Chimeric antigen receptors for binding dysfunctional P2X7 receptors

Abstract

The present invention relates to a chimeric antigen receptor for binding to a dysfunctional P2X7 receptor, a genetically modified cell containing the same, and uses thereof.

Description

[Technical Field]

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

[0002] Related Applications This application claims priority to Australian Provisional Application No. 2023900626, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0003] Current T cell therapies rely on enriched or modified human T cells to target and kill cancer cells in patients. To enhance the ability of T cells to target and kill specific cancer cells, methods have been developed to engineer T cells to express constructs that direct T cells to specific target cancer cells. Chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs), which contain binding domains that can interact with specific tumor antigens, enable T cells to target and kill cancer cells that express specific tumor antigens. However, some tumor types, particularly solid tumors, are resistant to T cell immunotherapy.

[0004] CARs have been designed to bind to a variety of tumor-specific antigens, but the architecture and binding affinity of CARs often require optimization to ensure appropriate levels of binding in the context of T cells that bind to cancer cells. In other words, antigen-binding domains derived directly from therapeutic antibodies may have inappropriate affinity for use in the context of a CAR, potentially resulting in the production of CAR T cells that cause toxicity in patients.

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

[0006] The reference to any prior art herein is not an admission or suggestion that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art could reasonably be expected to be understood, considered relevant, and / or combined with other pieces of prior art by a person skilled in the art. Summary of the Invention

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

[0008] In a first aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: i) an antigen recognition domain that recognizes a dysfunctional P2X7 receptor (nfP2X7 receptor) or an epitope derived therefrom; ii) a transmembrane domain; and iii) an intracellular domain, A chimeric antigen receptor (CAR) is provided, wherein the antigen recognition domain comprises a complementarity determining region (CDR) from a VH comprising the sequence set forth in SEQ ID NO:4, and a CDR from a VL comprising the sequence set forth in SEQ ID NO:12.

[0009] In a second aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: i) an antigen recognition domain that recognizes a dysfunctional P2X7 receptor (nfP2X7 receptor) or an epitope derived therefrom; ii) a transmembrane domain; and iii) an intracellular domain, A chimeric antigen receptor (CAR) is provided, wherein the antigen recognition domain comprises a complementarity determining region (CDR) from a VH comprising the sequence set forth in any of SEQ ID NOs: 135, 136, or 137.

[0010] According to a first aspect, the antigen recognition domain of the CAR comprises: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a, where: FR1, FR2, FR3, and FR4 are each a framework region; CDR1, CDR2, and CDR3 are each a complementarity determining region; FR1a, FR2a, FR3a, and FR4a are each a framework region; CDR1a, CDR2a, and CDR3a are each a complementarity determining region; The complementarity determining regions comprise the amino acid sequences set forth in Table 1 below.

[0011] According to a second aspect of the invention, the antigen recognition domain of the CAR comprises: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where: FR1, FR2, FR3, and FR4 are each a framework region; CDR1, CDR2, and CDR3 are each a complementarity determining region; The complementarity determining regions comprise the amino acid sequences set forth in Table 3 below.

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

[0013] In any embodiment of the first aspect of the invention, the antigen recognition domain comprises: (i) 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 the sequence set forth in SEQ ID NO: 5, 32, 39, or 46, and 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 the sequence set forth in SEQ ID NO: 6, 33, 40, or 47; a VH comprising 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 the 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 the sequence set forth in SEQ ID NO: 8, 35, 42, or 49; (ii) FR1 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 the sequence set forth in SEQ ID NO: 13, 53, 60, or 67; 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 the sequence set forth in SEQ ID NO: 14, 54, 61, or 68; a VL comprising an 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 the sequence set forth in SEQ ID NO: 15, 55, 62, or 69, and an 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 the sequence set forth in SEQ ID NO: 16, 56, 63, or 70; (iii) a VH comprising FR1 comprising the sequence set forth in SEQ ID NO: 5, 32, 39, or 46, FR2 comprising the sequence set forth in SEQ ID NO: 6, 33, 40, or 47, FR3 comprising the sequence set forth in SEQ ID NO: 7, 34, 41, or 48, and FR4 comprising the sequence set forth in SEQ ID NO: 8, 35, 42, or 49; (iv) a VL comprising FR1 comprising the sequence set forth in SEQ ID NO: 13, 53, 60, or 67, FR2 comprising the sequence set forth in SEQ ID NO: 14, 54, 61, or 68, FR3 comprising the sequence set forth in SEQ ID NO: 15, 55, 62, or 69, and FR4 comprising the sequence set forth in SEQ ID NO: 16, 56, 63, or 70; or (v) further comprising at least one of a VH comprising FR1 comprising the sequence set forth in SEQ ID NO: 5, 32, 39, or 46, FR2 comprising the sequence set forth in SEQ ID NO: 6, 33, 40, or 47, FR3 comprising the sequence set forth in SEQ ID NO: 7, 34, 41, or 48, and FR4 comprising the sequence set forth in SEQ ID NO: 8, 35, 42, or 49, and a VL comprising FR1 comprising the sequence set forth in SEQ ID NO: 13, 53, 60, or 67, FR2 comprising the sequence set forth in SEQ ID NO: 14, 54, 61, or 68, FR3 comprising the sequence set forth in SEQ ID NO: 15, 55, 62, or 69, and FR4 comprising the 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 SEQ ID NOs: 4 and 12 (in N-terminal to C-terminal or C-terminal to N-terminal order).

[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 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 an amino acid sequence set forth in SEQ ID NO:12, 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 a sequence that is 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, wherein the heavy and light chains do not contain any sequence variations within the CDRs compared to the sequence of SEQ ID NO: 4 or 12, respectively, and / or the antigen binding domain retains the ability to bind to the nfP2X7 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 set forth in SEQ ID NO: 4, and a light chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 12, and the heavy and light chain variable domains of the antigen binding domain comprise substitutions, deletions or additions of 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, or 20 or less amino acid residues compared to the amino acid sequence set forth in SEQ ID NO: 4 or 12, respectively, and preferably, the amino acid substitutions, deletions or additions are not within the CDRs, and / or the antigen binding domain retains the ability to bind to the nfP2X7 receptor.

[0017] Preferably, the antigen recognition domain comprises, consists essentially of, or consists of, in N-terminal to C-terminal order, SEQ ID NO: 12 and SEQ ID NO: 4 (i.e., 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 present invention, the antigen recognition domain competitively inhibits binding of an antibody comprising a VH comprising the sequence set forth in SEQ ID NO:4 and a VL comprising the sequence 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 comprises: It may comprise FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a.

[0020] As defined herein, a linker can be a chemical entity, one or more amino acids, or a disulfide 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 understood in the art, an scFv is a fusion protein comprising two portions that may share homology with, or be identical to, the variable heavy (VH) and variable light (VL) chains of an antibody, the two portions being connected together by a linker peptide.

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

[0023] Preferably, according to a second aspect of the present invention, there is provided a chimeric antigen receptor (CAR) comprising an antigen recognition domain, a transmembrane domain, and an intracellular domain, The antigen recognition domain is (i) 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 the sequence set forth in SEQ ID NO: 124, at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% identical to the sequence set forth in SEQ ID NO: 125, 126, or 127; a VH comprising a CDR2 comprising a sequence that is 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 the 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 the sequence set forth in SEQ ID NO: 135, 136, or 137; (iii) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 124, a CDR2 comprising the sequence set forth in SEQ ID NO: 125, 126, or 127, and a CDR3 comprising the sequence set forth in SEQ ID NO: 128, 129, or 130, wherein preferably, CDR1 comprises the sequence set forth in SEQ ID NO: 124, CDR2 comprises the sequence set forth in SEQ ID NO: 125, and CDR3 comprises the sequence set forth in SEQ ID NO: 128, or preferably, CDR1 comprises the sequence set forth in SEQ ID NO: 124, CDR2 comprises the sequence set forth in SEQ ID NO: 126, and CDR3 comprises the sequence set forth in SEQ ID NO: 129, or preferably, CDR1 comprises the sequence set forth in SEQ ID NO: 124, CDR2 comprises the sequence set forth in SEQ ID NO: 127, and CDR3 comprises the sequence set forth in SEQ ID NO: 130; or (iv) VH comprising the 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 comprises: (i) 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 the sequence set forth in SEQ ID NO: 131, and 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 the sequence set forth in SEQ ID NO: 132; a VH comprising 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 the 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 the sequence set forth in SEQ ID NO: 134. (ii) further comprising at least one of a VH comprising FR1 comprising the sequence set forth in SEQ ID NO: 131, FR2 comprising the sequence set forth in SEQ ID NO: 132, FR3 comprising the sequence set forth in SEQ ID NO: 133, and FR4 comprising the 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 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 mutations in the CDRs compared to the sequence of SEQ ID NO: 135, and / or the antigen binding domain retains the ability to bind to the nfP2X7 receptor.

[0027] In any embodiment, the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 135, and the heavy chain variable domain of the antigen binding domain comprises substitutions, deletions, or additions of 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, or 20 or less amino acid residues compared to the amino acid sequence set forth in SEQ ID NO: 135, preferably, the amino acid substitutions, deletions, or additions are not within the CDRs, and / or the antigen binding domain retains the ability to bind to the nfP2X7 receptor.

[0028] In any embodiment, the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence 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 mutations in the CDRs compared to the sequence of SEQ ID NO: 136, and / or the antigen binding domain retains the ability to bind to the nfP2X7 receptor.

[0029] In any embodiment, the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 136, and the heavy chain variable domain of the antigen binding domain comprises 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, or 20 or less amino acid residue substitutions, deletions, or additions compared to the amino acid sequence set forth in SEQ ID NO: 136, preferably, the amino acid substitutions, deletions, or additions are not within the CDRs, and / or the antigen binding domain retains the ability to bind to the nfP2X7 receptor.

[0030] In any embodiment, the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence 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 mutations in the CDRs compared to the sequence of SEQ ID NO: 137, and / or the antigen binding domain retains the ability to bind to the nfP2X7 receptor.

[0031] In any embodiment, the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 137, and the heavy chain variable domain of the antigen binding domain comprises substitutions, deletions, or additions of 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, or 20 or less amino acid residues compared to the amino acid sequence set forth in SEQ ID NO: 137, preferably, the amino acid substitutions, deletions, or additions are not within the CDRs, and / or the antigen binding domain retains the ability to bind to the nfP2X7 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 IgG4. 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 a preferred embodiment, 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 signaling domain of the CAR comprises a portion derived from an activating receptor. In some embodiments, the activating receptor is a member of the CD3 co-receptor complex. In some embodiments, the portion derived from the CD3 co-receptor complex is CD3-ζ (CD3-zeta), and optionally comprises the amino acid sequence set forth in SEQ ID NO:92.

[0035] In any embodiment of any aspect, the signaling domain of the CAR comprises a portion derived from a costimulatory receptor. In some embodiments, the costimulatory receptor is CD28, ICOS, CD27, OX40, and / or 4-1BB (CD137), and optionally comprises the amino acid sequence set forth in SEQ ID NOs: 90 and / or 91.

[0036] The signaling domain can comprise a portion derived from an activating receptor and a portion derived from a costimulatory receptor. In some embodiments, the activating receptor is a member of the CD3 co-receptor complex, and the costimulatory receptor is selected from CD28, ICOS, CD27, OX40, and / or 4-1BB. In some embodiments, the activating receptor is a member of the CD3 co-receptor complex, and the costimulatory receptor is CD28 and 4-1BB (CD137). Preferably, the portion derived from the CD3 co-receptor complex is CD3-zeta (CD3-zeta). In certain embodiments, the signaling domain comprises an amino acid sequence comprising the sequence set forth in SEQ ID NOs: 90, 91, and / or 92, and combinations thereof.

[0037] In one embodiment of any aspect, the CAR comprises (from N-terminus to C-terminus) an antigen-binding domain described herein, a linker, a CD8a hinge, a CD28 transmembrane domain, a CD28 signaling domain, a 4-1BB signaling domain, and a CD3ζ (zeta) signaling 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 the amino acid sequence 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%, or at least 99% identical thereto, wherein the functional variant preferably comprises an antigen recognition domain as defined herein, 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 a particularly preferred embodiment of the first aspect, the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:94.

[0040] In a preferred embodiment of the second aspect, the chimeric antigen receptor comprises the amino acid sequence 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 preferably comprises an antigen recognition domain as defined herein, including a VH comprising the amino acid sequence of SEQ ID NO: 135.

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

[0042] In a preferred embodiment of the second aspect, the chimeric antigen receptor comprises the amino acid sequence 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 preferably comprises an antigen recognition domain as defined herein, including a VH comprising the amino acid sequence of SEQ ID NO: 136.

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

[0044] In a preferred embodiment of the second aspect, the chimeric antigen receptor comprises the amino acid sequence 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 preferably comprises an antigen recognition domain as defined herein, including a VH comprising the amino acid sequence of SEQ ID NO: 137.

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

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

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

[0048] In another aspect, the present invention provides a nucleic acid construct comprising 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 transcription control sequence. In some embodiments, the transcription control sequence may be a constitutive promoter or an inducible promoter.

