain of the CAR to the nfP2X? receptor, or an epitope thereof. In another non-limiting example, the antigen recognition domain may directly bind to a processed form of nfP2X? receptor, which may be presented by antigen presenting molecules such as the major histocompatibility complex (MHC).

[0218] It will be appreciated that in certain circumstances, the CAR of the invention may indirectly recognise nfP2X7 receptor or another antigen on a target cell. In such embodiments, the CAR may bind to a target cell via an intermediate. As such, the present invention also provides a method of killing a target cell, the method including exposing the target cell to a genetically modified cell of the invention, thereby killing the target cell. In some embodiments of the invention, the method of killing a target cell further includes the step of exposing the target cell to an intermediate.

[0219] An intermediate may be a molecule such as a probe, polypeptide or fusion protein that binds or interacts directly with the CAR (such as via the antigen binding domain), and which also binds or interacts directly with an antigen on a target cell. Nonlimiting examples of such intermediates include peptides, antibodies, or fragments thereof, a Fab of an antibody, an scFv, a soluble engineered TCR or an aptamer. The antigen on the target cell may comprise nfP2X7 receptor, including an epitope of nfP2X7 receptor that is different to the epitope recognised by the antigen recognition domain ofthe CAR. The antigen on the target cell may comprise an antigen that is not nfP2X? receptor (such as any other tumour-associated or tumour-specific antigen present on the surface of a cancer cell, such as but not limited to CD33 (Siglec-3), CD123 (IL3RA), CD135 (FLT-3), CD44 (HCAM), CD44V6, CD47, CD184 (CXCR4), CLEC12A (CLL1 ), LeY, FRp, MICA / B, CD305 (LAIR-1 ), CD366 (TIM-3), CD96 (TACTILE), CD133, CD56, CD29 (ITGB1 ), CD44 (HCAM), CD47 (IAP), CD66 (CEA), CD1 12 (Nectin2), CD1 17 (c- Kit), CD133, CD146 (MCAM), CD155 (PVR), CD171 (LI CAM), CD200 (OX-2), CD221 (IGF1 ), CD227 (MUC1 ), CD243 (MRD1 ), CD246 (ALK), CD271 (LNGFR), CD19, CD20, GD2, and EGFRvlll).

[0220] The CAR may be able to directly recognise the intermediate or the intermediate may have a tag that is recognised by the CAR. In either regard the intermediate provides the specificity for the target cell whilst the genetically modified cell having the CAR provides the efficacy and directs an immune response against the target cell. Examples of a CAR that recognises cells by way of an intermediate are known in the art, for example European patent application EP 2651442. Such intermediates may also be referred to in the art as “bridging molecules”. Further examples of bridging molecules (also referred to as BRIDGE molecules) suitable for directing an nfP2X? receptor-binding CAR or a CAR of the present invention to a target cell are described in WO 2022 / 187906, incorporated herein by reference. Such bridging molecules typically comprise a polypeptide comprising an E200 sequence, or derivative thereof, that can be recognised by the antigen recognition domain of the CARs of the present invention.

[0221] The term “aptamer” as used throughout the specification refers to any oligonucleic acid, polynucleic acid, peptide or polypeptide which specifically binds to, or preferentially forms a complex with, a target (specifically mesothelin).

[0222] In some embodiments of the invention, the target cell is within the body of a subject. In some embodiments, the subject is a human. In some embodiments, the method further includes exposing the target cell to a genetically modified together with an exogenous cytokine.

[0223] In some embodiments of the invention, the genetically modified cell is a genetically modified cell autologous to the target cell from the subject.

[0224] In another aspect, the present invention provides a method of treating or preventing cancer in a subject, the method including providing a subject with a genetically modified cell of the invention, thereby treating or preventing cancer. Optionally, the method further comprises the step of exposing the target cell to an intermediate to enable binding of the genetically modified cell to a target cell.

[0225] The terms "treat", "treating" or "treatment," as used herein are to be understood to include within their scope one or more of the following outcomes: (i) inhibiting to some extent the growth of a primary tumour in a subject, including, slowing down and complete growth arrest, and including reducing the growth of the primary tumour after resection; (ii) inhibiting to some extent the growth and formation of one or more secondary tumours in a subject; (iii) reducing the number of tumour cells in a subject; (iv) reducing the size of a tumour in the subject; (v) inhibiting (i.e. reduction, slowing down or complete stopping) of tumour cell infiltration into peripheral organs; (vi) inhibiting (i.e. reduction, slowing down or complete stopping) of metastasis; (vii) improving the life expectancy of a subject as compared to the untreated state; (viii) improving the quality of life of a subject as compared to the untreated state; (ix) alleviating, abating or ameliorating at least one symptom of cancer in a subject; (x) causing regression or remission of cancer in a subject; (xi) relieving a condition in a subject that is caused by cancer; and (xii) stopping symptoms in a subject that are associated with cancer.

[0226] The terms "prevent" or "preventing" as used herein are to be understood to include within their scope inhibiting the formation of a primary tumour in a subject, inhibiting the formation of one or more secondary tumours in a subject, or reducing or eliminating the recurrence of cancer in a subject in remission.

[0227] The term “inhibiting” as used herein is taken to mean a decrease or reduction in the growth of a cancer, cancerous cell or tumour when compared to the growth in a control, such as an untreated cell or subject. In some embodiments, growth may be decreased or reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to an untreated control.

[0228] Inhibition of the growth of a cancer, tumour or cancerous cell may be assessed by a range of methods known in the art. For example, for a cancerous cell in vitro, the growth of the cell may be determined by a suitable proliferation assay, or by method which assess the extent of incorporation of tritiated thymidine into cellular DNA over a givenperiod of time. For a tumour or cancerous cell present in vivo, the growth of the tumour or cell may be determined for example by a suitable imaging method known in the art.

[0229] The term “subject” as used herein refers to any animal capable of suffering from cancer. Particular subjects of interest are human beings, and scientifically relevant species such as mice, rats, ferrets, guinea pigs, hamsters, non-human primates, dogs, pigs and sheep, or economically relevant animals such as horses, dogs, cats and cattle. In a preferred embodiment of the invention, the subject is a human.

