Chimeric Antigen Receptors for Binding to Dysfunctional P2X7 Receptor

Description

1 / 99 Chimeric Antigen Receptors for Connection to Dysfunctional P2X7 Receptor Field of invention

[001] 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. Related request

[002] This application claims priority over Australian provisional application AU 2023900626, the content of which is incorporated into the present invention by reference in its entirety. Background of the invention

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

[004] Although CARs have been designed for binding to various tumor-specific antigens, the architecture and binding affinity of CARs often require optimization to ensure appropriate binding levels in the context of T cells binding to cancer cells. In other words, antigen-binding domains derived directly from antibodies Petition 870250078796, dated 03 / 09 / 2025, pages 219 / 342 2 / 99 therapeutic agents may have an inappropriate affinity for use in the context of a CAR and may potentially result in the production of CAR T cells leading to toxicity in patients.

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

[006] Reference to any state of the art in the report is not an acknowledgment or suggestion that such state of the art forms part of common general knowledge in any jurisdiction or that such state of the art could reasonably be expected to be understood, considered relevant and / or combined with other state of the art passages by a person skilled in the art. Summary of the invention

[007] The present invention is based on the inventors' surprising discovery that particular antigen recognition domain sequences provide enhanced cell functionality and death in the context of chimeric antigen receptors.

[008] In a first aspect, the present invention provides a chimeric antigen receptor (CAR) comprising: i) an antigen recognition domain that recognizes a dysfunctional P2X7 receptor (nfP2X7 receptor) or an epitope derived from it; ii) a transmembrane domain; and iii) an intracellular domain, wherein the antigen recognition domain comprises complementarity-determining regions (CDRs) from a VH comprising a sequence as set out in SEQ ID NO: 4 and CDRs from a VL comprising a sequence as set out in SEQ ID NO: 12. Petition 870250078796, dated 03 / 09 / 2025, pages 220 / 342 3 / 99

[009] In a second aspect, the present invention provides a chimeric antigen receptor (CAR) comprising: i) an antigen recognition domain that recognizes a dysfunctional P2X7 receptor (nfP2X7 receptor) or an epitope derived from it; ii) a transmembrane domain; and iii) an intracellular domain, wherein the antigen recognition domain comprises complementarity-determining regions (CDRs) from a VH comprising a sequence as set out in any of the SEQ ID NOs: 135, 136 or 137. [01 0] According to the first aspect, the antigen recognition domain of 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 regions of structure; CDR1, CDR2, and CDR3 are each key regions of complementarity; FR1, FR2a, FR3a, and FR4a are each regions of structure; CDR1a, CDR2a, and CDR3a are each complementarity-determining regions, where the complementarity-determining regions comprise an amino acid sequence as described in Table 1 below. [0 11] According to the second aspect of the invention, the antigen recognition domain of the CAR comprises: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 Petition 870250078796, dated 03 / 09 / 2025, pages 221 / 342 4 / 99 where: FR1, FR2, FR3, and FR4 are each regions of structure; CDR1, CDR2, and CDR3 are each key regions of complementarity; where the complementarity-determining regions comprise an amino acid sequence as described in Table 3 below. [01 2] Preferably, and according to the first aspect of the invention, a chimeric antigen receptor (CAR) is provided comprising: an antigen recognition domain, a transmembrane domain; and an intracellular domain, wherein the antigen recognition domain comprises: (i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence established 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence established in SEQ ID NO: 2, 30, 37 or 44 and a CDR3 comprising a sequence at least approximately 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence established in SEQ ID NO: 3, 31, 38 or 45; Petition 870250078796, dated 03 / 09 / 2025, pp. 222 / 342 5 / 99 (ii) a VH comprising a sequence at least about 95% or 96% or 97% or 98% or 99% identical to a sequence established in SEQ ID NO: 4; (iii) a VL comprising a CDR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence established in SEQ ID NO: 9, 50, 57 or 64, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence established in SEQ ID NO: 10, 51, 58 or 65 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 a sequence established in SEQ ID NO: 11, 52, 59, or 66; (iv) a VL comprising a sequence that is at least about 95% or 96% or 97% or 98% or 99% identical to a sequence established in SEQ ID NO: 12; (v) a VH comprising a CDR1 comprising a sequence set out in SEQ ID NO: 1, 29, 36 or 43, a CDR2 comprising a sequence set out in SEQ ID NO: 2, 30, 37 or 44 and a CDR3 comprising a sequence set out in SEQ ID NO: 3, 31, 38 or 45; (vi) a VH comprising a sequence established in the SEQ ID Petition 870250078796, dated 03 / 09 / 2025, pages 223 / 342 6 / 99 NO: 4; (vii) a VL comprising a CDR1 comprising a sequence established in SEQ ID NO: 9, 50, 57 or 64, a CDR2 comprising a sequence established in SEQ ID NO: 10, 51, 58 or 65 and a CDR3 comprising a sequence established in SEQ ID NO: 11, 52, 59 or 66; (viii) a VL comprising a sequence established in SEQ ID NO: 12; (ix) a VH comprising a CDR1 comprising a sequence established in SEQ ID NO: 1, 29, 36 or 43, a CDR2 comprising a sequence established in SEQ ID NO: 2, 30, 37 or 44 and a CDR3 comprising a sequence established in SEQ ID NO: 3, 31, 38 or 45; and a VL comprising a CDR1 comprising a sequence established in SEQ ID NO: 9, 50, 57 or 64, a CDR2 comprising a sequence established in SEQ ID NO: 10, 51, 58 or 65 and a CDR3 comprising a sequence established in SEQ ID NO: 11, 52, 59 or 66; or (x) a VH comprising a sequence established in SEQ ID NO: 4 and a VL comprising a sequence established in SEQ ID NO: 12.