[0049] In some embodiments, the nucleic acid construct further comprises 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 immune cells, e.g., T cells, and induce expression of the CAR.

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

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

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

[0054] In some embodiments, the cell is an immune cell such as a leukocyte, hi 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 tumor-infiltrating lymphocyte (TIL).

[0055] In a preferred embodiment, the CAR-expressing immune cell is a T cell. Illustrative examples of suitable T cells include helper T cells (HTLV-1; CD4 + T cells), cytotoxic T cells (CTL; ​​CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - Other illustrative examples of suitable T cells include T cells that express 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 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 described herein, such that the transduced T cell expresses the CAR, thereby generating the CAR-T cell.

[0057] In another aspect, the present invention provides a method for killing a cell that expresses an nfP2X7 receptor, the method comprising exposing the cell that expresses the nfP2X7 receptor to a genetically modified cell of the invention described herein, thereby killing the cell that expresses the nfP2X7 receptor.

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

[0059] In some embodiments, the cells expressing the nfP2X7 receptor (i.e., the cells to which the CAR of the present invention binds) are cancer cells. In some embodiments, the cancer is selected from the group consisting of brain cancer, esophageal cancer, oral 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 cancer, skin cancer, leukemia, lymphoma, myeloma, breast cancer, ovarian cancer, endometrial cancer, and testicular cancer. In some embodiments, the cancer is selected from the group consisting of lung cancer, esophageal cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer, cervical cancer, vaginal cancer, epithelial cell cancer, skin cancer, blood-related cancer, breast cancer, endometrial cancer, uterine cancer, and testicular cancer.

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

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

[0062] In another aspect, the invention provides a method of expanding in vitro a genetically modified cell of the invention described herein, the method comprising exposing the cell to an antigen against the CAR, e.g., nfP2X7 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-β subfamily.

[0064] In some embodiments, the cytokine is selected from the group consisting of IFN-γ, IL-2, IL-5, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, TNF-α, TGF-β1, TGF-β2, TGF-β3, and GM-CSF, or a combination thereof.

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

[0066] In some embodiments, the agonist is immobilized on a beaded substrate. In one embodiment, the agonist may be immobilized on "human activator" Dynabeads™. In another preferred example, the agonist is immobilized on a colloidal polymer nanomatrix beaded substrate (e.g., on a "MACS GMP" TransAct™ beaded substrate) conjugated to recombinant humanized CD3 and CD28 agonists.

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

[0068] In some embodiments, the medium is TexMACS™ GMP medium, hi some embodiments, the medium is supplemented with interleukins, such as IL-7 and IL-15.

[0069] In some embodiments, the antibody is immobilized on the surface of a tissue culture vessel, such as the surface of a culture flask, plate, or bioreactor.

[0070] In another aspect, the present invention provides pharmaceutical compositions comprising the genetically modified cells of the invention described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition includes a suitable adjuvant, which may consist of a cytokine. In some embodiments, the pharmaceutical composition may also include an intermediate described herein.

[0071] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: Treating, preventing, or minimizing the progression of cancer in a subject; minimize, reduce, or prevent tumor growth in a subject; minimize, reduce, or prevent metastasis of cancer in a subject; or A method for increasing survival in a subject suffering from cancer, comprising: Optionally, the cancer or tumor is characterized by expression of the nfP2X7 receptor, and the method comprises administering to the subject a CAR of the invention described herein, a nucleic acid construct of the invention described herein, a genetically modified cell of the invention described herein, or a pharmaceutical composition of the invention described herein, whereby Treating, preventing, or minimizing the progression of cancer in a subject; minimize, reduce, or prevent tumor growth in a subject; minimize, reduce, or prevent metastasis in a subject; or -Provides a method for increasing survival of a subject suffering from cancer.

[0072] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: Treating, preventing, or minimizing the progression of cancer in a subject; minimize, reduce, or prevent tumor growth in a subject; minimize, reduce, or prevent metastasis of cancer in a subject; or Use of a CAR of the invention described herein, a nucleic acid construct of the invention described herein, a genetically modified cell of the invention described herein, or a pharmaceutical composition of the invention described herein in the manufacture of a medicament for increasing survival of a subject suffering from cancer, comprising: Optionally, the cancer or tumor is characterized by expression of the nfP2X7 receptor.

[0073] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: Treating, preventing, or minimizing the progression of cancer in a subject; minimize, reduce, or prevent tumor growth in a subject; minimize, reduce, or prevent metastasis of cancer in a subject; or - providing a CAR of the invention described herein, a nucleic acid construct of the invention described herein, a genetically modified cell of the invention described herein, or a pharmaceutical composition of the invention described herein for use in increasing survival of a subject suffering from cancer; Optionally, the cancer or tumor is characterized by expression of the nfP2X7 receptor.

[0074] As used herein, unless the context requires otherwise, the term "comprise" and variations of that term, such as "comprising," "comprises," and "comprised," are not intended to exclude additional additives, ingredients, elements, or steps.

[0075] Further aspects of the 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 referring to the accompanying drawings, in which: [Brief explanation of the drawings]

[0076] [Figure 1]MOLM-13 cell counts after contact with untransduced T cells (UTD) or T cells transduced with CAR12AV1 or CAR10A. NB = no bridging molecule (indicating direct cell killing). "With BRiDGE" = co-incubation with 200 ng / mL anti-CD33 BRiDGE molecule containing the E200 sequence for binding by the CAR antigen recognition domain. [Figure 2] JeKo-1 cell viability after 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 ranged from 0 ng / mL to 100 ng / mL. "BRiDGE" = fusion protein containing the E200 sequence for binding by the CAR antigen recognition domain and the anti-CD19 antigen-binding domain. Effector-target cell ratio = 2.77 / 1. Cell viability was read 21 hours after co-incubation. [Figure 3] MOLM-13 cell viability after contact with untransduced T cells (UTD), or T cells transduced with 3a-B9-CAR, or 4A7-CAR. 10:1 CAR-positive cell to cancer cell ratio. "BRiDGE" = co-incubation with 100 ng / mL of BRiDGE molecule containing E200 sequence for binding by the CAR antigen recognition domain and anti-CD33 binding domain or anti-CD19 binding domain. Cell viability was read 24 hours after co-incubation. [Figure 4] JeKo-1 cell viability after contact with untransduced T cells (UTD), or T cells transduced with 3a-B9-CAR, or 4A7-CAR. CAR-positive cell to cancer cell ratios of 10:1 in (A), and 10:1, 5:1, and 2.5:1 in (B). "BRiDGE" = co-incubation with 100 ng / mL of BRiDGE molecules containing the E200 sequence for binding by the CAR antigen recognition domain and either an anti-CD19 antigen-binding domain or an anti-CD33 binding domain. Cell viability was read 24 hours after co-incubation.

[0077] Sequence information [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 2-1] [Table 2-2] [Table 2-3] [Table 3-1] [Table 3-2] [Table 3-3] DETAILED DESCRIPTION OF THE INVENTION

[0078] Reference will now be made in detail to certain specific embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that it is not intended 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.

[0079] One skilled in the art will recognize 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.

[0080] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

[0081] All patents and publications referenced herein are incorporated by reference in their entirety.

[0082] The present invention aims to address one or more of the defects of the prior art, and is based on the inventors' recognition that specific antigen recognition domain provides improved anti-nfP2X7 receptor CAR function.More specifically, it is understood that the preferred CAR of the present invention provides higher affinity for binding to cancer cells that express nfP2X7 receptor, and therefore has stronger cell-killing ability (efficacy).The inventors have found that certain single-domain (sdAb-based) CAR and certain scFv-based CAR provide superior efficacy compared with the sdAb-based CAR of the prior art.

[0083] Definition - General Unless otherwise defined, 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.

[0084] For purposes of interpreting this specification, the following definitions shall generally apply and, whenever appropriate, terms used in the singular shall also include the plural and vice versa.

[0085] Throughout this specification, unless specifically stated otherwise or unless the context requires otherwise, a reference to a single step, composition of matter, group of steps, or group of compositions of matter is intended to include one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps, or group 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, a reference to "a" includes two or more and not just the singular, a reference to "an" includes two or more and not just the singular, a reference to "the" includes two or more and not just the singular, etc.

[0086] Those skilled in the art will appreciate that the present invention may be subject to variations and modifications other than those specifically described. The present invention is to be understood to include all such variations and modifications. The present invention also includes all steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, and any and all combinations of such steps or features, or any two or more thereof.

[0087] Any example or embodiment of the invention herein shall apply mutatis mutandis to any other example or embodiment of the invention unless specifically stated otherwise.

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0089] 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 in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M.G. Lover and B.D.H. Memes (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M.A. Usubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed. Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988), and J.E. Coligan et al. al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date), and other sources.

[0090] The term "and / or," e.g., "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be interpreted as providing explicit support for both meanings, or for either meaning.

[0091] As used herein, the term "derived from" should be construed to indicate that a specified element may be obtained from a particular source, but not necessarily directly from that source.

[0092] When comparing amino acid sequences, the sequences should be compared over a comparison window determined by the length of the polypeptide. The comparison window may include no more than about 20% additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning the comparison window can be performed by computerized implementations of algorithms such as the BLAST program family disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389-3402. Global alignment programs can also be used to align similar sequences of approximately 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 optimal 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).

[0093] "Purinergic receptor" generally refers to a receptor that uses a purine (such as ATP) as a ligand.

[0094] "P2X7 receptor" generally refers to a purinergic receptor formed from three protein subunits or monomers, at least one of which has the amino acid sequence substantially as set forth in SEQ ID NO: 141 below.

[0095] SEQ ID NO: 141 MPACCSCSDVFQYETNKVTRIQSMNYGTIKWFFHVIIFSYVCFALVSDKLYQRKEPVISSVHTKVKGIAEVKEEIVENGVKKLVHSVFDTADYTFPLQGNSFFVMTNFLKTEGQEQRLCPEYPTRRTLCSSDRGCKKGWMDPQSKGIQ TGRCVVYEGNQKTCEVSAWCPIEAVEEAPRPALLNSAENFTVLIKNNIDFPGHNYTTRNILPGLNITCTFHKTQNPQCPIFRLGDIFRETGDNFSDVAIQGGIMGIEIYWDCNLDRWFHHCRPKYSFRRLDDKTTNVSLYPGYNFRYAK YYKENNVEKRTLIKVFGIRFDILVFGTGKFDIIQLVVYIGSTLSYFGLAAVFIDFLIDTYSSNCCRSHIYPWCKCCQPCVVNEYYYRKKCESIVEPKPTLKYVSFVDESHIRMVNQQLLGRSLQDVKGQEVPRPAMDFTDLSRLPLAL HDTPPIPGQPEEIQLLRKEATPRSRDSPVWCQCGSCLPSQLPESHRCLEELCCRKKPGACITTSELFRKLVLSRHVLQFLLLYQEPLLALDVDSTNSRLRHCAYRCYATWRFGSQDMADFAILPSCCRWRIRKEFPKSEGQYSGFKSPY

[0096] Insofar as the P2X7 receptor is formed from three monomers, it is a "trimer" or "trimeric." "P2X7 receptor" encompasses naturally occurring variants of the P2X7 receptor, for example, P2X7 monomers are isoforms (e.g., forms consisting of the extracellular domain sequence or truncated forms thereof), naturally occurring variant forms (e.g., alternatively spliced ​​forms), and naturally occurring allelic variants, including splice variants, allelic variants, SNPs, and naturally occurring truncated or secreted forms of the monomers that form the P2X7 receptor. In certain embodiments of the present invention, the native sequence P2X7 monomer polypeptide disclosed herein is a mature or full-length native sequence polypeptide comprising the full-length amino acid sequence set forth in SEQ ID NO: 141. In certain embodiments, the P2X7 receptor may have a modified amino acid sequence, for example, various amino acids in the sequence set forth in SEQ ID NO: 141 may be substituted, deleted, or residues may be inserted.

[0097] "Functional P2X7 receptor" generally refers to the form of P2X7 receptor that has three intact binding sites or clefts for binding to ATP.When bound to ATP, functional receptor forms a non-selective sodium / calcium channel that converts into a pore-like structure that allows calcium ions and molecules up to 900 Da to enter the cell matrix, one of the consequences of which may be the induction of programmed cell death.In normal homeostasis, the expression of functional P2X7 receptor is generally limited to cells undergoing programmed cell death, such as thymocytes, dendritic cells, lymphocytes, macrophages, and monocytes.In addition, there may be some expression of functional P2X7 receptor on erythrocytes and other cell types.

[0098] "Dysfunctional P2X7 receptor" (also called "non-functional" or (nf)P2X7) is a P2X7 receptor that has an impaired response to ATP, such that it cannot form an apoptotic pore under normal physiological conditions. Dysfunctional P2X7 receptor (or nfP2X7 receptor) generally refers to a form of P2X7 receptor that has a different conformation from functional P2X7, whereby the receptor cannot form an apoptotic pore, but can still act as a non-selective channel by maintaining a single functional ATP-binding site located between adjacent monomers. One example occurs when one or more of the monomers have cis-isomerization at Pro210 (according to the sequence set forth in SEQ ID NO: 141). Isomerization can result from any molecular event that leads to misfolding of the monomer, including, for example, mutation of the monomer primary sequence or abnormal post-translational processing. One consequence of this isomerization is that the receptor cannot bind ATP at one, or more specifically, two, of the ATP-binding sites on the trimer, and as a result, cannot extend the opening of the channel. In such a situation, receptor cannot form pore, which limits the degree that calcium ions can enter into the cytosol.Dysfunctional P2X7 receptor is expressed on a wide range of epithelial cancers and hematopoietic cancers.As used herein, term " dysfunctional P2X7 receptor " can be used interchangeably with term " non-functional P2X7 receptor " or " nfP2X7 " receptor.