[0230] A reference to “providing a subject with” relates to administering to the subject the genetically modified cell. Alternatively, the genetically modified cell may be generated within the subject. For example, the genetically modified cell may be generated in vivo such that the subject has an endogenous population of genetically modified cells. Suitable means for such in vivo generation are known in the art and include gene therapy of a subject.

[0231] The provision of a genetically modified cell expressing a CAR directed against a target cell expressing or displaying nfP2X? receptor may be sufficient to provide effective immunotherapy against precancerous or cancerous cells, the provision of adjuvants together with the genetically modified cells may further enhance the induction of the immune response and may augment the immunotherapy. Cytokines, preferably proinflammatory cytokines, are particularly suitable adjuvants for provision to a subject together with genetically modified cells having CARs.

[0232] Therefore, in some embodiments of the invention, the genetically modified cell is administered to the subject together with a cytokine. It is to be understood that as used throughout the specification the term “together with” includes the genetically modified cell being administered simultaneously with a cytokine or administered in combination with a cytokine. Consequently, when administered in combination with a cytokine this may be considered to include a combination therapy whereby a subject’s immunotherapy includes both treatment with a cytokine and treatment with a genetically modified cell having a CAR directed against a target cell expressing or displaying nfP2X? receptor, or an epitope thereof. In some forms, the cytokine is administered on a different day (>24hrs) to the administration of the genetically modified cells. In other forms the cytokine is administered on the same day (within 24hrs) as the genetically modified cells. In further forms the cytokine(s) and the genetically modified cell is administered within 18hrs, 12hrs,6hrs, 4hrs, 2hrs, 1 hr, 45mins, 30mins, 15mins, 10mins, 5mins, 2mins or 1 min of each other.

[0233] Suitable cytokines for administration together with the genetically modified cell include IL-2, IL-4, IL-6, IL-7, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21 , IL-23, IFNa, IFN|3, IFNy, GM-CSF, TGF[3 and TNFa. Preferred cytokines include IL-7 and IL-15. Furthermore, the cytokines may be administered as recombinant forms, natural forms, or via delivery systems such as fusions with proteins, delivered as a nucleic acid sequence which is expressed in the genetically modified cell or conjugated with a polymer such a polyethylene glycol (PEG).

[0234] The cell to be genetically modified can be obtained from any suitable source. In some embodiments of the invention the cell to be genetically modified is an autologous cell, being a cell autologous to the cell expressing or displaying nfP2X? receptor, or epitope thereof. Advantageously, an autologous cell would not be recognised as ‘nonself’ by the subject’s immune system and would therefore be tolerated by the subject. However, in some forms of cancer suitable autologous cells may not be readily available. Therefore, in some embodiments of the invention the cell to be genetically modified is an allogeneic or heterologous cell.

[0235] It will be clearly understood that, although this specification refers specifically to applications in humans, the invention is also useful for veterinary purposes. Thus in all aspects the invention is useful for domestic animals such as cattle, sheep, horses and poultry; for companion animals such as cats and dogs; and for zoo animals. Therefore, the general term "subject” or “subject to be I being treated" is understood to include all animals (such as humans, apes, dogs, cats, horses, and cows).

[0236] The term "administered" means administration of a therapeutically effective dose of the aforementioned composition including the respective cells to an individual. By "therapeutically effective amount" is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localised delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.

[0237] Subjects requiring treatment include those already having a benign, pre- cancerous, or non-metastatic tumour as well as those in which the occurrence or recurrence of cancer is to be prevented. Subjects may have metastatic cells, including metastatic cells present in the ascites fluid and / or lymph node.

[0238] The objective or outcome of treatment may be to reduce the number of cancer cells; reduce the primary tumour size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumour metastasis; inhibit, to some extent, tumour growth; and / or relieve to some extent one or more of the symptoms associated with the disorder.

[0239] Efficacy of treatment can be measured by assessing the duration of survival, time to disease progression, the response rates (RR), duration of response, and / or quality of life.

[0240] The method is particularly useful for extending time to disease progression.

[0241] The method is particularly useful for extending survival of the human, including overall survival as well as progression free survival.

[0242] The method is particularly useful for providing a complete response to therapy whereby all signs of cancer in response to treatment have disappeared. This does not always mean the cancer has been cured.

[0243] The method is particularly useful for providing a partial response to therapy whereby there has been a decrease in the size of one or more tumours or lesions, or in the extent of cancer in the body, in response to treatment.

[0244] The objective or outcome of treatment may be any one or more of the following: to reduce the number of cancer cells; reduce the primary tumour size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumour metastasis;inhibit, to some extent, tumour growth; relieve to some extent one or more of the symptoms associated with the disorder.

[0245] In one embodiment, subjects requiring treatment include those having a benign, pre-cancerous, non-metastatic tumour.

[0246] In one embodiment, the cancer is pre-cancerous or pre-neoplastic.

[0247] In one embodiment, the cancer is a secondary cancer or metastasis. The secondary cancer may be located in any organ or tissue, and particularly those organs or tissues having relatively higher haemodynamic pressures, such as lung, liver, kidney, pancreas, bowel and brain. The secondary cancer may be detected in the ascites fluid and / or lymph nodes.

[0248] In one embodiment, the cancer may be substantially undetectable.

[0249] “Pre-cancerous" or “preneoplasia” generally refers to a condition or a growth that typically precedes or develops into a cancer. A "pre-cancerous" growth may have cells that are characterised by abnormal cell cycle regulation, proliferation, or differentiation, which can be determined by markers of cell cycle.

[0250] The cancer may be a solid or a “liquid” tumour. In other words, the cancer may be growth in a tissue (carcinoma, sarcoma, adenomas etc) or it may be a cancer present in bodily fluid such as in blood or bone marrow (e.g., lymphomas and leukaemias).

[0251] The term “cancer” will be understood to include benign, pre-cancerous, pre- neoplastic or non-metastatic tumours or metastatic tumours. In some embodiments, the cancer is metastatic cancer, such as stage III or stage IV cancer.

[0252] In some embodiments, the type of cancer to be treated includes those having a benign, pre-cancerous, pre-neoplastic or non-metastatic tumour. A benign tumour will be understood to not be a malignant tumour and to not invade nearby tissue or spread to other parts of the body. Similarly non-metastatic cancer will be understood to not invade nearby tissue or spread to other parts of the body. “Pre-cancerous" or “pre-neoplasia” generally refers to a condition or a growth that typically precedes or develops into a cancer. A "pre-cancerous" growth may have cells that are characterized by abnormal cellcycle regulation, proliferation, or differentiation, which can be determined by markers of cell cycle.