[013] In any embodiment of the first aspect of the invention, the antigen recognition domain further comprises at least one of: (i) a VH comprising a structure 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 Petition 870250078796, dated 03 / 09 / 2025, pages 224 / 342 7 / 99 98% or at least 99% identical to a sequence established in SEQ ID NO: 5, 32, 39, or 46; an FR2 comprising a sequence at least approximately 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence established in SEQ ID NO: 6, 33, 40, or 47; an FR3 comprising a sequence at least approximately 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence established in SEQ ID NO: 7, 34, 41 or 48 and an FR4 comprising a sequence at least approximately 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence established 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence established 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence established in SEQ ID NO: 14, 54, 61 or 68, an FR3 comprising a sequence of at least approximately 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 Petition 870250078796, dated 03 / 09 / 2025, pages 225 / 342 8 / 99 97%, at least 98%, or at least 99% identical to a sequence established in SEQ ID NO: 15, 55, 62, or 69 and an FR4 comprising a sequence at least approximately 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence established in SEQ ID NO: 16, 56, 63, or 70; (iii) a VH comprising an FR1 comprising a sequence established in SEQ ID NO: 5, 32, 39 or 46, an FR2 comprising a sequence established in SEQ ID NO: 6, 33, 40 or 47, an FR3 comprising a sequence established in SEQ ID NO: 7, 34, 41 or 48 and an FR4 comprising a sequence established in SEQ ID NO: 8, 35, 42 or 49; (iv) a VL comprising an FR1 comprising a sequence established in SEQ ID NO: 13, 53, 60 or 67, an FR2 comprising a sequence established in SEQ ID NO: 14, 54, 61 or 68, an FR3 comprising a sequence established in SEQ ID NO: 15, 55, 62 or 69 and an FR4 comprising a sequence established in SEQ ID NO: 16, 56, 63 or 70; or (v) a VH comprising an FR1 comprising a sequence established in SEQ ID NO: 5, 32, 39 or 46, an FR2 comprising a sequence established in SEQ ID NO: 6, 33, 40 or 47, an FR3 comprising a sequence established in SEQ ID NO: 7, 34, 41 or 48 and an FR4 comprising a sequence established in SEQ ID NO: 8, 35, 42 or 49;and a VL comprising an FR1 comprising a sequence established in SEQ ID NO: 13, 53, 60 or 67, an FR2 comprising a sequence established in SEQ ID NO: 14, 54, 61 or 68, an FR3 comprising a sequence established in SEQ ID NO: 15, 55, 62 or 69 and an FR4 comprising a sequence established in SEQ ID NO: 16, 56, 63 or 70.;

[014] In any modality of the first aspect, the domain of Petition 870250078796, dated 03 / 09 / 2025, pages 226 / 342 9 / 99 Antigen recognition comprises, consists essentially of, or consists of the amino acid sequence of (in order from N to C terminal or C to N terminal) SEQ ID NOs: 4 and 12.

[015] In any embodiment of the first aspect, the antigen recognition domain comprises a variable heavy chain domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 4, or a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto;and a variable light chain domain comprising or consisting of the amino acid sequence as 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; wherein the heavy and light chains do not comprise any sequence variation in the CDRs compared with the sequence of SEQ ID NO: 4 or 12, respectively, and / or wherein the antigen-binding domain retains the ability to bind to the nfP2Xy receptor.

[016] In any embodiment of the first aspect, the antigen recognition domain comprises a variable heavy chain domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 4 and a variable light chain domain comprising or consisting of the amino acid sequence as Petition 870250078796, dated 03 / 09 / 2025, pages 227 / 342 10 / 99 as established in SEQ ID NO: 12, wherein the variable heavy and light chain domain of the antigen-binding domain comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residue substitutions, deletions, or additions, compared to the amino acid sequence as established in SEQ ID NO: 4 or 12, respectively, preferably wherein amino acid substitutions, deletions, or additions are not in the CDRs and / or wherein the antigen-binding domain retains the ability to bind to the receptor. nfP2X7.

[017] Preferably, the antigen recognition domain comprises, consists essentially of, or consists of, in N- 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) - ligand - SEQ ID NO: 4 (VH).