[0099] A "cancer-associated P2X7 receptor" is a P2X7 receptor that is generally found on cancer cells (including pre-neoplastic cells, neoplastic cells, malignant cells, benign cells, or metastatic cells) but not on non-cancerous or normal cells.

[0100] "E200 epitope" generally refers to an epitope having the sequence GHNYTTNILPGLNITC (SEQ ID NO: 95) and variants thereof (e.g., SEQ ID NOs: 96-119). The antigen recognition domain of the CAR of the present invention is preferably capable of binding to E200 or a variant thereof.

[0101] "E300 epitope" generally refers to the epitope having the sequence KYYKENNVEKRTLIK and variants thereof (e.g., SEQ ID NOs: 120 and 121).

[0102] "Composite epitope" generally refers to an epitope formed from the juxtaposition of the E200 epitope and the E300 epitope, or portions of these epitopes. An example of a composite epitope comprising the E200 epitope and the E300 epitope is GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 122).

[0103] As used herein, the term "chimeric antigen receptor" or CAR refers to a protein artificially constructed for expression on the surface of immune cells, which comprises an extracellular domain (portion outside the cell) that includes 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 include a signal peptide.

[0104] With respect to the antigen-binding domain of a CAR, the terms "binds to," "specifically binds to," or "specifically for" refer to a domain that recognizes and binds to a specific antigen and does not substantially recognize or bind to other molecules in a sample. An antigen-binding domain that specifically binds to an antigen from one species may also bind to that antigen from another species. This cross-species reactivity is typical of many antibodies and, therefore, does not violate the definition of an antigen-binding domain being specific. An antigen-binding domain that specifically binds to an antigen may also bind to different allelic forms of the antigen (such as 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, does not violate the definition of an antigen-binding domain being specific.

[0105] As used herein, the terms "engineered cells" and "genetically modified cells" can be used interchangeably. These terms refer to the presence and / or expression of exogenous genes or nucleic acid sequences that, in turn, modify the genotype or phenotype of the cell or its progeny. The terms refer to the fact that cells, preferentially immune cells, can be engineered by recombinant methods well known in the art to stably or transiently express peptides or proteins not naturally expressed in these cells. For example, immune cells are engineered to express artificial constructs such as chimeric antigen receptors on their cell surface. For example, a nucleic acid sequence encoding a CAR can be delivered into cells using adenovirus, adeno-associated virus (AAV)-based, retrovirus, or lentivirus vectors, or any other pseudotyped variants thereof, or any other gene delivery mechanism such as electroporation or lipofection with CRISPR / Cas9, transposons (e.g., Sleeping Beauty), or variants thereof. Gene delivery can be in the form of mRNA (transient) or DNA (transient or permanent).

[0106] The term "immune cell" or "immune effector cell" refers to a cell that can be part of the immune system, either the adaptive (i.e., cellular or humoral) or innate immune system, and that performs a specific effector function, e.g., alpha-beta T cells, NK cells, NKT cells, B cells, Breg cells, Treg cells, innate lymphoid cells (ILCs), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, gamma-delta T cells, mesenchymal stem cells or mesenchymal stromal cells (MSCs), monocytes or macrophages, or any hematopoietic progenitor cell, such as an early progenitor cell subset that can mature or differentiate into pluripotent stem cells and somatic cells. The cells can be naturally occurring or generated by cytokine exposure, artificial / genetically modified cells (iPSCs and other artificial cell types). Immune cells may also be artificial cell subsets, including induced pluripotent stem cells and cells matured therefrom. Preferred immune cells are cells with cytotoxic effector function, such as α-β T cells, NK cells, NKT cells, ILCs, CIK cells, LAK cells, or γ-delta T cells. "Effector function" refers to a specific function of a cell; for example, in T cells, effector function can be cytolytic activity or helper cell activity, including secretion of cytokines.

[0107] As used herein, the term "treating a disorder" (treatment of a disorder) means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0108] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter within a cell.

[0109] An "antibody" or "immunoglobulin" or "Ig" is a gamma globulin protein found in the blood or other body fluids of vertebrates that functions in the immune system to bind to antigens, thus recognizing and / or neutralizing foreign substances.

[0110] Antibodies are generally heterotetrameric glycoproteins consisting of two identical light (L) chains and two identical heavy (H) chains. Each L chain is linked to an H chain by one covalent disulfide bond. Depending on the H chain isotype, the two H chains are linked to each other by one or more disulfide bonds. Each H and L chain also has regularly spaced intrachain disulfide bridges.

[0111] The heavy and light chains define specific Ig domains. More specifically, each heavy chain contains at its N-terminus a variable domain (V H ), followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has at its N-terminus a variable domain (V L ), followed at the other end by a constant domain (CL). L is V H Align with C L is aligned with the first constant domain of the heavy chain (CH1).

[0112] Antibodies can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains.

[0113] The constant domain comprises the Fc portion, which comprises the carboxy-terminal portions of both H chains held together by disulfides. Antibody effector functions, such as ADCC, are determined by sequences within the Fc region, which is also the region recognized by Fc receptors (FcRs) found on certain types of cells.

[0114] V H and V L The pairing of these together forms the "variable region" or "variable domain" which comprises the amino-terminal domain of the heavy or light chain of an antibody. The variable domain of the heavy chain is called the "V H The variable domain of the light chain is sometimes referred to as "V L The V domain contains the "antigen-binding site" that influences antigen binding and defines the specificity of a particular antibody for that particular antigen. The V region spans approximately 110 amino acid residues and consists of relatively invariant stretches called framework regions (FRs) (generally about 4 amino acids) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" (generally about 3 amino acids), each generally 9-12 amino acids long. The FRs adopt a primarily β-sheet configuration, and the hypervariable regions form loops that connect, and in some cases form part of, the β-sheet structure.

[0115] "Hypervariable region" refers to the region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops. Generally, antibodies contain six hypervariable regions (also called complementarity-determining regions, or CDRs), three of which are V H (H1, H2, H3), and three are V L (L1, L2, L3).

[0116] The complementarity determining region sequences (CDRs) of an antigen binding protein can be defined according to any one of several different numbering systems, including the IMGT numbering system, Kabat, or Chothia systems. Given the complete variable heavy and light sequences, it is well within the ability of one of ordinary skill in the art to be able to identify and determine the CDRs.

[0117] The descriptions and definitions of variable regions and portions thereof, immunoglobulins, antibodies, and fragments thereof herein may be further clarified by a 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 Al-Lazikani et al., J. Mol. Biol. 273, 927-948, 1997.

[0118] As used herein, the term "complementarity determining region" (synonym, CDR, i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues in an antibody variable region, the presence of which primarily contributes to specific antigen binding. Each variable region domain (V H or V L ) typically have three CDRs identified as CDR1, CDR2, and CDR3. H The CDRs of are also referred to herein as CDR H1, CDR H2, and CDR H3, respectively, and CDR H1 is H CDR1 corresponds to V, and CDR H2 corresponds to V H CDR2 corresponds to V, and CDR H3 corresponds to V H Similarly, V L The CDRs of V are referred to herein as CDR L1, CDR L2, and CDR L3, respectively, and CDR L1 is L CDR1 corresponds to V, and CDR L2 corresponds to V L CDR2 corresponds to V LIn one example, the amino acid positions assigned to the CDRs and FRs are defined according to the Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as the "Kabat numbering system"). In another example, the amino acid positions assigned to the 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 defined by the Kabat numbering system, but encompasses all numbering systems, including the standard numbering systems, or the numbering systems 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. 25:206-211, 1997.

[0119] "Framework regions" (FR) are variable region residues other than the CDR residues. The FRs of VH are also referred to herein as FR H1, FR H2, FR H3, and FR H4, respectively; FR H1 is the V H FR1 corresponds to V, FR H2 corresponds to V H FR2 corresponds to V, FR H3 corresponds to V H FR3 corresponds to V, FR H4 corresponds to V H Similarly, V L The FRs are referred to herein as FR L1, FR L2, FR L3, and FR L4, respectively, and FR L1 is V L FR1 corresponds to V, and FR L2 corresponds to V.L FR2 corresponds to V, and FR L3 corresponds to V. L FR3 corresponds to V, FR L4 corresponds to V L Compatible with FR4.

[0120] An "antigen-binding site" generally refers to a molecule comprising at least the hypervariable and framework regions required to confer antigen-binding function to a V domain. The antigen-binding site, in the methods described herein, may be in the form of an antibody or antibody fragment (such as a mAb, single-domain (SD)-mAb, dAb, Fab, SD-Fab, Fd, SD-Fv, Fv, F(ab')2, or scFv).

[0121] An "intact" or "whole" antibody comprises an antigen-binding site as well as a C L 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 variants thereof.

[0122] "Whole antibody fragments containing the variable domain" include SD-mAb, Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0123] "Fab fragment" is a fragment of the variable region domain of the heavy chain (V H ), and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.

[0124] "Fab' fragments" differ 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.

[0125] The "F(ab')2 fragment" roughly corresponds to two disulfide-linked Fab fragments that have divalent antigen-binding activity and are still capable of cross-linking antigen.

[0126] "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and binding site. This fragment consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association.

[0127] In single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a "dimeric" structure similar to a two-chain Fv species. The folding of these two domains results in six hypervariable loops (three loops each from the H and L chains) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody.

[0128] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is a Fv consisting of V chains joined to form a single polypeptide chain. H and V L Preferably, the scFv polypeptide is an antibody fragment containing an antibody domain. Preferably, the scFv polypeptide contains a V domain that enables the scFv to form the desired structure for antigen binding. H Domains and V L It further comprises a polypeptide linker between the domains.

[0129] A "single variable domain" is one half of an Fv (comprising only three CDRs specific for an antigen) that has the ability to recognize and bind to antigen, although generally with lower affinity than the entire binding site.

[0130] "Diabody" refers to an antibody fragment with two antigen-binding sites, which are bound to the same polypeptide chain (V H -V L ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ) Small antibody fragments include V HDomains and V L These fragments are prepared by constructing sFv fragments with a short linker (about 5-10 residues) between the V domains (see previous paragraph), which allows for inter-chain but not intra-chain pairing of the V domains and results in bivalent fragments, i.e., fragments with two antigen-binding sites.

[0131] Diabodies may be bivalent or bispecific. Bispecific diabodies are diabodies that combine the V H and V L Triabodies and tetrabodies, which are heterodimers of two "crossover" sFv fragments in which the domains are present on different polypeptide chains, are also commonly known in the art.

[0132] An "isolated antibody" is one that has been identified and separated and / or recovered from components of its original environment. Contaminating components are materials that would interfere with therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0133] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Human antibodies can be prepared by administering an antigen to transgenic animals that have been modified to produce such antibodies in response to antigen challenge, but whose endogenous gene loci have been disabled.

[0134] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) 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 capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. In general, humanized antibodies 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. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0135] A "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 an antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. Monoclonal antibodies may be prepared by the hybridoma method. "Monoclonal antibodies" may also be isolated from phage antibody libraries using molecular engineering techniques.

[0136] "Binding affinity" generally refers to the strength of the sum 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 otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an 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 antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigens more rapidly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which may be used for purposes of the present disclosure.

[0137] As used herein, the term "antigen" is intended to include a substance that binds to or induces the production of one or more antibodies, and may include, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates, and combinations thereof, such as glycosylated proteins or glycolipids. As used herein, the term "antigen" refers to a molecular entity that can be expressed on a target cell and recognized by the adaptive immune system, including, but not limited to, antibodies or TCRs, or engineered molecules, including, but not limited 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 achieve high affinity binding to a structure.

[0138] "Epitope" generally refers to the portion of an antigen 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 the primary protein structure. In certain embodiments, the epitope is a "conformational epitope," i.e., an epitope in which the hypervariable loops of the CDRs bind to residues as they appear in the tertiary or quaternary protein structure.

[0139] As used herein, the term "target cell" refers to a cell that expresses the nfP2X7 receptor. The target cell may be a cancer cell or any other diseased cell.

[0140] The term "disorder" or "condition" means a functional abnormality or malfunction in a subject, such as cancer, an autoimmune disorder, or a viral, bacterial, parasitic, or other infection.

[0141] For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may also exist in a non-native environment, such as, for example, a host cell.

[0142] As used herein, the term "autologous" refers to any material originating from the same subject into which it is subsequently reintroduced.

[0143] As used herein, the term "allogeneic" refers to any material derived from a different subject of the same species as the subject into which the material is being reintroduced.

[0144] The term "therapeutically effective amount" or "therapeutically effective population" refers, for example, to the amount of a cell population that provides a therapeutic benefit in a subject.