[0253] In one embodiment, the cancer is a secondary cancer or metastases. The secondary cancer may be located in any organ or tissue, and particularly those organs or tissues having relatively higher hemodynamic pressures, such as lung, liver, kidney, pancreas, bowel and brain. The secondary cancer may be detected in the ascites fluid and / or lymph nodes.

[0254] In certain embodiments, the cancer requiring treatment may be a cancer characterised by low levels of expression of dysfunctional P2X? receptor.

[0255] Cancers particularly suited for prevention or treatment are those that express or overexpress nfP2X? receptor. The expression or overexpression of nfP2X? receptor on a cancer or a specific tumour can be determined by detecting nfP2X? receptor protein present in or on a cancer cell.

[0256] Pre-neoplastic, neoplastic and metastatic cancers are particular examples to which the methods of the invention may be applied. Broad examples include breast tumours, colorectal tumours, adenocarcinomas, mesothelioma, bladder tumours, prostate tumours, germ cell tumour, hepatoma / cholongiocarcinoma, carcinomas, neuroendocrine tumours, pituitary neoplasm, small round cell tumour, squamous cell cancer, melanoma, atypical fibroxanthoma, seminomas, nonseminomas, stromal leydig cell tumours, Sertoli cell tumours, skin tumours, kidney tumours, testicular tumours, brain tumours, ovarian tumours, stomach tumours, pancreatic tumours, oral tumours, bladder tumours, bone tumours, cervical tumours, esophageal tumours, laryngeal tumours, liver tumours, lung tumours, vaginal tumours and Wilm's tumour. Preferred examples include pancreatic tumours, ovarian tumours, stomach tumours, lung tumours, liver tumours, colorectal tumours, cervical tumours, endometrial tumours, renal tumours, breast tumours, and testicular tumours.

[0257] Examples of particular cancers include but are not limited to adenocarcinoma, adenoma, adenofibroma, adenolymphoma, adontoma, AIDS related cancers, acoustic neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenocystic carcinoma, adrenocortical cancer, agnogenic myeloid metaplasia, alopecia, alveolar soft-part sarcoma, ameloblastoma, angiokeratoma, angiolymphoid hyperplasia with eosinophilia,angioma sclerosing, angiomatosis, apudoma, anal cancer, angiosarcoma, aplastic anaemia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancers, bowel cancer, brain stem glioma, brain and CNS tumours, breast cancer (preferably triple negative breast cancer), branchioma, CNS tumours, carcinoid tumours, cervical cancer, childhood brain tumours, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancers, cutaneous T-cell lymphoma, carcinoma (e.g. Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumour, Krebs 2, Merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell), carcinosarcoma, cervical dysplasia, cystosarcoma phyllodies, cementoma, chordoma, choristoma, chondrosarcoma, chondroblastoma, craniopharyngioma, cholangioma, cholangiocarincoma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, dermatofibrosarcoma- protuberans, desmoplastic-small-round-cell-tumour, ductal carcinoma, dysgerminoam, endocrine cancers, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extra-hepatic bile duct cancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tube cancer, fanconi anaemia, fibroma, fibrosarcoma, gall bladder cancer, gastric cancer, gastrointestinal cancers, gastrointestinal-carcinoid-tumour, genitourinary cancers, germ cell tumours, gestational trophoblastic-disease, glioma, gynaecological cancers, giant cell tumours, ganglioneuroma, glioma, glomangioma, granulosa cell tumour, gynandroblastoma, haematological malignancies, hairy cell leukemia, head and neck cancer, hepatocellular cancer, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole, hypercalcemia, hypopharynx cancer, hamartoma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, hemangiosarcoma, histiocytic disorders, histiocytosis malignant, histiocytoma, hepatoma, hidradenoma, immunoproliferative small, opoma, ontraocular melanoma, islet cell cancer, Kaposi's sarcoma, kidney cancer, langerhan's cell-histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, li-fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leigomyosarcoma, leukemia (e.g. B-cell, mixed cell, null-cell, T-cell, T-cell chronic, HTLV-llassociated, lymphangiosarcoma, lymphocytic acute, lymphocytic chronic, mast-cell and myeloid), leukosarcoma, leydig cell tumour, liposarcoma, leiomyoma, leiomyosarcoma, lymphangioma, lymphangiocytoma, lymphagioma,lymphagiomyoma, lymphangiosarcoma, male breast cancer, malignant- rhabdoid- tumour-of-kidney, medulloblastoma, melanoma, Merkel cell cancer, mesothelioma, metastatic cancer, mouth cancer, multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndromes, myeloma, myeloproliferative disorders, malignant carcinoid syndrome carcinoid heart disease, medulloblastoma, meningioma, melanoma, mesenchymoma, mesonephroma, mesothelioma, myoblastoma, myoma, myosarcoma, myxoma, myxosarcoma, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen breakage syndrome, non-melanoma skin cancer, non-small-cell-lung-cancer-(nsclc), neurilemmoma, neuroblastoma, neuroepithelioma, neurofibromatosis, neurofibroma, neuroma, neoplasms (e.g. bone, breast, digestive system, colorectal, liver), ocular cancers, oesophageal cancer, oral cavity cancer, oropharynx cancer, osteosarcoma, ostomy ovarian cancer, pancreas cancer, paranasal cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral- neuroectodermal-tumours, pituitary cancer, polycythemia vera, prostate cancer, osteoma, osteosarcoma, ovarian carcinoma, papilloma, paraganglioma, paraganglioma nonchromaffin, pinealoma, plasmacytoma, protooncogene, rare-cancers- and-associated- disorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, reticuloendotheliosis, rhabdomyoma, salivary gland cancer, sarcoma, schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (sclc), small intestine cancer, soft tissue sarcoma, spinal cord tumours, squamous-cell- carcinoma-(skin), stomach cancer, synovial sarcoma, sarcoma (e.g. Ewing's experimental, Kaposi's and mast-cell sarcomas), Sertoli cell tumour, synovioma, testicular cancer, thymus cancer, thyroid cancer, transitional-cell-cancer-(bladder), transitional-cell-cancer-(renal-pelvis- / -ureter), trophoblastic cancer, teratoma, theca cell tumour, thymoma, trophoblastic tumour, urethral cancer, urinary system cancer, uroplakins, uterine sarcoma, uterus cancer, vaginal cancer, vulva cancer, Waldenstrom' s-macroglobulinemia and Wilms' tumour. Preferred particular examples include pleural or peritoneal mesothelioma, gastric cancer, endometrial cancers, colorectal cancer, nonsmall cell lung adenocarcinoma, cholangiocarcinoma, ovarian carcinoma, esophageal cancer, pancreatic ductal adenocarcinoma, triple negative breast cancer and any other mesothelin-positive cancers.