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

[019] Optionally, the variable heavy and variable light regions of the antigen recognition domain are joined via a linker. In either embodiment, the antigen recognition domain may comprise: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 - ligand - FR1a - CDR1a - FR2a CDR2a - FR3a - CDR3a - FR4a. Petition 870250078796, dated 03 / 09 / 2025, pages 228 / 342 11 / 99

[020] As defined in the present invention, the linker can be a chemical, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

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

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

[023] Preferably, and according to the second aspect of the invention, a chimeric antigen receptor (CAR) is provided comprising: an antigen recognition domain, a transmembrane domain; and an intracellular domain, wherein the antigen recognition domain comprises: (i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence established in SEQ ID NO: 124, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, by Petition 870250078796, dated 03 / 09 / 2025, pages 229 / 342 12 / 99 less 98% or at least 99% identical to a sequence established in SEQ ID NO: 126, 126 or 127 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence established in SEQ ID NO: 128, 129 or 130; (ii) a VH comprising a sequence that is at least about 95% or 96% or 97% or 98% or 99% identical to a sequence established in SEQ ID NO: 135, 136 or 137; (iii) a VH comprising a CDR1 comprising a sequence established in SEQ ID NO: 124, a CDR2 comprising a sequence established in SEQ ID NO: 125, 126, or 127 and a CDR3 comprising a sequence established in SEQ ID NO: 128, 129 or 130; preferably, the CDR1 comprises a sequence established in SEQ ID NO: 124, the CDR2 comprises a sequence established in SEQ ID NO: 125 and the CDR3 comprises a sequence established in SEQ ID NO: 128; or preferably CDR1 comprises a sequence established in SEQ ID NO: 124, CDR2 comprises a sequence established in SEQ ID NO: 126 and CDR3 comprises a sequence established in SEQ ID NO: 129; or preferably CDR1 comprises a sequence established in SEQ ID NO: 124, CDR2 comprises a sequence established in SEQ ID NO: 127 and CDR3 comprises a sequence established in SEQ ID NO: 130; or (iv) a VH comprising a sequence established in SEQ ID NO: 135, 136 or 137. [02 4] In any embodiment of the second aspect of the invention, the antigen recognition domain further comprises Petition 870250078796, dated 03 / 09 / 2025, pages 230 / 342 13 / 99 minus one of: (i) a VH comprising a structure region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence established in SEQ ID NO: 131, 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 a sequence established in SEQ ID NO: 132, 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 a sequence established in SEQ ID NO: 133 and an FR4 comprising a sequence at least approximately 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence established in SEQ ID NO: 134;, (ii) a VH comprising an FR1 comprising a sequence established in SEQ ID NO: 131, an FR2 comprising a sequence established in SEQ ID NO: 132, an FR3 comprising a sequence established in SEQ ID NO: 133 and an FR4 comprising a sequence established in SEQ ID NO: 134. [ 025] 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. Petition 870250078796, dated 03 / 09 / 2025, pages 231 / 342 14 / 99

[026] In any embodiment, the antigen recognition domain comprises a variable heavy chain domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 135, or a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; where the heavy chain does not comprise any sequence variation in the CDRs compared to the sequence of SEQ ID NO: 135, and / or where the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

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

[028] In any modality, the domain of recognition of Petition 870250078796, dated 03 / 09 / 2025, pp. 232 / 342 The 15 / 99 antigen comprises a variable heavy chain domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 136, or a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; wherein the heavy chain does not comprise any sequence variation in the CDRs compared with the sequence in SEQ ID NO: 136, and / or wherein the antigen-binding domain retains the ability to bind to the nfP2Xy receptor.

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

[030] In any embodiment, the antigen recognition domain comprises a variable heavy chain domain comprising Petition 870250078796, dated 03 / 09 / 2025, pages 233 / 342 16 / 99 or consisting of the amino acid sequence as set forth in SEQ ID NO: 137, or a sequence at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; wherein the heavy chain does not comprise any sequence variation in the CDRs compared to the sequence in SEQ ID NO: 137, and / or wherein the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

[031] In any embodiment, the antigen recognition domain comprises a variable heavy chain domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 137, wherein the variable heavy chain domain of the antigen-binding domain comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions or additions, compared to the amino acid sequence as set forth in SEQ ID NO: 137, preferably wherein the substitutions,Deletions or additions of amino acids are not present in CDRs and / or the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

[032] In any embodiment of any aspect, the CAR further comprises a hinge region (also referred to in the present invention as a spacer region). The hinge region can be derived from Petition 870250078796, dated 03 / 09 / 2025, pages 234 / 342 17 / 99 derived from CD28, CD8a, or from 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 situated between the antigen recognition domain and the transmembrane domain. In preferred embodiments, the hinge region comprises or consists of the amino acid sequence shown in SEQ ID NO: 88.

[033] In any embodiment of any aspect, the transmembrane domain of 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.

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

[035] In any embodiment of any aspect, the CAR signaling domain comprises a portion derived from a co-stimulatory receptor. In some embodiments, the co-stimulatory receptor is CD28, ICOS, CD27, OX40 and / or 4-1BB (CD137) and optionally comprises an amino acid sequence as set out in SEQ ID NO: 90 and / or 91.