[0145] Chimeric Antigen Receptor Generally, "antigen-binding domain" or "antigen recognition domain" refers to the region of a CAR that specifically binds to an antigen (and thus can target cells containing the antigen). The CAR of the present invention may comprise one or more antigen-binding domains, at least one of which is for binding to the nfP2X7 receptor. The antigen-binding domain may comprise an antibody or an antibody-binding fragment thereof. The antigen-binding domain may comprise, for example, a full-length heavy chain, a Fab fragment, a single-chain Fv (scFv) fragment, a bivalent single-chain antibody, or a bispecific antibody. Any molecule that specifically binds to a given antigen, such as an affibody or a ligand-binding domain from a naturally occurring receptor, may be used as the antigen-binding domain. In many cases, the antigen-binding domain is an scFv. Typically, in an scFv, the variable regions of immunoglobulin heavy and light chains are fused by a flexible linker to form the scFv. Such a linker may be, for example, a "(G4 / S1)3-linker" and variations thereof, although those skilled in the art will understand that various linker sequences and formats may be used.

[0146] In some cases, it is beneficial for the antigen-binding domain to be derived from the same species as the CAR will be used in. For example, if it is planned to be used therapeutically in humans, it may be beneficial for the antigen-binding domain of the CAR to comprise a human or humanized antibody or its antigen-binding fragment. Human or humanized antibodies or their antigen-binding fragments can be produced by various methods well known in the art.

[0147] "Signal peptide" refers to a peptide sequence that directs the transport and localization of a protein within a cell, for example, to a specific organelle (such as the endothelial endoplasmic reticulum) and / or to the cell surface.

[0148] As used herein, "spacer" or "hinge" refers to a hydrophilic region located between the antigen-binding domain and the transmembrane domain. The CAR of the present invention may include an extracellular spacer domain, but may also exclude such a spacer. The spacer may include, for example, an Fc fragment or fragment thereof of an antibody, a hinge region or fragment thereof of an antibody, a CH2 or CH3 region of an antibody, an accessory protein, an artificial spacer sequence, or a combination thereof. A notable example of a spacer is the CD8 alpha hinge.

[0149] The transmembrane domain of a CAR can be derived from any desired natural or synthetic source for such a domain. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane domain can be derived from, for example, CD8α or CD28. If the main signaling and antigen recognition modules (domains) are located on two (or more) polypeptides, the CAR can have two (or more) transmembrane domains. The division of the main signaling and antigen recognition modules allows for small molecule-dependent, titratable, and reversible control of CAR cell expression due to the small molecule-dependent heterodimerization domains in each polypeptide of the CAR (Wu et al., 2015, Science 350:293-303).

[0150] The cytoplasmic domain (or intracellular signaling domain) of a CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. "Effector function" refers to a specific function of a cell; for example, in T cells, effector function can be cytolytic activity or helper cell activity, including secretion of cytokines. The intracellular signaling domain refers to the portion of a protein that transmits an effector function signal and instructs the cell expressing the CAR to perform a specific function. The intracellular signaling domain can include any complete, mutated, or truncated portion of the intracellular signaling domain of a given protein sufficient to transduce a signal that initiates or blocks immune cell effector function.

[0151] The signaling domain of a CAR can be any suitable domain that can induce or participate in the induction of an intracellular signaling cascade upon activation of the CAR as a result of antigen recognition by the antigen recognition domain of the CAR. The signaling domain of a CAR is specifically selected depending on the cellular outcome desired after CAR activation. Although there are many possible signaling domains, when used in immunotherapy and cancer therapy, signaling domains can be classified into two general categories based on the receptors from which they are derived: activating receptors and costimulatory receptors. Thus, in some embodiments, the signaling domain of a CAR comprises a portion derived from an activating receptor. In some embodiments, the signaling domain comprises a portion derived from a costimulatory receptor.

[0152] As used throughout this specification, the term "activating receptor" relates to a receptor or co-receptor that forms a component of or participates in the formation of a T cell receptor (TCR) complex, or receptor that is involved in the specific activation of an immune cell as a result of recognition of an antigen or other immunogenic stimulus.

[0153] The function of the intracellular domain may be pro-inflammatory or anti-inflammatory and / or immunomodulatory, or a combination thereof.

[0154] Some examples of intracellular signaling domains for use in CARs include the cytoplasmic signaling sequence of the T cell receptor (TCR) and co-receptors that initiate signal transduction after antigen receptor binding.

[0155] Generally, T cell activation can be mediated by two different classes of cytoplasmic signaling sequences: first, those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and second, those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences, costimulatory signaling domains). Thus, the intracellular signaling domain of a CAR can comprise one or more primary cytoplasmic signaling domains and / or one or more secondary cytoplasmic signaling domains.

[0156] Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain ITAM (immunoreceptor tyrosine-based activation motif) signaling motifs.

[0157] Examples of ITAMs containing primary cytoplasmic signaling sequences commonly 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 notably, the sequence derived from CD3 zeta.

[0158] The cytoplasmic domain of the CAR can be designed to contain a CD3-zeta signaling domain, either alone or in combination with any desired cytoplasmic domain. The cytoplasmic domain of the CAR can include a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to the portion of the CAR that contains the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is necessary for efficient and sufficient lymphocyte response to antigens. Examples of costimulatory molecules are CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0159] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other in a random or specified order, with or without a linker. A short oligo- or polypeptide linker, preferably 2-10 amino acids in length, can form the linkage. A prominent linker is a glycine-serine duplex.

[0160] As another example, the cytoplasmic domain may comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another example, the cytoplasmic domain may comprise the signaling domain of CD3-zeta and the signaling domain of CD27. In a further example, the cytoplasmic domain may comprise the signaling domain of CD3-zeta, the signaling domain of CD28, and the signaling domain of CD27.

[0161] Either the extracellular portion of the CAR, or the transmembrane domain, or the cytoplasmic domain may also contain a heterodimerization domain for the purpose of separating the main signaling and antigen recognition modules of the CAR.

[0162] CARs for use in accordance with the present invention, i.e., CARs comprising an nfP2X7E200 binding domain, may be designed to include any portion or parts of the above-mentioned domains described herein in any order and / or combination that results in a functional CAR.

[0163] A CAR disclosed herein, or a polypeptide derived therefrom, a nucleic acid molecule encoding the CAR, or a recombinant expression vector cell, or a population of cells expressing the CAR, can be isolated and / or purified. The term "isolated" means changed or removed from its natural state. For example, an isolated population of cells refers to the enrichment of such cells and their separation from other cells that normally associate with the isolated cells in their natural state. An isolated population of cells refers to a substantially purified population of cells, which is a more homogeneous population of cells than that found in nature. Preferably, an enriched cell population contains at least about 90% of a selected cell type. In certain embodiments, a cell population contains at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% of a selected cell type.

[0164] The affinity with which the antigen recognition domain of a CAR binds to the nfP2X7 recognition site E200 on a cell (or the E200 sequence contained in a "bridging molecule" as described elsewhere herein) can vary, but generally the binding affinity can be in the range of 100 μM, 1 nM, 10 nM, or 100 nM, preferably at least about 1 pM or 10 pM, and even more preferably at least about 100 pM.

[0165] Linker The linker can be a peptide having a length of up to 20 amino acids. The terms "linked to" or "fused to" refer to a covalent bond, e.g., a peptide bond, formed between two moieties. Thus, in the context of the present invention, a linker can have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids. For example, the CAR provided herein may include a linker between the VH and VL of the antigen-binding domain and / or a linker between the antigen-binding domain and the hinge. Such linkers have the advantage that they can increase the likelihood that different polypeptides of the fusion protein will fold independently and behave predictably.

[0166] Those skilled in the art will be familiar with the design and use of various peptide linkers composed of various amino acids and of various lengths suitable for use as linkers in accordance with the present invention. Linkers may contain various combinations of repeated amino acid sequences. Linkers may be flexible linkers (e.g., those containing repeats of glycine and serine residues), rigid linkers (e.g., those containing glutamic acid and lysine residues, adjacent alanine repeats), and / or cleavable linkers (e.g., sequences susceptible to protease cleavage).

[0167] 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 GGGGSGGGSGGGGGS, i.e., (GS) (SEQ ID NO: 93).

[0168] In any embodiment, the peptide linker can comprise the amino acid sequence GGGGGS (a linker six amino acids in length, SEQ ID NO: 19), or even longer. The linker can 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 can be (GS)3 (i.e., GSGSGS) (SEQ ID NO: 20) or longer. 11 (SEQ ID NO: 142) or longer. It will be understood that n can be any number, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more.

[0169] nucleic acid In another aspect, the present invention provides a nucleic acid molecule, such as a recombinant nucleic acid molecule, comprising a nucleotide sequence encoding a chimeric antigen receptor according to the present invention.

[0170] In some embodiments of the invention, a nucleic acid molecule comprises a nucleotide sequence that encodes the amino acid sequence set forth in SEQ ID NO: 4 and / or 12, or encodes a functional variant thereof, such as SEQ ID NO: 94, wherein the functional variant retains the ability to bind to the nfP2X7 receptor. In some embodiments, the nucleic acid molecule encodes a functional variant comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 94 and that retains the ability to bind to the nfP2X7 receptor. In some embodiments, the nucleic acid molecule encodes a functional variant comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 94, that retains the ability to bind to nfP2X7, and that comprises an antigen recognition domain comprising the amino acid sequence of SEQ ID NO: 4 and 12.

[0171] Nucleic acid molecules can contain any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified or modified, RNA or DNA. For example, nucleic acid molecules can include single-stranded 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 a mixture of single- and double-stranded regions, and hybrid molecules containing DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, nucleic acid molecules can contain triple-stranded regions containing RNA or DNA, or both RNA and DNA. Nucleic acid molecules can also contain one or more modified bases or DNA or RNA backbones modified for stability or other reasons. Because various modifications can be made to DNA and RNA, the term "nucleic acid molecule" encompasses chemically, enzymatically, or metabolically modified forms.

[0172] In some embodiments of the invention, the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:74 and / or SEQ ID NO:82.

[0173] Those skilled in the art will understand that any nucleotide sequence encoding a chimeric antigen receptor comprising the amino acid sequence set forth in SEQ ID NO:4 and / or 12, e.g., SEQ ID NO:94, or a functional variant thereof, is contemplated by the present invention. For example, variants of SEQ ID NO:94 that contain one or more nucleic acids that differ from SEQ ID NO:74 and / or 82 but still encode the same amino acid sequence are contemplated. Due to the degeneracy of the genetic code, a large number of nucleic acids can encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, all nucleotide sequences herein encoding a chimeric antigen receptor having the amino acid sequence set forth in SEQ ID NO:94, or a functional variant thereof, also represent all possible silent variations of the nucleotide sequence. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to result in a functionally identical molecule. Thus, each silent variation of a nucleotide sequence encoding a polypeptide is implicit in each described sequence.

[0174] In another aspect, the present invention provides a nucleic acid construct comprising a nucleic acid molecule according to the present 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 active in one or more hosts, and / or one or more transcription control sequences.

[0175] As used herein, the term "selectable marker gene" includes any gene that confers a phenotype on cells in which it is expressed, to facilitate the identification and / or selection of cells that have been transfected or transformed with the construct.

[0176] A "selectable marker gene" includes any nucleotide sequence that, when expressed by cells transformed with a construct, confers a phenotype on the cells that facilitates identification and / or selection of those transformed cells. A series of nucleotide sequences encoding suitable selectable markers are known in the art (e.g., Mortesen, RM. and Kingston RE. Curr Protoc Mol Biol, 2009; Unit 9.5). Exemplary nucleotide sequences encoding selectable markers include the following: adenosine deaminase (ADA) gene; cytosine deaminase (CDA) gene; dihydrofolate reductase (DHFR) gene; histidinol dehydrogenase (hisD) gene; puromycin-N-acetyltransferase (PAC) gene; thymidine kinase (TK) gene; xanthine-guanine phosphoribosyltransferase (XGPRT) gene, or antibiotic resistance genes such as ampicillin resistance gene, puromycin resistance gene, bleomycin resistance gene, hygromycin resistance gene, kanamycin resistance gene, and ampicillin resistance gene; fluorescent reporter genes such as genes encoding green, red, yellow, or blue fluorescent proteins; and luminescence-based reporter genes such as luciferase genes, particularly those that allow optical selection of cells using techniques such as fluorescence-activated cell sorting (FACS).

[0177] Furthermore, it should be noted that the selectable marker gene can be a separate open reading frame in the construct, or can be expressed as a fusion protein with another polypeptide (e.g., CAR).

[0178] As described above, the nucleic acid construct may also contain one or more transcription control sequences. The term "transcription control sequence" should be understood to include any nucleic acid sequence that affects the transcription of an operably connected nucleic acid. Transcription control sequences may include, for example, a leader, a polyadenylation sequence, a promoter, an enhancer, or an upstream activation sequence, and a transcription terminator. Typically, a transcription control sequence includes at least a promoter. As used herein, the term "promoter" refers to any nucleic acid that confers, activates, or enhances the expression of a nucleic acid in a cell.