[0258] In some embodiments of the invention the method is used for the prevention or treatment of a cancer selected from one or more of; brain cancer, oesophageal cancer, mouth cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreaticcancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cervical cancer, epithelial cell cancers, skin cancer, leukaemia, lymphoma, myeloma, breast cancer, ovarian cancer, endometrial cancer, testicular cancer. Preferably the cancer is selected from one or more of lung cancer, oesophageal cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer, cervical cancer, vaginal cancers, epithelial cell cancers, skin cancer, blood-related cancers, breast cancer, endometrial cancer, uterine cancer testicular cancer.

[0259] The existence of, improvement in, treatment of, or minimisation of progression of cancer may be determined by any clinically or biochemically relevant method as described herein or known in the art. A positive response to treatment or a minimisation of progression of a cancer may be determined by any method known in the art and may include the determination of:- a reduction in the number of cancer cells;- a reduction in the tumour size;- an inhibition (i.e. , slow to some extent and preferably stop) of cancer cell infiltration into peripheral organs;- an inhibition (i.e., slow to some extent and preferably stop) of tumour metastasis;- a reduction or complete prevention of tumour metastasis following removal of the primary tumour;- an inhibition, to some extent, of tumour growth;- relieving, to some extent, of one or more of the symptoms associated with the cancer; and / or- increased survival of the subject.

[0260] The determination of any of the above may be considered to be a positive response to a treatment as described herein.

[0261] The subject who has received the treatment for cancer may be in partial or complete remission. In other words, the subject, having received a treatment for cancer, as described above, may have a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% orgreater reduction in the measurable parameters of tumour growth as may be found on physical examination, radiologic study, or by biomarker levels from a blood or urine test. Alternatively, where the subject is in complete remission, there is a complete disappearance of all detectable manifestations of disease, such that the subject does not have any detectable signs of cancer. The subject may have substantially undetectable signs of cancer. A cancer that is “substantially undetectable” generally refers to a circumstance where therapy has depleted the size, volume or other physical measure of a cancer so that using relevant standard detection techniques such as in vivo imaging, the cancer, as a consequence of the therapy, is not clearly detectable.

[0262] The objective or outcome of treatment may be to reduce the number of cancer cells; reduce the primary tumour size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumour metastasis; inhibit, to some extent, tumour growth; and / or relieve to some extent one or more of the symptoms associated with the disorder.

[0263] Efficacy of treatment can be measured by assessing the duration of survival, time to disease progression, the response rates (RR), duration of response, and / or quality of life.

[0264] In one embodiment, the method is particularly useful for delaying cancer progression. In one embodiment, the method is particularly useful for extending survival of the subject, including overall survival as well as progression free survival. It will be understood that overall survival is the length of time from either the date of diagnosis or the start of treatment of a cancer, that patients diagnosed with the cancer are still alive. It will be understood that progression free survival is the length of time during and after the treatment of a cancer that a patient lives with the disease but it does not get worse.

[0265] Survival analysis can be performed using well known techniques in the art including the Kaplan-Meier method. The Kaplan-Meier method estimates the survival function from life-time data. In medical research, it can be used to measure the fraction of patients living for a certain amount of time after treatment. A plot of the Kaplan-Meier method of the survival function is a series of horizontal steps of declining magnitude which, when a large enough sample is taken, approaches the true survival function for that population. The value of the survival function between successive distinct sampled observations ("clicks") is assumed to be constant.

[0266] An important advantage of the Kaplan-Meier curve is that the method can take into account "censored" data- losses from the sample before the final outcome is observed (for instance, if a patient withdraws from a study). On the plot, small vertical tick-marks indicate losses, where patient data has been censored. When no truncation or censoring occurs, the Kaplan-Meier curve is equivalent to the empirical distribution.

[0267] In one embodiment, the method is particularly useful for providing a complete response to therapy whereby all signs of cancer in response to treatment have disappeared. This does not always mean the cancer has been cured. In one embodiment, the method is particularly useful for providing a partial response to therapy whereby there has been a decrease in the size of one or more tumours or lesions, or in the extent of cancer in the body, in response to treatment.Compositions and administration

[0268] The delivery or administration of the genetically modified cell according to the invention may be delivery or administration of the cell alone, or delivery or administration of the cell formulated into a suitable pharmaceutical composition. Accordingly, the present invention provides a pharmaceutical composition including a genetically modified cell of the invention, and a pharmaceutically acceptable carrier.

[0269] Methods are known in the art for providing CAR-containing cells for immunotherapy (see for example Kershaw, MH. et al. Clin Cancer Res. 2006;12(20): 6106-15; Parker LL. et al. Hum Gene Ther 2000;1 1 : 2337-87). Furthermore, protocols and methods are known in the art for the preparation, expansion and assessment of mammalian CAR-expressing cells (see for example Cheadle, EJ. et al. Antibody Engineering: Methods and Protocols, Second Edition, Methods in Molecular Biology, vol. 907: 645-66) and are summarised in the Examples below.

[0270] The pharmaceutical composition may also include one or more pharmaceutically acceptable additives, including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients and bulking agents, taking into consideration the particular physical and chemical characteristics of the cell to be administered. In some embodiments, the pharmaceutical composition includes a suspension of genetically modified cells of the invention in a suitable medium, such as isotonic saline solution. In some embodiments, the pharmaceutical composition mayinclude suitable adjuvants such as one or more cytokines as described above. In some embodiments, the pharmaceutical composition may also include an intermediate as described above.