[036] The signaling domain may comprise a portion derived from an activating receptor and a portion derived from a co-stimulatory receptor. In some modalities, the activating receptor is a member of the CD3 co-receptor complex and the co-stimulatory receptor is Petition 870250078796, dated 03 / 09 / 2025, pp. 235 / 342 18 / 99 selected from CD28, ICOS, CD27, OX40 and / or 4-1BB. In some embodiments, the activating receptor is a member of the CD3 coreceptor complex and the co-stimulatory receptor is CD28 and 4-1BB (CD137). Preferably, the portion derived from the CD3 coreceptor complex is CD3-Z (CD3-zeta). In certain embodiments, the signaling domain comprises an amino acid sequence comprising the sequence established in SEQ ID NOs: 90, 91 and / or 92 and combinations thereof.

[037] In any aspect embodiment, the CAR comprises (from the N to the C terminal) an antigen-binding domain as described in the present invention, a linker, a CD8a hinge, a CD28 transmembrane domain, a CD28 signaling domain, a 4-1BB signaling domain and a CD3z (zeta) signaling domain. Optionally, the CAR may further comprise an epitope or affinity tag.

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

[039] In a particularly preferred embodiment of the first Petition 870250078796, dated 03 / 09 / 2025, pp. 236 / 342 19 / 99 aspect, the chimeric antigen receptor comprises an amino acid sequence as set forth in SEQ ID NO: 94.

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

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

[042] In a preferred embodiment of the second aspect, the chimeric antigen receptor comprises an amino acid sequence as set forth in SEQ ID NO: 139, or a functional variant thereof comprising a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; wherein the functional variant comprises an antigen recognition domain as defined in the present invention, Petition 870250078796, dated 03 / 09 / 2025, pp. 237 / 342 20 / 99 preferably comprising a VH comprising the amino acid sequence of SEQ ID NO: 136.

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

[044] In a preferred embodiment of the second aspect, the chimeric antigen receptor comprises an amino acid sequence as set forth in SEQ ID NO: 140, or a functional variant thereof comprising a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; wherein the functional variant comprises an antigen recognition domain as defined in the present invention, preferably comprising a VH comprising the amino acid sequence of SEQ ID NO: 137.

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

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

[047] In a further aspect, a nucleic acid encoding a chimeric antigen receptor is provided according to the first or second aspects of the invention. Petition 870250078796, dated 03 / 09 / 2025, pp. 238 / 342 21 / 99

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

[049] In some embodiments, the nucleic acid construct additionally includes an internal ribosome entry site (IRES) that allows translation initiation on mRNA after expression from the nucleic acid construct.

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

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

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

[053] In another aspect, the present invention provides a method of generating a genetically modified cell, said method comprising transducing the cell, preferably an immune cell, with a nucleic acid construct encoding a CAR of the invention as described herein, such that the transduced cell expresses the CAR, thereby generating a genetically modified cell. Petition 870250078796, dated 03 / 09 / 2025, pages 239 / 342 22 / 99

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

[055] In a preferred embodiment, the immune cell expressing CAR is a T cell. Illustrative examples of suitable T cells include helper T cells (HTL; CD4+ T cell), a cytotoxic T cell (CTL; ​​CD8+ T cell), CD4+CD8+ T cell, CD4-CD8- T cell, or any other subset of T cells. Other illustrative examples of suitable T cells include T cells expressing one or more of the following markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, CD197, and HLA-DR.

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

[057] In another aspect, the present invention provides a method of killing a cell expressing nfP2X7 receptor, the method comprising exposing the cell expressing nfP2X7 receptor to a genetically modified cell of the invention described in the present invention, thereby killing a cell expressing an nfP2X7 receptor.

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

[059] In some forms, the cell that expresses nfP2X7 receptor Petition 870250078796, dated 03 / 09 / 2025, pages 240 / 342 23 / 99 (i.e., the cell being linked by the CAR of the invention) is a cancerous cell. In some embodiments, the cancer is selected from the group consisting of: brain cancer, esophageal cancer, mouth cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cervical cancer, epithelial cell cancers, skin cancer, 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 cancers, epithelial cell cancers, skin cancer, blood-related cancers, breast cancer, endometrial cancer, uterine cancer, and testicular cancer.

[060] In some forms, the cancer is metastatic. In some forms, the cancer is stage III cancer or stage IV cancer.

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

[062] In another aspect, the present invention provides a method of expanding in vitro the genetically modified cell of the invention described in the present invention, the method comprising the step of exposing the cell to an antigen for the CAR, for example, nfP2X7 receptor and simultaneously exposing the cell to a cytokine.

[063] In some forms, the cytokine is a member of the IL-2 subfamily, the interferon subfamily, the IL-10 subfamily, the IL-1 subfamily, the IL-1 subfamily, or the TGF-β subfamily. Petition 870250078796, dated 03 / 09 / 2025, pp. 241 / 342 24 / 99

[064] 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-βΙ, TGF-β2, TGF^3 and GM-CSF, or a combination thereof.

[065] In another aspect, the present invention provides a method of expanding in vitro the genetically modified cell of the invention described in the present invention, the method comprising - expose the cell to immobilized CD3 and CD28 agonists; and - To place the cells in contact with a medium under conditions that allow cell proliferation, preferably T cells, preferably human T cells.