[0179] In some embodiments, at least one transcription control sequence is operably linked to a nucleic acid molecule of the present invention. For purposes of this specification, a transcription control sequence is considered to be "operably linked" to a given nucleic acid molecule if the transcription control sequence is capable of promoting, inhibiting, or otherwise regulating the transcription of the nucleic acid molecule. Thus, 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.

[0180] A "nucleic acid construct" can be in any suitable form, such as in the form of a plasmid, phage, transposon, cosmid, chromosome, vector, etc., which, when associated with the appropriate control elements, is capable of replicating and transferring the genetic 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.

[0181] A promoter can constitutively or differentially regulate expression of an operably linked nucleic acid molecule with respect to the cell, tissue, or organ in which expression occurs. Thus, a promoter can 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 particular environmental or physiological conditions. The present invention contemplates the use of any promoter that is active in the cell of interest. As such, a wide range of promoters will be readily identified by one of ordinary skill in the art.

[0182] Mammalian constitutive promoters may include, but are not limited to, Simian Virus 40 (SV40), cytomegalovirus (CMV), P-actin, ubiquitin C (UBC), elongation factor-1 alpha (EF1A), phosphoglycerate kinase (PGK), and CMV early enhancer / chicken beta actin (CAGG).

[0183] Inducible promoters may include, but are not limited to, chemically inducible promoters and physically inducible promoters. Chemically inducible promoters include promoters whose activity is regulated by chemical compounds such as alcohol, antibiotics, steroids, metal ions, or other compounds. Examples of chemically inducible promoters include steroid-responsive promoters such as tetracycline-regulated promoters (see, e.g., U.S. Pat. Nos. 5,851,796 and 5,464,758), glucocorticoid receptor promoters (see, e.g., U.S. Pat. No. 5,512,483), and ecdysone receptor promoters (see, e.g., U.S. Pat. No. 6,379,945), and metal-responsive promoters such as metallothionein promoters (see, e.g., U.S. Pat. Nos. 4,940,661, 4,579,821, and 4,601,978), among others.

[0184] As mentioned above, the control sequence may also include a terminator. The term "terminator" refers to a DNA sequence at the end of a transcription unit that signals the termination of transcription. A terminator is generally a 3' untranslated DNA sequence containing a polyadenylation signal, which promotes the addition of a polyadenylation sequence to the 3' end of a primary transcript. Similar to a promoter sequence, a terminator may be any terminator sequence that is operable in the cell, tissue, or organ in which it is intended to be used. Suitable terminators are known to those skilled in the art.

[0185] As will be appreciated, the nucleic acid constructs of the present invention can further comprise additional sequences, such as sequences that allow for enhanced expression, cytoplasmic or membrane transport, and location signals. Specific, non-limiting examples include internal ribosome entry sites (IRES).

[0186] The present invention extends to essentially all genetic constructs as described herein, which may further comprise nucleotide sequences intended for the maintenance and / or replication of the genetic construct in eukaryotic organisms and / or for the integration of the genetic construct, or parts thereof, into the genome of eukaryotic cells.

[0187] Methods for intentionally introducing (transfecting / transducing) exogenous genetic material, such as the nucleic acid constructs of the present invention, into eukaryotic cells are known in the art. As will be appreciated, the most suitable method for introducing a nucleic acid construct into a desired host cell will depend on many factors, such as the size of the nucleic acid construct, the type of host cell, the desired rate of transfection / transduction efficiency, and the ultimate desired or required viability of the transfected / transduced cells. Non-limiting examples of such methods include chemical transfection using chemicals such as cationic polymers, calcium phosphate, or structures such as liposomes and dendrimers; non-chemical methods such as electroporation, sonoporation, heat shock, or phototransfection; particle-based methods such as "gene gun" delivery, magnetofection, or impalefection; or viral transduction.

[0188] The nucleic acid construct is 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 the host cell is viral transduction. Methods for utilizing viral transduction to induce expression of CAR in PBMCs (Parker, LL. et al. Hum Gene Ther. 2000;11:2377-87), and more generally, for utilizing 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) are known in the art. In other embodiments, the nucleic acid construct is a plasmid, cosmid, artificial chromosome, etc., and can be transfected into cells by any suitable method known in the art.

[0189] Genetically modified cells As described herein, in certain embodiments, the present invention includes therapeutic methods involving the use of cells expressing the chimeric antigen receptors (CARs) of the present invention.

[0190] The cells may be "engineered cells," "genetically modified cells," "immune cells," or "immune effector cells" as described herein. Furthermore, the cells may be capable of differentiating into immune cells. Cells capable of differentiating into immune cells (e.g., T cells expressing a dysfunctional P2X7CAR) may be stem cells, multilineage progenitor cells, or induced pluripotent stem cells.

[0191] In any embodiment, the cell may be a T cell, and optionally the T cell does not express TcRαβ, PD1, CD3, or CD96 (e.g., by knocking down or knocking out one of these genes at the genetic or functional level).

[0192] In any embodiment, the cell may be an immune cell, and optionally the cell does not express an accessory molecule, which may be a checkpoint, exhaustion, or apoptosis-related signaling receptor, and ligands such as PD-1, LAG-3, TIGIT, CTLA-4, FAS-L, and FAS-R (e.g., by knocking out one of these genes at the genetic or functional level).

[0193] In some embodiments, the genetically modified cells comprise two or more different CARs. For example, the genetically modified cells may comprise CARs with different architectures (e.g., different signaling domains) but the same antigen recognition sequence. Alternatively, the genetically modified cells may comprise CARs with different antigen recognition domains but binding to the nfP2X7 receptor. The CARs may bind to the same or different epitopes of the nfP2X7 receptor. In alternative embodiments, the genetically modified cells may comprise CARs for binding to different antigens (e.g., nfP2X7 receptor and different antigens on cancer cells).

[0194] In some embodiments of the invention, the genetically modified cell comprises a nucleic acid molecule or nucleic acid construct encoding two or more different CARs. In some embodiments of the invention, the genetically modified cell comprises two or more nucleic acid molecules or two or more nucleic acid constructs, each of which encodes a different CAR.

[0195] As referred to herein, a "genetically modified cell" includes any cell that contains a non-native 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 within the cell as a discreet DNA molecule or it may be integrated into the genomic DNA of the cell.

[0196] 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 the cell. Thus, genomic DNA of a cell should be understood to include chromosomal, mitochondrial DNA, etc. Thus, the term "genomically integrated" contemplates chromosomal integration, mitochondrial DNA integration, etc. The "genomically integrated form" of a construct may be all or a portion of the construct. However, in some embodiments, the genomically integrated form of a construct comprises at least a nucleic acid molecule of the second aspect of the invention.

[0197] As used herein, the term "different CARs" or "different chimeric antigen receptors" refers to any two or more CARs that have either non-identical antigen recognition and / or non-identical signal transduction domains.In one example, "different CARs" includes two CARs that have the same antigen recognition domain (e.g., both CARs can recognize dysfunctional P2X7 receptors), but have different signal transduction domains, such as one CAR with a signal transduction domain that has a part of an activating receptor and the other CAR with a signal transduction domain that has a part of a costimulatory receptor.As will be understood, at least one of the two or more CARs in this embodiment has an antigen recognition domain that recognizes dysfunctional P2X7 receptors, and the other CAR can take any suitable form and be directed against any suitable antigen.

[0198] Thus, in some embodiments of the invention, two or more different CARs have different signaling domains and may have the same or different antigen recognition domains. Specifically, a genetically modified cell of the invention may comprise a first chimeric antigen receptor having a signaling domain comprising a portion derived from an activating receptor, and a second chimeric antigen receptor having a signaling domain comprising a portion derived from a costimulatory receptor.

[0199] In some embodiments, the activating receptor (from which a portion of the signaling domain is derived) is a CD3 co-receptor complex or an Fc receptor.

[0200] In some embodiments, the costimulatory receptor (from which a portion of the signaling domain is derived) is selected from the group consisting of CD27, CD28, CD-30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS.

[0201] In some embodiments, the costimulatory receptor (from which a portion of the signaling domain is derived) is selected from the group consisting of CD28, OX40, or 4-1BB.

[0202] In some embodiments, the genetically modified cells are further modified to constitutively express a costimulatory receptor.

[0203] As mentioned above, cellular immune responses are typically induced only when an activating signal (typically in response to an antigen) and a costimulatory signal are simultaneously experienced. Therefore, by having genetically modified cells according to some of the above embodiments, which contain two or more CARs that provide both an intracellular activation signal and an intracellular costimulatory signal in combination, it is ensured that a sufficient immune response can be induced in response to the recognition of their cognate antigen by the CARs. Alternatively, the genetically modified cells may contain only one CAR that has an antigen recognition domain that recognizes a dysfunctional P2X7 receptor and can constitutively express a costimulatory receptor, thereby increasing the likelihood that costimulation is simultaneously provided when the CAR is activated. Alternatively, the genetically modified cells may be further modified to constitutively express both the costimulatory receptor and its / their ligand. In this way, the cells continuously experience costimulation, and only the activation of the CAR with a signaling domain that includes a portion from the activating receptor is required for immune activation of the cells.

[0204] Thus, in some embodiments, the genetically modified cells expressing the CAR are further modified to constitutively express a costimulatory receptor. In a further embodiment, the genetically modified cells are further modified to express a ligand for the costimulatory receptor, thereby promoting self-stimulation of the cells. Examples of CAR-expressing T cells that also express both costimulatory receptors and their cognate ligands (to induce self-stimulation) are known in the art, including, inter alia, those disclosed in Stephen MT. et al. Nat Med, 2007;13:1440-9.

[0205] The efficacy of genetically modified cells containing a CAR can be enhanced by further modifying the cells to secrete cytokines, preferably pro-inflammatory or pro-proliferative cytokines. This secretion of cytokines provides autocrine support to the CAR-expressing cells and alters the local environment surrounding the CAR-expressing cells such that other cells of the immune system are recruited and activated. Thus, in some embodiments of the fourth or fifth aspect of the invention, the genetically modified cells are further modified to secrete cytokines. This secretion may be constitutive or inducible upon recognition of the CAR by its cognate antigen of a ligand.

[0206] While any one or more cytokines can be selected depending on the desired immune response, preferred cytokines and / or chemokines include IL-2, IL-7, IL-12, IL-15, IL-17, IL-18, and IL-21, CCL19, CCL21, or combinations thereof.

[0207] The immune cells may be any suitable immune cell or progenitor thereof, or may be a homogeneous or heterogeneous population of cells. In some embodiments, the cells are leukocytes, peripheral blood mononuclear cells (PBMCs), lymphocytes, T cells, CD4+ T cells, CD8+ T cells, natural killer cells, natural killer T cells, or γδ T cells.

[0208] The immune cell may be a T cell, and optionally the T cell does not express TcRαβ, PD1, CD3, or CD96 (e.g., by knocking down or knocking out one of these genes at the genetic or functional level).

[0209] The immune cells do not express accessory molecules, which may be checkpoint, exhaustion, or apoptosis-related signaling receptors, and ligands such as PD-1, LAG-3, TIGIT, CTLA-4, FAS-L, and FAS-R (e.g., by knocking out or knocking down one of these genes at the genetic or functional level).

[0210] Methods of Treatment and Administration As discussed further herein, the present invention is applicable to the treatment of a variety of conditions, but preferably to the treatment of cancer.

[0211] The present invention also contemplates various scenarios for the use of the CARs of the present invention, including the administration of genetically modified immune cells that express a nucleic acid encoding a chimeric antigen receptor of the present invention, such that the cells present the CAR on their cell surface. Preferably, the genetically modified immune cells are for killing target cells.

[0212] In a preferred embodiment, the target cell is a cancer cell, and the cell surface molecule of the cancer cell is an antigen associated with the cancer. The antigen can be a tumor-specific antigen or a tumor-associated antigen. The antigen may be associated with a particular type of cancer. For example, overexpression of an antigen may be associated with a particular cancer or a particular class of cancer. For example, if the cancer is breast cancer, the antigen may be associated with breast cancer but not with other forms of cancer. Alternatively, the antigen may be associated with a class of cancer, such as solid tumors, but not with hematological (i.e., "liquid") tumors, or vice versa. The antigen may be associated with a particular lineage of cancer but not with other lineages of cancer. For example, the antigen may be associated with sarcomas but not with lymphomas or carcinomas. As used herein, the term "associated with" in reference to cancer will be understood to mean that expression of the antigen (whether increased or decreased) is considered a marker of the cancer. It will be understood that there may be low levels of antigen expression, but this is not synonymous with the antigen being "associated" with a given cancer.

[0213] In a preferred embodiment, the present invention provides a method for killing a target cell expressing an nfP2X7 receptor, the method comprising exposing a cell expressing an nfP2X7 receptor to a genetically modified cell of the present invention, thereby killing the target cell.

[0214] According to the preferred embodiment of the present invention, the antigen recognition domain of CAR of the present invention is for directly recognizing nfP2X7 receptor.As used herein, the term " directly recognize " includes the direct binding of the antigen recognition domain of CAR to nfP2X7 receptor or its epitope.In another non-limiting example, antigen recognition domain can directly bind to the processed form of nfP2X7 receptor, which can be presented by antigen-presenting molecules such as major histocompatibility complex (MHC).