[0271] Administration of the pharmaceutical composition may also be via parenteral means which include intravenous, intraventricular, intraperitoneal, intramuscular, intrapleurally or intracranial injection, or local injections to the site of a tumour or cancerous mass.

[0272] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.ExamplesExample 1 : Identification of alternative sdAb-sequences for inclusion in CARs

[0273] Single domain CARs for binding to nfP2X? are known, including CARs which comprise the sdAb designated “BIL03s”.

[0274] The inventors sought to identify alternative sdAb-based CARs for targeting cancer cells. A number of different anti-nfP2X? sdAb sequences were tested in the context of a CAR.

[0275] Three suitable sdAb sequences (4A7, 3aB9 and 3aD9, as defined herein in table 3), were identified as providing efficacy in the context of a CAR including in the context of a CAR T cell / BRiDGE system (as disclosed in WO 2022 / 187906), preferably wherein the CAR has the hinge region of CD8A, the transmembrane domain of CD28, and the co- stimulatory / signalling domains of CD28, 41 BB (CD137) and CD3zeta.

[0276] Interestingly, the inventors found that none of these sdAb sequences provided for suitable CAR constructs when the sdAbs were paired with different light chain sequences.Example 2: Identification of light chain pairing for BIL03 heavy chain

[0277] Although single domain antigen binding domains for inclusion in anti-nfP2X? receptor CARs are known, it is not evident whether binding affinity could be improved by addition of a paired variable light chain. As indicated in Example 1 , in some instances, light chain pairings to heavy chains in the context of a CAR, did not result in improved CAR function. Moreover, the selection of an appropriate light chain for pairing to a known heavy chain is not evident from the prior art.

[0278] The inventors sought to identify a suitable light chain pairing for the single domain heavy chain protein BIL03 (as defined herein in Table 1 ) which is disclosed in the prior art in the context of a sdAb-based CAR.

[0279] More than 10 different light chain pairings were tested, including the variable light chain defined as G B1 in Table 1 herein.

[0280] The inventors found that the specific light chain pairing with WT B1 (defined in Table 1 ), provided for stable expression and reduced aggregation. Surprisingly, this light-heavy chain pairing also significantly improved binding affinity to target antigen, as demonstrated in the below table.

[0281] Table 2: binding affinity of BIL03 heavy chain compared to affinity when paired with various light chain variable domains

[0282] Binding affinity was assessed in relation to two different antigens derived from dysfunctional P2X7receptor: E200 peptide (GHNYTTRNILPGLNITC) (SEQ ID NO: 143) and Ext peptide 17 (GHNYTTRNILPGLNITSTFHKTSGSGK) (SEQ ID NO: 102).Example 3: scFv CAR design and productionCAR construction:

[0283] Two CARs were designed. The first (CAR10A) was designed containing the single domain antibody BIL03 (2-2-1 ) (sequence provided in Table 1 ) and the hinge region of CD8A, the transmembrane domain of CD28, and the co-stimulatory / signalling domains of CD28, 41 BB (CD137) and CD3zeta.

[0284] The second (CAR 12AV1 ) was designed containing the scFv light-heavy chain pairing identified in Example 2: WTB1 -BIL03 (sequence provided in Table 1 ) and the hinge region of CD8A, the transmembrane domain of CD28, and the co- stimulatory / signalling domain of CD28, 41 BB and CD3zeta.

[0285] Both CARs comprise a C terminal tEGFR to facilitate detection of the CAR.

[0286] Nucleic acids encoding the CARs were designed in silica, synthesised, cloned into the transfer vector, and packaged into self-inactivating (SIN) lentiviral particles (3rdgeneration LV system) in HEK293T cells. The supernatant was concentrated by ultracentrifugation according to standard protocols.CAR T cell production

[0287] CAR T cells were generated by lentiviral transduction of CD4 / CD8 positive selected T cells (1 :1 ratio) via magnetic activated cell sorting (MACS) stimulated with TransAct (all according to manufacturer’s instructions) cultivated in IL7 / IL15 supplemented TexMACS media (both 10 ng / mL). The donor source was a buffy coat.

[0288] Activated untransduced T cells (aUT) do not express any receptor that can either engage with the EGFR.

[0289] In luciferase-based kill assays, reporter cell lines were incubated with effector cells with or without a BRIDGE molecule (for example as disclosed in WO 2022 / 187906). Relevant controls were used to calculate viability and / or specific lysis, e.g. only target cells at 100%, 75%, 50%, 25%, 10% and 0% to calculate the standard curve for final determination of target cell lysis.

[0290] In flow-based kill assays, reporter cell lines were incubated with effector cells a BRIDGE molecule (for example as disclosed in WO 2022 / 187906) at the indicated concentrations. Relevant controls were used to calculate viability and / or specific lysis.

[0291] Results indicate that cells transduced with CAR12AV1 were capable of greater direct cell killing compared to cells transduced with CAR10A, although both CARs enabled cells to kill cells indirectly, when combined with BRIDGE molecule.

[0292] Figure 1 shows a direct comparison of the cell-killing capacity of T cells expressing CAR10A compared to cells expressing CAR12AV1. Target cells used were the AML cell line MOLM-13. ET 5:1 , CAR to target ratio of 2.1 to 1 . Cells were deprived of cytokine for 3 days prior to exposure to CAR T cells.

[0293] The results also show significantly greater direct cell killing (as shown by reduced MOL-13 cell count) by CAR12AV1 -T cells compared to CAR10A-T cells. Co-incubation with 200 ng / mL of Fab-based anti-CD33 BRIDGE molecule (as disclosed in WO2022 / 187906) enabled T cells expressing either CAR to kill MOLM-13 cells with greater efficacy.Example 4: Cell killing by T cells expressing CAR molecule of the invention

[0294] CAR T cells expressing CAR10A and CAR12A were produced according to the methods described in Example 3 above. In addition, T cells were produced expressing the single domain CARs 3a-B9-CAR and 4A7-CAR.

[0295] The CAR T cells were assessed for their ability to kill JeKo-1 and MOLM-13 cells directly, or in the presence of anti-CD19 or anti-CD33 BRIDGE molecules (as disclosed in WO 2022 / 187906).