[066] In some embodiments, the agonists are immobilized on a substrate with microspheres. In one embodiment, the agonists can be immobilized on “Human Activator” Dynabeads™. In another preferred embodiment, the agonists are immobilized on a substrate with colloidal polymeric nanomatrix microspheres conjugated with recombinant humanized CD3 and CD28 agonist (e.g., on a substrate with “MACS GMP” TransAct™ microspheres).

[067] In some modalities, CD3 and CD28 agonists are anti-CD3 and anti-CD28 antibodies.

[068] In some embodiments the medium is GMP TexMACS™ medium. In some embodiments, the medium is supplemented with interleukins, for example, IL7 and IL-15.

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

[070] In another aspect, the present invention provides a pharmaceutical composition including a genetically modified cell of the invention. Petition 870250078796, dated 03 / 09 / 2025, pages 242 / 342 25 / 99 described in the present invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises suitable adjuvants which may consist of cytokines. In some embodiments, the pharmaceutical composition may also comprise an intermediate as described in the present invention.

[071] In another aspect, the invention provides a method of: • treat, prevent or minimize the progression of cancer in an individual, • minimize, reduce or prevent the growth of a tumor in an individual, • minimize, reduce or prevent cancer metastasis in an individual, or • increase the survival of an individual suffering from cancer, optionally wherein the cancer or tumor is characterized by the expression of nfP2X7 receptor; the method comprising administering to the individual a CAR of the invention described in the present invention, a nucleic acid construct of the invention as described in the present invention, a genetically modified cell of the invention as described in the present invention or a pharmaceutical composition of the invention as described in the present invention, thus: • To treat, prevent, or minimize the progression of cancer in an individual, • To minimize, reduce, or prevent the growth of a tumor in an individual, • To minimize, reduce, or prevent metastasis in an individual, or • To increase the survival of an individual suffering from cancer.

[072] In another aspect, the present invention provides the use of a CAR of the invention described in the present invention, a nucleic acid construct of Petition 870250078796, dated 03 / 09 / 2025, pages 243 / 342 26 / 99 invention as described in the present invention, a genetically modified cell of the invention as described in the present invention, or a pharmaceutical composition of the invention as described in the present invention, in the manufacture of a medicament for: • to treat, prevent, or minimize the progression of cancer in an individual, • to minimize, reduce, or prevent the growth of a tumor in an individual, • to minimize, reduce, or prevent cancer metastasis in an individual, or • to increase the survival of an individual suffering from cancer, optionally where the cancer or tumor is characterized by the expression of the nfP2X7 receptor.

[073] In another aspect, the present invention provides a CAR of the invention described in the present invention, a nucleic acid construct of the invention as described in the present invention, a genetically modified cell of the invention as described in the present invention, or a pharmaceutical composition of the invention as described in the present invention for use in: • to treat, prevent, or minimize the progression of cancer in an individual, • to minimize, reduce, or prevent the growth of a tumor in an individual, • to minimize, reduce, or prevent cancer metastasis in an individual, or • to increase the survival of an individual suffering from cancer, optionally where the cancer or tumor is characterized by the expression of the nfP2X7 receptor.

[074] As used in the present invention, except where the context requires otherwise, the term includes and variations of the term, such as Petition 870250078796, dated 03 / 09 / 2025, pages 244 / 342 27 / 99 “compreendendo”, “compreende” and “compreendo”, are not intended to exclude additives, components, whole numbers or additional steps.

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

[076] Figure 1: MOLM-13 cell count after contact with non-transduced T cells (UTD) or T cells transduced with CAR12AV1 or CAR10A. NB = no binding molecule (indicative of direct cell death). “With BRiDGE” = co-incubation with 200 ng / mL of anti-CD33 BRiDGE molecule comprising E200 sequence for binding via CAR antigen recognition domain.

[077] Figure 2: Viability of JeKo-1 cells after contact with non-transduced T cells (UTD) or T cells transduced with CAR10A, CAR12A, 3a-B9CAR or 4A7-CAR, supplemented with anti-CD19 BRiDGE. BRiDGE concentrations range from 0 ng / mL to 100 ng / mL. “BRiDGE” = fusion protein comprising E200 sequence for binding via CAR antigen recognition domain and anti-CD19 antigen binding domain. Effector target cell ratio = 2.77 / 1. Cell viability was read 21 hours after co-incubation.

[078] Figure 3: Viability of MOLM-13 cells after contact with non-transduced T cells (UTD) or T cells transduced with 3a-B9-CAR or 4A7-CAR. Ratio of CAR-positive cells to cancer cells of 10:1. “BRiDGE” = co-incubation with 100 ng / mL of BRiDGE molecule comprising E200 sequence for CAR antigen recognition domain binding and an anti-CD33 binding domain or an anti-CD19 binding domain. Cell viability was read 24 hours after co-incubation. Petition 870250078796, dated 03 / 09 / 2025, pages 245 / 342 28 / 99