[0215] It will be understood that in certain circumstances, CAR of the present invention can indirectly recognize nfP2X7 receptor or another antigen on target cell.In such an embodiment, CAR can bind to target cell via intermediate.Therefore, the present invention also provides a method for killing target cell, comprising exposing target cell to genetically modified cell of the present invention, thereby killing target cell.In some embodiments of the present invention, the method for killing target cell further comprises exposing target cell to intermediate.

[0216] The intermediate may be a molecule such as a probe, polypeptide, or fusion protein, which binds to CAR or directly interacts with CAR (for example, via the antigen-binding domain), and also binds to or directly interacts with an antigen on target cells.Non-limiting examples of such intermediates include peptides, antibodies or fragments thereof, antibody Fab, scFv, soluble engineered TCR, or aptamers.The antigen on target cells may comprise an nfP2X7 receptor, which comprises an epitope of the nfP2X7 receptor that is different from the epitope recognized by the antigen-recognition domain of CAR. The antigen on the target cell may be an antigen that is not a nfP2X7 receptor (e.g., but is 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), CD112 (Nectin2), CD117 (c-Kit), CD133, CD146 (MCAM), CD155 (PVR), CD171 (LI The antigens may include any other tumor-associated or tumor-specific antigens present on the surface of cancer cells, such as CD19, CD20, GD2, and EGFRvIII.

[0217] The CAR may be capable of directly recognizing the intermediate, or the intermediate may have a tag that is recognized by the CAR. In either case, the intermediate provides specificity to target cells, while the genetically modified cells with the CAR provide efficacy and direct immune responses to target cells. Examples of CARs that recognize cells through intermediates are known in the art, for example, in European Patent Application No. 2651442. Such intermediates can also be referred to in the art as "bridging molecules." Further examples of bridging molecules (also referred to as BRiDGE molecules) suitable for directing the nfP2X7 receptor-binding CAR or CAR of the present invention to target cells are described in WO2022 / 187906, which is incorporated herein by reference. Such bridging molecules typically comprise a polypeptide comprising an E200 sequence or a derivative thereof that can be recognized by the antigen recognition domain of the CAR of the present invention.

[0218] As used throughout this specification, the term "aptamer" refers to any oligonucleic acid, polynucleic acid, peptide, or polypeptide that specifically binds to or preferentially forms a complex with a target (specifically mesothelin).

[0219] In some embodiments of the present invention, the target cells are present in a subject. In some embodiments, the subject is a human. In some embodiments, the method further comprises exposing the target cells to genetic modification in conjunction with an exogenous cytokine.

[0220] In some embodiments of the present invention, the genetically modified cells are genetically modified cells that are autologous to the target cells from the subject.

[0221] In another aspect, the invention provides a method of treating or preventing cancer in a subject, the method comprising providing to the subject a genetically modified cell of the invention, thereby treating or preventing cancer. Optionally, the method further comprises exposing the target cell to an intermediate to allow binding of the genetically modified cell to the target cell.

[0222] As used herein, the terms "treat," "treating," or "treatment" should be understood to include within their scope one or more of the following outcomes. (i) inhibiting to some extent the growth of a primary tumor in a subject, including slowing or completely stopping growth, including reducing the growth of a primary tumor after resection; (ii) inhibiting to some extent the growth and formation of one or more secondary tumors in a subject; (iii) reducing the number of tumor cells in a subject; (iv) reducing the size of a tumor in a subject; (v) inhibiting (i.e., reducing, slowing, or completely stopping) tumor cell invasion into peripheral organs; (vi) inhibiting (i.e., reducing, slowing, or completely stopping) metastasis; (vii) improving the life expectancy of a subject compared to an untreated state; (viii) improving the quality of life of a subject compared to an untreated state; (ix) alleviating, reducing, 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 caused by cancer; and (xii) arresting a symptom in a subject that is associated with cancer.

[0223] As used herein, the terms "prevention" or "preventing" should be understood to include within their scope inhibiting the formation of a primary tumor in a subject, inhibiting the formation of one or more secondary tumors in a subject, or reducing or eliminating the recurrence of cancer in a subject in remission.

[0224] As used herein, the term "inhibiting" is intended to mean a decrease or reduction in the proliferation of cancer, cancerous cells, or tumors compared to proliferation in a control, such as an untreated cell or subject. In some embodiments, proliferation may be decreased or reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to an untreated control.

[0225] The inhibition of cancer, tumor or cancerous cell proliferation can be evaluated by a series of methods known in the art.For example, for in vitro cancerous cells, cell proliferation can be determined by a suitable proliferation assay or by evaluating the degree of tritiated thymidine incorporation into cellular DNA over a given period of time.For in vivo tumors or cancerous cells, tumor or cell proliferation can be determined, for example, by a suitable imaging method known in the art.

[0226] As used herein, the term "subject" refers to any animal that may be affected by cancer. Particular subjects of interest are humans, as well as 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 cows. In a preferred embodiment of the invention, the subject is a human.

[0227] Reference to "providing to a subject" relates to administering the genetically modified cells to a subject. Alternatively, the genetically modified cells can be generated within a subject. For example, the genetically modified cells can 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 the subject.

[0228] Providing genetically modified cells expressing a CAR to target cells that express or display the nfP2X7 receptor can be sufficient to provide effective immunotherapy for precancerous or cancerous cells, and providing an adjuvant together with the genetically modified cells can further enhance the induction of an immune response and augment the immunotherapy. Cytokines, preferably pro-inflammatory cytokines, are particularly suitable adjuvants for providing to a subject with genetically modified cells bearing a CAR.

[0229] Therefore, in some embodiments of the present invention, the genetically modified cells are administered to a subject together with a cytokine. As used throughout this specification, the term "together" should be understood to include genetically modified cells administered simultaneously with or in combination with a cytokine. Thus, when administered in combination with a cytokine, this can be considered to include a combination therapy in which the subject's immunotherapy includes both treatment with a cytokine and treatment with target cells that express or present the nfP2X7 receptor, or genetically modified cells with a CAR directed against its epitope. In some embodiments, the cytokine is administered on a different day (more than 24 hours) from the administration of the genetically modified cells. In other embodiments, the cytokine is administered on the same day (within 24 hours) as the genetically modified cells. In further embodiments, the cytokine and the genetically modified cells are administered within 18 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 2 minutes, or 1 minute of each other.

[0230] Suitable cytokines for administration with the genetically modified cells include IL-2, IL-4, IL-6, IL-7, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, IFNα, IFNβ, IFNγ, GM-CSF, TGFβ, and TNFα. Preferred cytokines include IL-7 and IL-15. Furthermore, cytokines may be administered in recombinant form, natural form, or via delivery systems such as fusions with proteins, expressed in the genetically modified cells, or delivered as nucleic acid sequences conjugated to polymers such as polyethylene glycol (PEG).

[0231] The cells to be genetically modified can be obtained from any suitable source.In some embodiments of the present invention, the cells to be genetically modified are autologous cells that are autologous to the cells that express or present nfP2X7 receptor, or its epitope.Advantageously, autologous cells will not be recognized as "non-self" by the subject's immune system, and therefore will 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 present invention, the cells to be genetically modified are allogeneic or xenogeneic cells.

[0232] Although this specification refers specifically to human application, it will be clearly understood that the invention is also useful for veterinary purposes. Thus, the invention in all its aspects is useful for livestock animals such as cattle, sheep, horses, and poultry, companion animals such as cats and dogs, and zoo animals. The general term "subject" or "subject to be treated / being treated" will therefore be understood to include all animals (such as humans, apes, dogs, cats, horses, and cattle).

[0233] The term "administered" means administering to an individual a therapeutically effective amount of the aforementioned composition comprising the respective cells. A "therapeutically effective amount" means a dose that produces the effect for which it is administered. The exact dose will depend on the purpose of the treatment and will be ascertainable by one of ordinary skill in the art using known techniques. As is known in the art and discussed above, adjustments for systemic versus local delivery, age, weight, general health, sex, diet, time of administration, drug interactions, and severity of the condition may be necessary and will be ascertainable by one of ordinary skill in the art using routine experimentation.

[0234] Subjects in need of treatment include those already having a benign, precancerous, or non-metastatic tumor and those in whom the development or recurrence of cancer is to be prevented. A subject may have metastatic cells, including metastatic cells present in ascites and / or lymph nodes.

[0235] The goal or outcome of treatment may be to reduce the number of cancer cells, to reduce the size of the primary tumor, to inhibit (i.e., slow to some extent, and preferably stop) cancer cell invasion into peripheral organs, to inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis, to inhibit to some extent tumor growth, and / or to alleviate to some extent one or more symptoms associated with the disorder.

[0236] The efficacy of treatment can be measured by assessing survival, time to disease progression, response rate (RR), duration of response, and / or quality of life.

[0237] The methods are particularly useful for extending the time to disease progression.

[0238] The methods are particularly useful for extending human survival, including overall survival and progression-free survival.

[0239] The methods are particularly useful for providing a complete response to treatment, whereby all signs of cancer have disappeared in response to treatment, which does not necessarily mean that the cancer has been cured.

[0240] The methods are particularly useful for providing a partial response to treatment, whereby there has been a decrease in the size of one or more tumors or lesions, or a decrease in the extent of cancer in the body, in response to treatment.

[0241] The goal or outcome of treatment may be any one or more of the following: -reducing the number of cancer cells; - reducing the size of the primary tumor; - inhibiting (i.e. slowing to some extent, preferably stopping) the invasion of cancer cells into peripheral organs, - inhibiting (i.e. slowing to some extent, preferably stopping) tumor metastasis, - inhibiting tumor growth to some extent, -Relieving to some extent one or more of the symptoms associated with the disorder.

[0242] In one embodiment, the subject in need of treatment includes a subject with a benign tumor, a pre-cancerous tumor, or a non-metastatic tumor.

[0243] In one embodiment, the cancer is precancerous or preneoplastic.

[0244] In one embodiment, the cancer is a secondary cancer or metastasis. The secondary cancer may be located in any organ or tissue, particularly an organ or tissue with relatively high hemodynamic pressure, such as the lung, liver, kidney, pancreas, intestine, and brain. The secondary cancer may be detected in ascites and / or lymph nodes.

[0245] In one embodiment, the cancer may be substantially undetectable.

[0246] "Precancerous" or "preneoplastic" generally refers to a condition or growth that typically precedes or develops into cancer. A "precancerous" growth can have cells characterized by aberrant cell cycle regulation, proliferation, or differentiation, as can be determined by cell cycle markers.

[0247] Cancers can be solid or "liquid" tumors. In other words, cancers can be growths in tissue (carcinomas, sarcomas, adenomas, etc.) or cancers present in body fluids such as blood or bone marrow (e.g., lymphomas and leukemias).

[0248] The term "cancer" will be understood to include benign, precancerous, preneoplastic, or non-metastatic tumors, or metastatic tumors. In some embodiments, the cancer is a metastatic cancer, e.g., a stage III or stage IV cancer.

[0249] In some embodiments, the types of cancer to be treated include those having benign, precancerous, pre-neoplastic, or non-metastatic tumors. It will be understood that a benign tumor is not a malignant tumor and does not invade nearby tissues or spread to other parts of the body. Similarly, it will be understood that a non-metastatic cancer does not invade nearby tissues or spread to other parts of the body. "Precancerous" or "pre-neoplastic" generally refers to a condition or growth that typically precedes or develops into cancer. A "precancerous" growth may have cells characterized by aberrant cell cycle regulation, proliferation, or differentiation, as can be determined by cell cycle markers.

[0250] In one embodiment, the cancer is a secondary cancer or metastasis. The secondary cancer may be located in any organ or tissue, particularly an organ or tissue with relatively high hemodynamic pressure, such as the lung, liver, kidney, pancreas, intestine, and brain. The secondary cancer may be detected in ascites and / or lymph nodes.

[0251] In certain embodiments, the cancer in need of treatment may be a cancer characterized by low levels of expression of a dysfunctional P2X7 receptor.

[0252] Cancers particularly suitable for prevention or treatment are those that express or overexpress the nfP2X7 receptor. The expression or overexpression of the nfP2X7 receptor on a cancer or a particular tumor can be determined by detecting the nfP2X7 receptor protein present in or on the cancer cells.

[0253] Preneoplastic, neoplastic, and metastatic cancers are specific examples to which the methods of the present invention can be applied. Broad examples include breast tumors, colorectal tumors, adenocarcinomas, mesothelioma, bladder tumors, prostate tumors, germ cell tumors, hepatocellular carcinoma / cholangiocarcinoma, carcinomas, neuroendocrine tumors, pituitary tumors, small round cell tumors, squamous cell carcinomas, melanomas, atypical fibroxanthoma, seminomas, nonseminomas, stromal Leydig cell tumors, Sertoli cell tumors, skin tumors, kidney tumors, testicular tumors, brain tumors, ovarian tumors, stomach tumors, pancreatic tumors, oral tumors, bladder tumors, bone tumors, cervical tumors, esophageal tumors, laryngeal tumors, liver tumors, lung tumors, vaginal tumors, and Wilm's tumors. Preferred examples include pancreatic tumors, ovarian tumors, stomach tumors, lung tumors, liver tumors, colorectal tumors, cervical tumors, endometrial tumors, kidney tumors, breast tumors, and testicular tumors.