[0296] Figure 2 shows that T cells expressing any of CAR10A, CAR12A, 3a-B9-CAR and 4A7-CAR induced cell killing in the absence of a BRIDGE molecule indicating that these CAR T cells are all capable of direct cell killing. Co-incubation with various concentrations of anti-CD19 BRIDGE molecule enabled the T cells to kill JeKo-1 cells with greater efficacy. The results also show that the cell killing efficacy increases in a dose-dependent manner in response to increasing concentrations of the BRIDGE molecule.

[0297] Figure 3 shows that T cells expressing 3a-B9-CAR and 4A7-CAR were also capable of direct and indirect killing of MOLM-13 cells when co-incubated with an anti- CD33 BRIDGE molecule. Similar results were achieved with CAR positive cell and cancer cell ratios of 5:1 and 2.5:1 (data not shown).

[0298] Figure 4 (A) and (B) further shows that T cells expressing 3a-B9-CAR and 4A7- CAR were capable of direct and indirect killing of JeKo-1 cells when co-incubated with an anti-CD19 BRIDGE molecule. The results also show that the effect is BRiDGE-molecule specific with cells co-incubated with an anti-CD33 BRIDGE unable to elicit greater cell killing. JeKo-1 cells do not express CD33 but do express CD19 and therefore the use of an anti-CD19 BRIDGE molecule to induce greater cell killing shows that the effect is controlled by the specific CAR / BRiDGE interaction. Similar results were achieved with CAR positive cell and cancer cell ratios of 5:1 and 2.5:1 (Figure 4 (B)).

[0299] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentionedor evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Claims

CLAIMS1 . A chimeric antigen receptor (CAR) comprising: i) an antigen recognition domain that recognises dysfunctional P2X? receptor or epitope derived therefrom; ii) a transmembrane domain; and iii) an intracellular domain, wherein the antigen recognition domain comprises the complementary determining regions (CDRs) from a VH comprising a sequence as set forth in SEQ ID NO: 4 and the CDRs from a VL comprising a sequence as set forth in SEQ ID NO: 12.

2. The CAR of claim 1 , wherein the antigen recognition domain comprises:(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to a sequence set forth in SEQ ID NO: 1 , 29, 36 or 43, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to a sequence set in SEQ ID NO: 2, 30, 37 or 44 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to a sequence set forth in SEQ ID NO: 3, 31 , 38 or 45;(ii) a VH comprising a sequence at least about 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in SEQ ID NO: 4;(iii) a VL comprising a CDR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 9, 50, 57 or 64, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 10, 51 , 58 or 65, and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 11 , 52, 59 or 66;(iv) a VL comprising a sequence at least about 95% identical to a sequence set forth in SEQ ID NO: 12;(v) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 1 , 29,36 or 43, a CDR2 comprising a sequence set forth in SEQ ID NO: 2, 30, 37 or 44, and aCDR3 comprising a sequence set forth in SEQ ID NO: 3, 31 , 38 or 45;(vi) a VH comprising a sequence set forth in SEQ ID NO: 4;(vii) a VL comprising a CDR1 comprising a sequence set SEQ ID NO: 9, 50, 57 or 64, a CDR2 comprising a sequence set forth in SEQ ID NO: 10, 51 , 58 or 65 and a CDR3 comprising a sequence set forth in SEQ ID NO: 1 1 , 52, 59 or 66;(viii) a VL comprising a sequence set forth in SEQ ID NO: 12;(ix) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 1 , 29, 36 or 43, a CDR2 comprising a sequence set forth in SEQ ID NO: 2, 30, 37 or 44 and a CDR3 comprising a sequence set forth in SEQ ID NO: 3, 31 , 38 or 45; and a VL comprising a CDR1 comprising a sequence set SEQ ID NO: 9, 50, 57 or 64, a CDR2 comprising a sequence set forth in SEQ ID NO: 10, 51 , 58 or 65 and a CDR3 comprising a sequence set forth in SEQ ID NO: 11 , 52 59 or 66; or(x) a VH comprising a sequence set forth in SEQ ID NO: 4 and a VL comprising a sequence set forth in SEQ ID NO: 12.

3. The CAR of claim 2, wherein the antigen recognition domain further comprises at least one of:(i) a VH comprising a framework region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 5, 32, 39, or 46, a FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set in SEQ ID NO: 6, 33, 40 or 47, a FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 7, 34, 41 or 48, and a FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 8, 35, 42 or 49;(ii) a VL comprising a FR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 13, 53, 60 or 67, a FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 14, 54, 61 or 68, a FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 15, 55, 62 or 69, and a FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 16, 56, 63 or 70;(iii) a VH comprising a FR1 comprising a sequence set forth in SEQ ID NO: 5, 32, 39 or 46, a FR2 comprising a sequence set forth in SEQ ID NO: 6, 33, 40 or 47, a FR3 comprising a sequence set forth in SEQ ID NO: 7, 34, 41 or 48, and a FR4 comprising a sequence set forth in SEQ ID NO: 8, 35, 42 or 49;(iv) a VL comprising a FR1 comprising a sequence set forth in SEQ ID NO: 13, 53, 60 or 67, a FR2 comprising a sequence set forth in SEQ ID NO: 14, 54, 61 or 68, a FR3 comprising a sequence set forth in SEQ ID NO: 15, 55, 62 or 69, and a FR4 comprising a sequence set forth in SEQ ID NO: 16, 56, 63 or 70; or(v) a VH comprising a FR1 comprising a sequence set forth in SEQ ID NO: 5, 32, 39 or 46, a FR2 comprising a sequence set forth in SEQ ID NO: 6, 33, 40 or 47, a FR3 comprising a sequence set forth in SEQ ID NO: 7, 34, 41 or 48, and a FR4 comprising a sequence set forth in SEQ ID NO: 8, 35, 42 or 49; and a VL comprising a FR1 comprising a sequence set forth in SEQ ID NO: 13, 53, 60 or 67, a FR2 comprising a sequence set forth between in SEQ ID NO: 14, 54, 61 or 68, a FR3 comprising a sequence set forth in SEQ ID NO: 15, 55, 62 or 69, and a FR4 comprising a sequence set forth in SEQ ID NO: 16, 56, 63 or 70.