[079] Figure 4: Viability of JeKo-1 cells after contact with non-transduced T cells (UTD) or T cells transduced with 3a-B9-CAR or 4A7-CAR. Ratio of CAR-positive cells to cancer cells 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 molecule comprising E200 sequence for CAR antigen recognition domain binding and also an anti-CD19 antigen binding domain or an anti-CD33 binding domain. Cell viability was read 24 hours after co-incubation. Sequence information Table 1: Sequence information Identifier Region SEQ ID NO: Amino Acid Sequence BIL03 HCDR1 (Kabat) 1 NHDMG HCDR2 (Kabat) 2 AISGSGGSTYYANSVKG HCDR3 (Kabat) 3 PKPMDTEFDY VH 4 EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHDMG WVRQAPGKGLEWVSAISGSGGSTYYANSVKGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTEFD YRSPGTLVTVSS HFR1 (Kabat) 5 EVQLLESGGGLVQPGGSLRLSCAASGFTFR HFR2 (Kabat) 6 WVRQAPGKGLEWVS HFR3 (Kabat) 7 RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAE HFR4 (Kabat) 8 RSPGTLVTVSS WT B1 LCDR1 (Kabat) 10 LFR1 13 DIQMTQSPSSLSASVGDRVTITC Petition 870250078796, dated 03 / 09 / 2025, pages 246 / 342 29 / 99 Region Identifier SEQ ID NO: Aminoacid Sequence (Kabat) LFR2 (Kabat) 14 WYQQKPGKAPKLLIY LFR3 (Kabat) 15 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LFR4 (Kabat) 16 FGQGTKVEIKR G B1 LCDR1 21 RASQSISSYLN LCDR2 22 AASSLQS LCDR3 23 QQFDYMPLT VL 24 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQ QKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTIS SLQPEDFATYYCQQFDYMPLTFGQGTKVEIKR LFR1 25 DIQMTQSPSSLSASVGDRVTITC HFR1 (Chothia) 32 EVQLLESGGGLVQPGGSLRLSCAAS HFR2 (Chothia) 33 DMGWVRQAPGKGLEWVSAI HFR3 (Chothia) 34 TYYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YCAE HFR4 (Chothia) 35 RSPGTLVTVSS HCDR1 (IMGT) 36 GFTFRNHD HCDR2 (IMGT) 37 ISGSGGST HCDR3 (IMGT) 38 AEPKPMDTEFDY HFR1 (IMGT) 39 EVQLLESGGGLVQPGGSLRLSCAAS HFR2 (IMGT) 40 MGWVRQAPG KGLEWVSA HFR3 (IMGT) 41 YYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY C HFR4 (IMGT) 42 RSPGTLVTVSS Petition: 870250078796, on September 3, 2025, page. 247 / 342 30 / 99 Identificador Região SEQ ID NO: Sequência de Aminoácidos HCDR1 (Martin) 43 NHDMG HCDR2 (Martin) 44 AISGSGGSTYYANSVKG HCDR3 (Martin) 45 PKPMDTEFDY HFR1 (Martin) 46 EVQLLESGGGLVQPGGSLRLSCAASGFTFR HFR2 (Martin) 47 WVRQAPGKGLEWVS HFR3 (v) 48 RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAE HFR4 (Martin) 49 RSPGTLVTVSS WTB1 LCDR1 (Chothia) 50 RASQYIYDYLN LCDR2 (Chothia) 51 AASYLQS LCDR3 (Chothia) 52 QQYHHPST LFR1 (Chothia) 53 DIQMTQSPSSLSASVGDRVTITC LFR2 (Chothia) 54 WYQQKPGKAPKLLIY LFR3 (Chothia) 55 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LFR4 (Chothia) 56 FGQGTKVEIK LCDR1 (IMGT) 57 QYIYDY LCDR2 (IMGT) 58 AAS LCDR3 (IMGT) 59 QQYHHPST LFR1 (IMGT) 60 DIQMTQSPSSLSASVGDRVTITCRAS LFR2 (IMGT) 61 LNWYQQKPGKAPKLLIY LFR3 (IMGT) 62 YLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LFR4 (IMGT) 63 FGQGTKVEIK LCDR1 (Martin) 64 QYIYDY LCDR2 (Martin) 65 AAS Petition: 870250078796, on September 3, 2025, page. 248 / 342 31 / 99 SEQ Region Identifier ID NO: Amino Acid Sequence LCDR3 (Martin) 66 QQYHHPST LFR1 (Martin) 67 DIQMTQS PSSLSASVGDRVTITCRAS LFR2 (Martin) 68 LNWYQQKPGKAPKLLIY LFR3 (Martin) 69 YLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LFR4 (Martin) 70 FGQGTKVEIK Region Identifier SEQ ID NO: Nucleic Acid Sequence BIL03 HCDR1 (Kabat) 71 aaccatgatatgggc HCDR2 (Kabat) 72 gcgattagcggcagcggcggcagcacctattatgcgaacagcgtg aaggc HCDR3 (Kabat) 73 ccgaaaccgatggataccgaatttgattat VH 74 gaagtgcagctgctggaaagcggcggcggcgcgcgcgc ggcagcctgcgcctgagctgcgcggcgcggctttaccttcgca accatgatatgggc tgggtcgccaggcgcggcaaaggcctggaatgggtgagcgc gattagcggcagcggcgcacctattatgcgaacagcgtgaa aggccgctttaccattagccgcgataacagcaaaaacaccctgtat ctgcagatgaacagcctgcgcgaagataccgcggtgtatt gcgcggaaccgaaccgatggataccgaatttgattatcgcagcc cgggcaccctggtgaccgtgagcagc HFR175 gaagtgcagctgctggaaagcggcggcggcctgcgccgggc ggcagcctgcgctgagctgcgcggcgcggctttaccttcgc HFR2 (Kabat) 76tgggtgcgccaggcgccgggcaaaggcctggaatgggtgagc HFR3 (Kabat) 77 cgctttaccattagccgcgataacagcaaaaacaccctgtatctgc