[0254] Examples of specific cancers include adenocarcinoma, adenoma, adenofibroma, adenolymphoma, odontoma, AIDS-related cancer, acoustic neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenocystic carcinoma, adrenocortical carcinoma, primary myelofibrosis, alopecia, alveolar soft part sarcoma, ameloblastoma, angiokeratoma, eosinophilic angiolymphocytosis, sclerosing hemangioma, hemangiomatosis, apodoma, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancer, intestinal cancer, brain stem glioma, brain and CNS tumors, breast cancer (preferably triple-negative breast cancer), gill cancer, tumors, CNS tumors, carcinoid tumors, cervical cancer, childhood brain tumors, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, cutaneous T-cell lymphoma, carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pierce, ductal, Ehrlich, Krebs2, Merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell), carcinosarcoma, cervical dysplasia, cystosarcoma phyllodes, cementoma, chordoma, choristoma, chondrosarcoma, chondroblastoma , craniopharyngioma, cholangiocarcinoma, bile duct carcinoma, cholesteatoma, cystadenocarcinoma, cystadenoma, dermatofibrosarcoma protuberans, desmoplastic small round cell tumor, ductal carcinoma, dysgerminoma, endocrine carcinoma, endometrial carcinoma, ependymoma, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct carcinoma, eye cancer, ocular melanoma, retinoblastoma, fallopian tube cancer, Fanconi anemia, fibroma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, genitourinary cancer, germ cell tumor, gestational trophoblastic disease, glioma, gynecological cancer, giant cell tumor, ganglioneuroma, glioma, glomus angiomas, granulosa cell tumor, ginandrobras Thoma, hematological malignancies, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole, hypercalcemia, hypopharyngeal cancer, hamartoma, hemangioendothelioma, hemangioma, hemangiopericytoma, angiosarcoma, histiocytic disorders, malignant histiocytosis, histiocytoma, liver cancer, hidradenoma, immunoproliferative small intestine, opoma, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, Langerhans 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-II-associated, lymphangiosarcoma, lymphocytic acute, lymphocytic chronic, mast cell, and myeloid), leukemia, Leydig cell tumor, liposarcoma, leiomyoma, leiomyosarcoma, lymphangioma, lymphangiocytoma, lymphangiomas, lymphangiosarcomas, male breast cancer, malignant rhabdoid tumor of the kidney, medulloblastoma, melanoma, melanocyt ... Kell cell carcinoma, mesothelioma, metastatic cancer, oral cancer, multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndrome, myeloma, myeloproliferative disorder, malignant carcinoid syndrome, carcinoid heart disease, medulloblastoma, meningioma, melanoma, mesenchymoma, mesothelioma, myoblastoma, fibroid, myosarcoma, myxoma, myxosarcoma, nasal cavity cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen destruction syndrome, non-melanoma skin cancer, non-small cell lung cancer (NSCLC), schwannoma, neuroblastoma, neuroepithelioma, neurofibromatosis, neurofibroma, neuroma, neoplasms (e.g., bone, breast, digestive system) , colorectal, liver), eye cancer, esophageal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ostomy ovarian cancer, pancreatic cancer, sinus cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral neuroectodermal tumors, pituitary cancer, polycythemia vera, prostate cancer, osteoma, osteosarcoma, ovarian cancer, papilloma, paraganglioma, nonchromaffin paraganglioma, pinealoma, plasmacytoma, proto-oncogenes, rare cancers and related disorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, reticuloendotheliosis, rhabdomyoma, salivary gland cancer, sarcoma, schwannoma, Sézary syndrome, skin cancer, small cell lung cancer (sclc), small intestine cancer, soft tissue sarcoma, spinal tumor, squamous cell carcinoma (skin), gastric cancer, synovial sarcoma, sarcoma (e.g., Ewing's experimental, Kaposi's, and mast cell sarcoma), Sertoli cell tumor, synovioma, testicular cancer, thymic cancer, thyroid cancer, transitional cell carcinoma (bladder), transitional cell carcinoma (renal pelvis / ureter), choriocarcinoma, teratoma, theca cell tumor, thymoma, trophoblastic tumor, urethral cancer, urinary system cancer, uroplakin, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor. Preferred specific examples include pleural or peritoneal mesothelioma, gastric cancer,Endometrial cancer, colorectal cancer, non-small cell lung adenocarcinoma, bile duct cancer, ovarian cancer, esophageal cancer, pancreatic ductal adenocarcinoma, triple-negative breast cancer, and any other mesothelin-positive cancer.

[0255] In some embodiments of the invention, the method is used for the prevention or treatment of cancer selected from one or more of brain cancer, esophageal cancer, oral 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 cancer, skin cancer, leukemia, lymphoma, myeloma, breast cancer, ovarian cancer, endometrial cancer, and testicular cancer. Preferably, the cancer is selected from one or more of lung cancer, esophageal cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer, cervical cancer, vaginal cancer, epithelial cell cancer, skin cancer, blood-related cancer, breast cancer, endometrial cancer, uterine cancer, and testicular cancer.

[0256] The presence, amelioration, treatment, or minimization of cancer progression may be determined by any clinically or biochemically relevant method described herein or known in the art. A positive response to treatment or minimization of cancer progression may be determined by any method known in the art and may include determining: -Reduction of the number of cancer cells, - Reduction in tumor size, - inhibiting (i.e. slowing to some extent, preferably stopping) cancer cell invasion of peripheral organs, - inhibiting (i.e. slowing to some extent, preferably stopping) tumor metastasis, - Reduction or complete prevention of tumor metastasis after removal of the primary tumor, - some inhibition of tumor growth, - Relief to some extent of one or more of the symptoms associated with cancer, and / or -Increased subject survival.

[0257] Any of the above determinations may be considered a positive response to the treatment described herein.

[0258] A subject treated for cancer may be in partial or complete remission. In other words, as described above, a subject treated for cancer may have a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in measurable parameters of tumor growth, which may be detected by physical examination, radiological studies, or biomarker levels from blood or urine tests. Alternatively, when a subject is in complete remission, there is a complete disappearance of all detectable symptoms of the disease, such that the subject no longer has any detectable signs of cancer. A subject may have substantially undetectable signs of cancer. A "substantially undetectable" cancer generally refers to a situation in which the treatment results in a reduction in the size, volume, or other physical measures of the cancer, such that the cancer cannot be clearly detected using relevant standard detection techniques, such as in vivo imaging, as a result of the treatment.

[0259] The goal or outcome of treatment may be to reduce the number of cancer cells, to reduce the size of the primary tumor, to inhibit (i.e., slow to some extent, and preferably stop) cancer cell invasion into peripheral organs, to inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis, to inhibit to some extent tumor growth, and / or to alleviate to some extent one or more symptoms associated with the disorder.

[0260] The efficacy of treatment can be measured by assessing survival, time to disease progression, response rate (RR), duration of response, and / or quality of life.

[0261] In one embodiment, the method is particularly useful for slowing the progression of cancer. In one embodiment, the method is particularly useful for extending the survival of a subject, including overall survival and progression-free survival. Overall survival will be understood to be the length of time from either the date of cancer diagnosis or the start of treatment that a patient diagnosed with cancer remains alive. Progression-free survival will be understood to be the length of time during and after cancer treatment that a patient remains alive with the disease but does not worsen.

[0262] Survival analysis can be performed using techniques well known in the art, including the Kaplan-Meier method. The Kaplan-Meier method estimates a survival function from survival data. In medical research, this can be used to measure the proportion of patients who survive a certain period of time after treatment. The Kaplan-Meier plot of the survival function is a series of horizontal steps of decreasing magnitude that, when a large enough sample is taken, approaches the true survival function of the population. The value of the survival function between successive different sampled observations ("clicks") is assumed to be constant.

[0263] A key advantage of the Kaplan-Meier curve is that the method can account for "censored" data loss from the sample (e.g., when a patient drops out of the study) before the final outcome is observed. In the plot, small vertical checkmarks indicate censored loss of patient data. If no truncation or censoring occurs, the Kaplan-Meier curve is equivalent to the empirical distribution.

[0264] In one embodiment, the method is particularly useful for providing a complete response to treatment, whereby all signs of cancer disappear in response to treatment. This does not necessarily mean that the cancer has been cured. In one embodiment, the method is particularly useful for providing a partial response to treatment, whereby there is a decrease in the size of one or more tumors or lesions, or a decrease in the extent of cancer in the body, in response to treatment.

[0265] Compositions and Administration Delivery or administration of the genetically modified cells according to the present invention can be the delivery or administration of the cells alone or the delivery or administration of the cells formulated in a suitable pharmaceutical composition. Accordingly, the present invention provides a pharmaceutical composition comprising the genetically modified cells of the present invention and a pharmaceutically acceptable carrier.

[0266] Methods for providing CAR-containing cells for immunotherapy are known in the art (see, e.g., Kershaw, M.H. et al. Clin Cancer Res. 2006; 12(20):6106-15; Parker L.L. et al. Hum Gene Ther 2000; 11:2337-87). Additionally, protocols and methods for the preparation, propagation, and evaluation of mammalian CAR-expressing cells are known in the art (see, e.g., Cheadle, E.J. et al. Antibody Engineering: Methods and Protocols, Second Edition, Methods in Molecular Biology, vol. 907:645-66) and are outlined in the Examples below.

[0267] 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 account the specific physical and chemical characteristics of the cells to be administered. In some embodiments, the pharmaceutical composition comprises a suspension of the genetically modified cells of the present invention in a suitable medium, such as isotonic saline. In some embodiments, the pharmaceutical composition may also include a suitable adjuvant, such as one or more cytokines described above. In some embodiments, the pharmaceutical composition may also include intermediates, such as those described above.

[0268] Administration of the pharmaceutical composition may also be via parenteral means, including intravenous, intraventricular, intraperitoneal, intramuscular, intrapleural, or intracranial injection, or local injection at the site of a tumor or cancerous mass.

[0269] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention. [Example]

[0270] Example 1: Identification of alternative sdAb sequences for inclusion in CARs Single domain CARs for binding to nfP2X7 are known, including a CAR containing an sdAb designated "BIL03."

[0271] We sought to identify alternative sdAb-based CARs for targeting cancer cells. Several different anti-nfP2X7 sdAb sequences were tested in the context of CARs.

[0272] Three suitable sdAb sequences (4A7, 3aB9, and 3aD9, as defined herein in Table 3) have been identified as providing efficacy in the context of a CAR, including in the context of the CAR T cell / BRiDGE system (disclosed in WO2022 / 187906), preferably wherein the CAR has the hinge region of CD8A, the transmembrane domain of CD28, and the costimulatory / signaling domains of CD28, 41BB (CD137), and CD3 zeta.

[0273] Interestingly, we found that none of these sdAb sequences resulted in suitable CAR constructs when the sdAbs were paired with different light chain sequences.

[0274] Example 2: Identification of light chain pairings for BIL03 heavy chains Although the single domain antigen binding domain for inclusion in anti-nfP2X7 receptor CAR is known, it is not clear whether the addition of paired variable light chain can improve binding affinity.As shown in Example 1, in some cases, light chain pairing with heavy chain in the context of CAR does not result in improved CAR function.In addition, the selection of suitable light chain for pairing with known heavy chain is not clear from the prior art.

[0275] The inventors sought to identify suitable light chain pairings of the single domain heavy chain protein BIL03 (defined herein in Table 1) disclosed in the prior art in the context of sdAb-based CARs.

[0276] More than 10 different light chain pairings were tested, including the variable light chain defined as G B1 in Table 1 herein.

[0277] We found that a specific light chain pairing with WT B1 (defined in Table 1) resulted in stable expression and reduced aggregation. Surprisingly, this light-heavy chain pairing also significantly improved binding affinity to the target antigen, as shown in the table below.

[0278] [Table 4]

[0279] Binding affinity was assessed in the context of two different antigens derived from dysfunctional P2X7 receptors: E200 peptide (GHNYTTRNILPGLNITC) (SEQ ID NO: 143) and Ext peptide 17 (GHNYTTRNILPGLNITSTFHKTSGSGK) (SEQ ID NO: 102).

[0280] Example 3: Design and production of scFv CARs CAR construction: Two CARs were designed: the first (CAR10A) was designed to contain 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 costimulatory / signaling domains of CD28, 41BB (CD137), and CD3 zeta.

[0281] The second (CAR 12AV1) was designed to contain 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 costimulatory / signaling domains of CD28, 41BB, and CD3 zeta.

[0282] Both CARs contain a C-terminal tEGFR to facilitate detection of the CAR.

[0283] The nucleic acid encoding the CAR was designed in silico, synthesized, cloned into a transfer vector, and packaged into self-inactivating (SIN) lentiviral particles (third-generation LV system) in HEK293T cells. The supernatant was concentrated by ultracentrifugation according to standard protocols.

[0284] CAR T cell production 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) cultured in TexMACS medium supplemented with IL7 / IL15 (both at 10 ng / mL). The donor source was buffy coat.

[0285] Activated untransduced T cells (aUT) do not express any receptors that can engage EGFR.