4. The CAR of any one of claims 1 to 3, wherein antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 4, or a functional variant thereof comprising a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%,at least 98%, at least 99% identical thereto; and a light chain variable domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 12, or a functional variant thereof comprising a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; and wherein the antigen recognition domain retains the ability to bind to nfP2X? receptor.

5. The CAR of any one of claims 1 to 3, wherein antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 4, and a light chain variable domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 12; wherein the heavy and / or light chain variable domain comprise no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 1 1 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions or additions compared to the amino acid sequence as set forth in SEQ ID NO: 4 or 12, respectively, and wherein the antigen recognition domain retains the ability to bind to nfP2X? receptor.

6. The CAR of claim 4 wherein the functional variant does not comprise amino acid substitutions deletions or additions in the CDRs or the CAR of claim 5, wherein the amino acid substitutions deletions or additions are not in the CDRs.

7. The CAR of any one of claims 1 to 6, wherein antigen recognition domain comprises, consists essentially of or consists of the amino acid sequence of (in order of N to C terminus or C to N terminus) SEQ ID NOs: 4 and 12.

8. The CAR of any one of claims 1 to 7, wherein the antigen recognition domain comprises, consists essentially of or consists of, in order N to C terminus, SEQ ID NO: 12 and SEQ ID NO: 4 (ie VL to VH).

9. The CAR of claim 7, wherein the antigen binding protein comprises SEQ ID NO: 12 (VL) - linker - SEQ ID NO: 4 (VH).

10. The CAR of any one of claims 1 to 9, wherein the antigen recognition domain is a single-chain variable fragment (scFv).1 1. A chimeric antigen receptor (CAR) comprising: i) an antigen recognition domain that recognises dysfunctional P2X? receptor or epitope derived therefrom; ii) a transmembrane domain; and iii) an intracellular domain, wherein the antigen recognition domain comprises the complementary determining regions (CDRs) from a VH comprising a sequence as set forth in SEQ ID NO: 135, 136 or 137.

12. The CAR of claim 1 1 , wherein the antigen recognition domain comprises:(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 124, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 125, 126 or 127 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 128, 129 or 130;(ii) a VH comprising a sequence at least about 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in SEQ ID NO: 135, 136 or 137;(iii) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 124, a CDR2 comprising a sequence set forth in SEQ ID NO: 125, 126, or 127, and a CDR3 comprising a sequence set forth in SEQ ID NO: 128, 129 or 130; or(iv) a VH comprising a sequence set forth in SEQ ID NO: 135, 136 or 137.

13. The CAR of claim 1 1 or 12, wherein the antigen recognition domain further comprises at least one of:(i) a VH comprising a framework region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 131 , a FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set in SEQ ID NO: 132, a FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 133, and a FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 134;(ii) a VH comprising a FR1 comprising a sequence set forth in SEQ ID NO: 131 , a FR2 comprising a sequence set forth in SEQ ID NO: 132, a FR3 comprising a sequence set forth in SEQ ID NO: 133, and a FR4 comprising a sequence set forth in SEQ ID NO:134.

14. The CAR of any one of claims 1 1 to 13, wherein the antigen recognition domain comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO:135, 136 or 137.

15. The CAR of any one of claims 1 1 to 13, wherein the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 135, or a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; wherein the heavy chain does not comprise any sequence variation in the CDRs compared to the sequence of SEQ ID NO: 135, and / or wherein the antigen binding domain retains the ability to bind to nfP2X? receptor.

16. The CAR of any one of claims 1 1 to 13 wherein the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 135, wherein the heavy chain variable domain of the antigen binding domain comprises no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 1 1 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions or additions, compared to the amino acid sequence as set forth in SEQ ID NO: 135; preferably wherein the amino acid substitutions, deletions or additions are not in the CDRs and / or wherein the antigen binding domain retains the ability to bind to nfP2X? receptor.

17. The CAR of any one of claims 1 1 to 13 wherein the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 136, or a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; wherein the heavy chain does not comprise any sequence variation in the CDRs compared to the sequence of SEQ ID NO: 136, and / or wherein the antigen binding domain retains the ability to bind to nfP2X? receptor.

18. The CAR of any one of claims 1 1 to 13, wherein the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 136, wherein the heavy chain variable domain of the antigen binding domain comprises no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 1 1 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions or additions, compared to the amino acid sequence as set forth in SEQ ID NO: 136; preferably wherein the amino acid substitutions, deletions or additions are not in the CDRs and / or wherein the antigen binding domain retains the ability to bind to nfP2X? receptor.

19. The CAR of any one of claims 1 1 to 13, wherein the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acidsequence as set forth in SEQ ID NO: 137, or a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; wherein the heavy chain does not comprise any sequence variation in the CDRs compared to the sequence of SEQ ID NO: 137, and / or wherein the antigen binding domain retains the ability to bind to nfP2X? receptor.

20. The CAR of any one of claims 1 1 to 13, wherein the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 137, wherein the heavy chain variable domain of the antigen binding domain comprises no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 1 1 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions or additions, compared to the amino acid sequence as set forth in SEQ ID NO: 137; preferably wherein the amino acid substitutions, deletions or additions are not in the CDRs and / or wherein the antigen binding domain retains the ability to bind to nfP2X? receptor.21 . The CAR of any one of claims 1 to 20, wherein the CAR further comprises a hinge region.

22. The CAR of claim 21 , wherein the hinge region is derived from CD8a, CD28 or an lgG4 hinge region.

23. The CAR of claim 22, wherein the hinge region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 88.

24. The CAR of any one of claims 21 to 23, wherein the hinge is C-terminal to the antigen recognition domain.

25. The CAR of claim 24, wherein the hinge is located between the antigen recognition domain and the transmembrane domain of the CAR.

26. The CAR of any one of claims 1 to 26, wherein the transmembrane domain of the CAR comprises a portion from CD8a or CD28.

21. The CAR of claim 26, wherein the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 89.

28. The CAR of any one of claims 1 to 27, wherein the signalling domain of the CAR comprises a portion derived from an activation receptor.

29. The CAR of claim 28 wherein activation receptor is a member of the CD3 coreceptor complex.

30. The CAR of claim 29, wherein the portion derived from the CD3 co-receptor complex is CD3- (CD3-zeta).

31. The CAR of claim 30, wherein the signalling domain comprises an amino acid sequence as set forth in SEQ ID NO: 92.

32. The CAR of any one of claims 1 to 31 , wherein the signalling domain of the CAR comprises a portion derived from a co-stimulatory receptor.