agatgaacagcctgcgcgcggaagataccgcggtgtattattgcgc ggaa HFR4 (Kabat) 78 cgcagcccgggcaccctggtgaccgtgagcagc WTB1 LCDR1 (Kabat) 79 cgcgcgagccagagcattagcagctatctgaac LCDR2 (Kabat) 80 gcggcgagcagcctgcagagc LCDR3 (Kabat) 81 cagcagtttgattatatgccgctgacc VL 82 gatattcagatgacccagagcccgagcagcctgagcgcgagcgtg ggcgatcgcgtgaccattacctgccgcgcgagccagagcattagc agctatctgaactggtatcagcagaaaccgggcaaagcgccgaaa ctgctgatttatgcggcgagcagcctgcagagcggcgtgccgagcc gctttagcggcagcggcagcggcaccgattttaccctgaccattag Petição 870250078796, de 03 / 09 / 2025, pág. 249 / 342 32 / 99 Region Identifier SEQ ID NO: Amino Acid Sequence cagcctgcagccggaagattttgcgacctattgccagcagtttg attatat gccgctgaccttttggccagggcaccaaagtggaaattaa acgc LFR1 (Kabat) 83 gatattcagatgacccagagcccgagc ggcgatcgcgtgaccattacctgc LFR2 (Kabat) 84 tggtatcagcagaaaccgggcaaagcgccgaaactgctgattat LFR3 (Kabat) 85 ggcgtgccgagccgctttagcggcagcggcagcggc LFR4 (Kabat) 86 tttggccagggcaccaaagtggaaattaaacgc Table 2: Additional sequence information Description SEQ ID NO: Amino Acid Sequence Signal Peptide CD8A 87 MALPVTALLLPLALLLHAARP Spacer / Hinge CD8A 88 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHT RGLDFACD Transmembrane Domain CD28 89 FWVLVVVGGVLACYSLLVTVAFIIFWV Co-stimulatory Domain CD28 90 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRD FAAYRS Co-stimulatory Domain 4-1BB 91 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCEL Zeta Signaling Domain CD2 92 VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR 4GS x 3 ligand 93 GGGGSGGGGSGGGGS CAR exemplary Comprising: VL (WTB1) and VH (BIL03) joined by ligand CD8A_WTB1_3*G4S linker_BIL03_CD8as pacer_CD28TM_CD2 8costimulatory domain_41 BBcostim ulatory domain_ CD3zeta signaling 94 MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVG DRVTITCRASQYIYDYLNWYQQKPGKAPKLLIYAASY LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQY HHPSTFGQGTKVEIKRGGGGSGGGGSGGGGSEVQL LESGGGLVQPGGSLRLSCAASGFTFRNHDMGWVR QAPGKGLEWVSAISGSGGSTYYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTEFDYRS PGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVT VAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQE EDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPR Petition 870250078796, dated 03 / 09 / 2025, pages 250 / 342 33 / 99 Description SEQ ID NO: Amino Acid Sequence G B1 123 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQ QKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQFDYMPLTFGQGTKVEIKR Example nfP2X7 receptor epitope sequences E200 epitope 95 GHNYTTRNILPGLNITC E200 epitope variant (E200') 96 GHNYTTRNILPGLNIT E200 epitope Cys to Ser modification 97 GHNYTTRNILPGLNITS Extended E200 Cys to Ser modification 98 GHNYTTRNILPGLNITSTFHK Extended E200' Cys to Ser modification (22 aa) 99 GHNYTTRNILPGLNITSTFHKT E200” extended Cys modification to Ser 100 GHNYTTRNILPGLNITSTFHKTC Pep17 101 GHNYTTRNILPGLNITSTFHKTS Extended Pep17 (27 aa) 102 GHNYTTRNILPGLNITSTFHKTSGSGK E200 + G4S ligand 103 GHNYTTRNILPGLNITSGGGGS E200 + 2xG4S linker 104 GHNYTTRNILPGLNITSTFHKTGS Peptide E200_extended 17v4 (22 aa) 107 GHNYTTRNILPGLNITSTFHGS Peptide E200_extended 17v5 (19aa) 108 GHNYTTRNILPGLNITSGS E200_extended 17v6 peptide (22 aa) (extended E200 epitope in N-terminal region) 109 DFPGHNYTTRNILPGLNITSGS E200_extended 17v7 peptide (25 aa) + ligand 110 DFPGHNYTTRNILPGLNITSGGGGS Peptide 111 DFPGHNYTTRNILPGLNITSGGGGSGGGGS Petition 870250078796, dated 03 / 09 / 2025, pages 251 / 342 34 / 99 Description SEQ ID NO: Amino Acid Sequence E200_extended 17v8 (30 aa) + ligand Peptide E200_extended 17v9 (35 aa) + ligand 112 DFPGHNYTTRNILPGLNITSGGGGSGGGGSGGGGGS Peptide E200_extended 17v10 (30 aa) 113 DFPGHNYTTRNILPGLNITSTFHKTSGSGK Peptide E200_extended 17v11 (32 aa) + ligand 114 DFPGHNYTTRNILPGLNITSTFHKTSGSGKGS Peptide E200_extended 17v12 (35 aa) + ligand 115 DFPGHNYTTRNILPGLNITSTFHKTSGSGKGGGGS Peptide E200_extended 17v13 (25 aa) + linker 116 DFPGHNYTTRNILPGLNITSTFHGGGGS Peptide E200_extended 17v14 (22 aa) + linker 117 GHNYTTRNILPGLNITSTFHGGGGS Peptide E200_extended 17v15 (30 aa) + linker 118 DFPGHNYTTRNILPGLNITSTFHKTGGGGS Peptide E200_extended 