[0286] In luciferase-based killing assays, reporter cell lines were incubated with effector cells with or without BRiDGE molecules (e.g., as disclosed in WO2022 / 187906). Relevant controls were used to calculate viability and / or specific lysis of target cells only, e.g., 100%, 75%, 50%, 25%, 10%, and 0%, and a standard curve for the final determination of target cell lysis was calculated.

[0287] In the flow-based killing assay, reporter cell lines were incubated with BRiDGE molecules at the indicated concentrations (e.g., as disclosed in WO2022 / 187906) along with effector cells. Viability and / or specific lysis were calculated using relevant controls.

[0288] The results show that cells transduced with CAR12AV1 enabled greater direct cell killing compared to cells transduced with CAR10A, but both CARs enabled cells to indirectly kill cells when combined with the BRiDGE molecule.

[0289] Figure 1 shows a direct comparison of the cell killing ability of T cells expressing CAR10A compared to cells expressing CAR12AV1. The target cells used were the AML cell line MOLM-13. ET5:1, a CAR to target ratio of 2.1 to 1. Cells were cytokine-deprived for 3 days prior to exposure to CAR T cells.

[0290] The results also show significantly greater direct cell killing (as indicated by reduced MOL-13 cell numbers) by CAR12AV1-T cells compared to CAR10A-T cells. Co-incubation with 200 ng / mL of a 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.

[0291] Example 4: Cell killing by T cells expressing the CAR molecule of the present invention CAR T cells expressing CAR10A and CAR12A were generated according to the methods described above in Example 3. In addition, T cells expressing the single domain CARs 3a-B9-CAR and 4A7-CAR were generated.

[0292] 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 WO2022 / 187906).

[0293] Figure 2 shows that T cells expressing either CAR10A, CAR12A, 3a-B9-CAR, or 4A7-CAR induced cell killing in the absence of BRiDGE molecules, indicating that these CAR T cells are all capable of direct cell killing. Co-incubation with various concentrations of anti-CD19 BRiDGE molecules enabled the T cells to kill JeKo-1 cells with higher efficacy. The results also show that cell-killing efficacy increased in a dose-dependent manner in response to increasing concentrations of BRiDGE molecules.

[0294] 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 anti-CD33 BRiDGE molecules. Similar results were achieved at CAR-positive cell to cancer cell ratios of 5:1 and 2.5:1 (data not shown).

[0295] Figures 4(A) and (B) further show that T cells expressing 3a-B9-CAR and 4A7-CAR were able to directly and indirectly kill JeKo-1 cells when co-incubated with anti-CD19 BRiDGE molecules. The results also show that this effect is BRiDGE molecule-specific, with cells co-incubated with anti-CD33 BRiDGE unable to induce greater cell killing. Because JeKo-1 cells do not express CD33 but do express CD19, the use of anti-CD19 BRiDGE molecules to induce greater cell killing indicates that the effect is controlled by the specific CAR / BRiDGE interaction. Similar results were achieved with CAR-positive cell to cancer cell ratios of 5:1 and 2.5:1 (Figure 4(B)).

[0296] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

Claims

1. i) Dysfunctional P2X ７ an antigen recognition domain that recognizes a receptor or an epitope derived therefrom; ii) a transmembrane domain; and iii) an intracellular domain, A chimeric antigen receptor (CAR), wherein the antigen recognition domain comprises a complementarity determining region (CDR) from a VH comprising the sequence set forth in SEQ ID NO: 4, and a CDR from a VL comprising the sequence set forth in SEQ ID NO:

12.

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

12.

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

4. A heavy chain variable domain in which the antigen recognition domain comprises or consists of the amino acid sequence 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%, or at least 99% identical thereto. and a light chain variable domain comprising, or consisting of, the amino acid sequence 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; The antigen recognition domain is nfP2X ７ The CAR according to any one of claims 1 to 3, which retains the ability to bind to a receptor.

5. the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4, and a light chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 12; The heavy and / or light chain variable domains comprise substitutions, deletions, or additions of 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, or 20 or less amino acid residues compared to the amino acid sequence set forth in SEQ ID NO: 4 or 12, respectively, and the antigen recognition domain is nfP2X ７ The CAR according to any one of claims 1 to 3, which retains the ability to bind to a receptor.

6. 6. The CAR of claim 4, wherein the functional variant does not comprise an amino acid substitution, deletion, or addition in the CDR or the CAR of claim 5, and wherein the amino acid substitution, deletion, or addition is not within the CDR.

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

8. The CAR according to any one of claims 1 to 7, wherein the antigen recognition domain comprises, consists essentially of, or consists of, in N-terminus to C-terminus, SEQ ID NO: 12 and SEQ ID NO: 4 (i.e., 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 according to any one of claims 1 to 9, wherein the antigen recognition domain is a single-chain variable fragment (scFv).

11. i) Dysfunctional P2X ７ an antigen recognition domain that recognizes a receptor or an epitope derived therefrom; ii) a transmembrane domain; and iii) an intracellular domain, A chimeric antigen receptor (CAR), wherein the antigen recognition domain comprises a complementarity-determining region (CDR) from a VH comprising the sequence set forth in SEQ ID NO: 135, 136, or 137.

12. The antigen recognition domain is (i) 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 the sequence set forth in SEQ ID NO: 124, at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% identical to the sequence set forth in SEQ ID NO: 125, 126, or 127; a VH comprising 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 the 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 the sequence set forth in SEQ ID NO: 135, 136, or 137; (iii) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 124, a CDR2 comprising the sequence set forth in SEQ ID NO: 125, 126, or 127, and a CDR3 comprising the sequence set forth in SEQ ID NO: 128, 129, or 130; or (iv) a VH comprising the sequence set forth in SEQ ID NO: 135, 136, or 137.

13. The antigen recognition domain is (i) 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 the sequence set forth in SEQ ID NO: 131, and 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 the sequence set forth in SEQ ID NO: 132; a VH comprising an 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 the 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 the sequence set forth in SEQ ID NO:

134. (ii) a VH comprising an FR1 comprising the sequence set forth in SEQ ID NO: 131, an FR2 comprising the sequence set forth in SEQ ID NO: 132, an FR3 comprising the sequence set forth in SEQ ID NO: 133, and an FR4 comprising the sequence set forth in SEQ ID NO:

134.

14. The CAR according to any one of claims 11 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 antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence 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 mutations within the CDRs compared to the sequence of SEQ ID NO: 135, and / or the antigen binding domain is ７ The CAR according to any one of claims 11 to 13, which retains the ability to bind to a receptor.

16. The antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 135, and the heavy chain variable domain of the antigen binding domain comprises substitutions, deletions, or additions of 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, or 20 or less amino acid residues compared to the amino acid sequence set forth in SEQ ID NO: 135, preferably, the amino acid substitutions, deletions, or additions are not within the CDRs, and / or the antigen binding domain is ７ The CAR according to any one of claims 11 to 13, which retains the ability to bind to a receptor.

17. the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence 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 mutations within the CDRs compared to said sequence of SEQ ID NO: 136, and / or the antigen binding domain is ７ The CAR according to any one of claims 11 to 13, which retains the ability to bind to a receptor.

18. The antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 136, and the heavy chain variable domain of the antigen binding domain comprises substitutions, deletions, or additions of 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, or 20 or less amino acid residues compared to the amino acid sequence set forth in SEQ ID NO: 136, preferably, the amino acid substitutions, deletions, or additions are not within the CDRs, and / or the antigen binding domain is selected from the group consisting of nfP2X ７ The CAR according to any one of claims 11 to 13, which retains the ability to bind to a receptor.

19. the antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence 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 mutations within the CDRs compared to the sequence of SEQ ID NO: 137, and / or the antigen binding domain is selected from the group consisting of nfP2X ７ The CAR according to any one of claims 11 to 13, which retains the ability to bind to a receptor.

20. The antigen recognition domain comprises a heavy chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 137, and the heavy chain variable domain of the antigen binding domain comprises substitutions, deletions, or additions of 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, or 20 or less amino acid residues compared to the amino acid sequence set forth in SEQ ID NO: 137, preferably, the amino acid substitutions, deletions, or additions are not within the CDRs, and / or the antigen binding domain is ７ The CAR according to any one of claims 11 to 13, which retains the ability to bind to a receptor.

21. The CAR according to any one of claims 1 to 20, further comprising a hinge region.

22. 22. The CAR of claim 21 , wherein the hinge region is derived from a CD8a, CD28, or IgG4 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 according to any one of claims 21 to 23, wherein the hinge is C-terminal to the antigen recognition domain.

25. 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.

27. 27. 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 signaling domain of the CAR comprises a portion derived from an activating receptor.

29. The CAR of claim 28, wherein the activating receptor is a member of the CD3 co-receptor complex.

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

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

92.

32. The CAR of any one of claims 1 to 31, wherein the signaling domain of the CAR comprises a portion derived from a costimulatory receptor.

33. The CAR according to claim 32, wherein the costimulatory receptor is CD28 and / or 4-1BB (CD137).

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

35. The CAR according to any one of claims 1 to 34, wherein the signaling domain comprises a portion derived from an activating receptor and a portion derived from a costimulatory receptor.

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

37. 37. The CAR of claim 36, wherein the signaling domain comprises an amino acid sequence comprising the sequence set forth in SEQ ID NO: 90, 91, and / or 92, and combinations thereof.

38. The CAR according to any one of claims 1 to 37, comprising (from the N-terminus to the C-terminus) the antigen recognition domain of any one of claims 1 to 20, a CD8a hinge, a CD28 transmembrane domain, a CD28 signaling domain, a 4-1BB signaling domain, and a CD3ζ (zeta) signaling domain.

39. 39. The CAR of claim 38, wherein the CAR comprises the amino acid sequence 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%, or at least 99% identical thereto, wherein the functional variant comprises an antigen recognition domain as defined herein, 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. 39. The CAR of claim 38, wherein the CAR comprises the amino acid sequence 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 defined herein, preferably comprising a VH comprising the amino acid sequence of SEQ ID NO:

135.

41. 39. The CAR of claim 38, wherein the CAR comprises the amino acid sequence 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 defined herein, preferably comprising a VH comprising the amino acid sequence of SEQ ID NO:

136.

42. 39. The CAR of claim 38, wherein the CAR comprises an amino acid sequence 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 defined herein, preferably comprising a VH comprising the amino acid sequence of SEQ ID NO:

137.

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

44. A nucleic acid construct comprising the nucleic acid molecule of claim 43.

45. A genetically modified cell comprising the CAR of any one of claims 1 to 42.

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

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

48. 48. The genetically modified cell of claim 47, wherein the immune cell is a leukocyte.

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

50. 50. The genetically modified cell of claim 49, wherein the T cell is a CD4+ T cell or a CD8+ T cell.

51. 45. A method of generating a genetically modified cell, said method comprising transducing said cell, preferably an immune cell, with the nucleic acid construct of claim 44, such that said transduced cell expresses said CAR, thereby generating a genetically modified cell.

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

53. nfP2X ７ A method of killing a cell that expresses a nfP2X receptor, the method comprising: ７ Cells expressing the receptor are exposed to the genetically modified cells of any one of claims 45 to 50, thereby ７ killing a cell that expresses the receptor.

54. The nfP2X ７ 54. The method of claim 53, wherein the cell expressing the receptor is a cancer cell.

55. 55. The method of claim 54, wherein the cancer is selected from the group consisting of brain cancer, esophageal cancer, oral 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 cancer, skin cancer, leukemia, lymphoma, myeloma, breast cancer, ovarian cancer, endometrial cancer, thymic cancer, and testicular cancer.

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

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

58. Treating, preventing, or minimizing the progression of cancer in a subject; minimize, reduce, or prevent tumor growth in a subject; minimize, reduce, or prevent metastasis in a subject; or A method of increasing survival in a subject, comprising: The method comprises administering to the subject a CAR according to any one of claims 1 to 42, a nucleic acid according to claim 43, a nucleic acid construct according to claim 44, a genetically modified cell according to any one of claims 45 to 50, or a pharmaceutical composition according to claim 57, thereby Treating, preventing, or minimizing the progression of cancer in a subject; minimize, reduce, or prevent tumor growth in a subject; minimize, reduce, or prevent metastasis in a subject; or - A method for increasing the survival of a subject.

59. Treating, preventing, or minimizing the progression of cancer in a subject; minimize, reduce, or prevent tumor growth in a subject; minimize, reduce, or prevent metastasis in a subject; or - Use of a CAR described in any one of claims 1 to 42, a nucleic acid described in claim 43, a nucleic acid construct described in claim 44, a genetically modified cell described in any one of claims 45 to 50, or a pharmaceutical composition described in claim 57 in the manufacture of a medicament for increasing survival of a subject.

60. Treating, preventing, or minimizing the progression of cancer in a subject; minimize, reduce, or prevent tumor growth in a subject; minimize, reduce, or prevent metastasis in a subject; or - A CAR described in any one of claims 1 to 42, a nucleic acid described in claim 43, a nucleic acid construct described in claim 44, a genetically modified cell described in any one of claims 45 to 50, or a pharmaceutical composition described in claim 57, for use in increasing the survival of a subject.

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