33. The CAR of claim 32, wherein the co-stimulatory receptor is CD28 and / or 4-1 BB (CD137).

34. The CAR of claim 33, wherein the co-stimulatory receptor comprises an amino acid sequence as set forth in SEQ ID NO: 90 and / or 91 .

35. The CAR of any one of claims 1 to 34, wherein the signalling domain comprises a portion derived from an activation receptor and a portion derived from a co-stimulatory receptor.

36. The CAR of claim 35, wherein the activation receptor is a member of the CD3 coreceptor complex and the co-stimulatory receptor is selected from CD28 and / or 4-1 BB.

37. The CAR of claim 36, wherein the signalling domain comprises an amino acid sequence comprising the sequences set forth in SEQ ID NOs: 90, 91 and / or 92 and combinations thereof.

38. The CAR of any one of claims 1 to 37, wherein the CAR comprises (from N to C terminus) an antigen recognition domain as defined in any of claims 1 to 20, a CD8a hinge, a CD28 transmembrane domain, a CD28 signalling domain, a 4-1 BB signalling domain, and a CD3 (zeta) signalling domain.

39. The CAR of claim 38 wherein the CAR comprises an amino acid sequence as set forth in SEQ ID NO: 94, or a functional variant thereof comprising a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; wherein the functional variant comprises an antigen recognition domain as herein defined, preferably comprising a VH comprising the amino acid sequence of SEQ ID NO: 4; and a VL comprising the amino acid sequence of SEQ ID NO: 12.

40. The CAR of claim 38 wherein the CAR comprises an amino acid sequence as set forth in SEQ ID NO: 138, or a functional variant thereof comprising a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; wherein the functional variant comprises an antigen recognition domain as herein defined, preferably comprising a VH comprising the amino acid sequence of SEQ ID NO: 135.41 . The CAR of claim 38 wherein the CAR comprises an amino acid sequence as set forth in SEQ ID NO: 139, or a functional variant thereof comprising a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; wherein the functional variant comprises an antigen recognition domain as herein defined, preferably comprising a VH comprising the amino acid sequence of SEQ ID NO: 136.

42. The CAR of claim 38 wherein the CAR comprises an amino acid sequence as set forth in SEQ ID NO: 140, , or a functional variant thereof comprising a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; wherein the functional variant comprises an antigen recognition domain as herein defined, preferably comprising a VH comprising the amino acid sequence of SEQ ID NO: 137.

43. A nucleic acid encoding a chimeric antigen receptor of any one of claims 1 to 42.

44. A nucleic acid construct that includes a nucleic acid molecule of claim 43.

45. A genetically modified cell that comprises a CAR of any one of claims 1 to 42.

46. A genetically modified cell comprising a nucleic acid molecule of claim 43 or construct of claim 44.

47. A genetically modified cell of claim 45 or 46, wherein the cell is an immune cell.

48. A genetically modified cell of claim47 wherein the immune cell is a leukocyte.

49. A genetically modified cell of claim 48, wherein the leukocyte is a lymphocyte, aT cell, a natural killer (NK) cell, a natural killer T cell or a tumour infiltrating lymphocyte (TIL).

50. A genetically modified cell of claim 49, wherein the T cell is a CD4+ T cell or a CD8+ T cell.51 . A method of generating a genetically modified cell, said method comprising transducing the cell, preferably an immune cell, with a nucleic acid construct of claim 44, so that the transduced cell expresses the CAR, thereby generating a genetically modified cell.

52. A method of claim 51 , wherein the immune cell is a T cell.

53. A method of killing a cell expressing nfP2X? receptor, the method comprising exposing the cell expressing nfP2X? receptor to a genetically modified cell of any one of claims 45 to 50, thereby killing a cell expressing nfP2X? receptor.

54. A method of claim 53, wherein the cell expressing nfP2X? receptor is a cancer cell.

55. A method of claim 54, wherein the cancer is selected from the group consisting of; brain cancer, oesophageal cancer, mouth cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cervical cancer, epithelial cell cancers, skincancer, leukaemia, lymphoma, myeloma, breast cancer, ovarian cancer, endometrial cancer, thymic and testicular cancer.

56. A method of claim 55, wherein the cancer is selected from the group consisting of; lung cancer, oesophageal cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer, cervical cancer, vaginal cancers, epithelial cell cancers, skin cancer, blood-related cancers, breast cancer, endometrial cancer, uterine cancer and testicular cancer.

57. A pharmaceutical composition including a genetically modified cell of any one of claims 45 to 50 and a pharmaceutically acceptable carrier.

58. A method of:• treating, preventing or minimising progression of cancer in a subject,• minimising, reducing or preventing growth of a tumour in a subject,• minimising, reducing or preventing metastasis in a subject, or• increasing survival of a subject, the method comprising, administering to the subject a CAR of any one of claims 1 to 42, a nucleic acid of claims 43, a nucleic acid construct of claim 44, a genetically modified cell of any one of claims 45 to 50, or a pharmaceutical composition of claim 57, thereby:• treating, preventing or minimising progression of cancer in a subject,• minimising, reducing or preventing growth of a tumour in a subject,• minimising, reducing or preventing metastasis in a subject, or• increasing survival of a subject,59. Use of a CAR of any one of claims 1 to 42, a nucleic acid of claim 43, a nucleic acid construct of claim 44, a genetically modified cell of any one of claims 45 to 50, or a pharmaceutical composition of claim 57, in the manufacture of a medicament for: treating, preventing or minimising progression of cancer in a subject,• minimising, reducing or preventing growth of a tumour in a subject,• minimising, reducing or preventing metastasis in a subject, or• increasing survival of a subject.

60. A CAR of any one of claims 1 to 42, a nucleic acid of claim 43, a nucleic acid construct of claim 44, a genetically modified cell of any one of claims 45 to 50, or a pharmaceutical composition of claim 57 for use in:• treating, preventing or minimising progression of cancer in a subject,• minimising, reducing or preventing growth of a tumour in a subject,• minimising, reducing or preventing metastasis in a subject, or• increasing survival of a subject.

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