17v16 (27 aa) + 119 GHNYTTRNILPGLNITSTFHKTGGGGS E300 120 KYYKENNVEKRTLIK E300 variant 121 KYYKENNVEKRTLIKVF Composite epitope 122 GHNYTTRNILPGAGAKYYKENNVEK Table 3: Sequence information for sdAb-based CARs of the invention Clone Region SEQ ID NO: Amino Acid Sequence 4A7 3aB9 3aD9 HCDR1 124 DEDMG 4A7 HCDR2 125 AIDDAGDSTYYADSVKG 3aB9 HCDR2 126 AISSDGYSTYYADSVKG 3aD9 HCDR2 127 AIDSPGYSTYYADSVKG 4A7 HCDR3 128 VYPYDTVFDY 3aB9 HCDR3 129 YPYDTPFDY 3aD9 HCDR3 130 VYPYDSYFDY Petition 870250078796, dated 03 / 09 / 2025, pages 252 / 342 35 / 99 Clone Região SEQ ID NO: Sequência de Aminoácidos 4A7 3aB9 3aD9 HFR1 131 EVQLLESGGGLVQPGGSLRLSCAASGFRIS 4A7 3aB9 3aD9 HFR2 132 WVRQAPGKGLEWVS 4A7 3aB9 3aD9 HFR3 133 RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK 4A7 3aB9 3aD9 HFR4 134 WGQGTLVTVSS 4A7 VH 135 EVQLLESGGGLVQPGGSLRLSCAASGFRISDEDMG WVRQAPGKGLEWVSAIDDAGDSTYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCAKVYPYDT VFDYWGQGTLVTVSS 3aB9 VH 136 EVQLLESGGGLVQPGGSLRLSCAASGFRISDEDMG WVRQAPGKGLEWVSAISSDGYSTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKVYPYDTP FDYWGQGTLVTVSS 3aD9 VH 137 EVQLLESGGGLVQPGGSLRLSCAASGFRISDEDMG WVRQAPGKGLEWVSAIDSPGYSTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKVYPYDSY FDYWGQGTLVTVSS 4A7-CAR 138 MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPG GSLRLSCAASGFRISDEDMGWVRQAPGKGLEWVS AIDDAGDSTYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCAKVYPYDTVFDYWGQGTLVTVSS TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH TRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWV RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR DFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR 3aB9-CAR 139 MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPG GSLRLSCAASGFRISDEDMGWVRQAPGKGLEWVS AISSDGYSTYYADSVKGRFTISRDNSKNTLYLQMNS LRAEDTAVYYCAKVYPYDTPFDYWGQGTLVTVSST TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHT RGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWV RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR DFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY Petition 870250078796, dated 03 / 09 / 2025, pages 253 / 342 36 / 99 Clone Region SEQ ID NO: Amino Acid Sequence NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR 3aD9-CAR 140 MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPG GSLRLSCAASGFRISDEDMGWVRQAPGKGLEWVS AIDSPGYSTYYADSVKGRFTISRDNSKNTLYLQMNS LRAEDTAVYYCAKVYPYDSYFDYWGQGTLVTVSST TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHT RGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWV RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR DFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR Detailed description of the modalities

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

[081] A person skilled in the art will recognize that many methods and materials similar or equivalent to those described in the present invention can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

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

[083] All patents and publications mentioned in the present invention Petition 870250078796, dated 03 / 09 / 2025, pages 254 / 342 37 / 99 are incorporated by reference in full.

[084] The present invention seeks to address one or more deficiencies in the prior art and is based on the inventors' recognition that a specific antigen recognition domain provides an anti-nfP2X7 receptor CAR function. More specifically, preferred CARs of the invention are understood to provide higher affinity for binding to cancer cells expressing nP2X7 receptor and therefore have a stronger ability to kill cells (potency). The inventors have found that particular single-domain CARs (based on sdAb) and a particular scFv-based CAR provide superior efficacy compared to prior art sdAb-based CARs. Definitions - general

[085] Unless otherwise defined, the technical and scientific terms used in the present invention have the same meaning as commonly understood by a person skilled in the art to which this invention pertains.

[086] For the purposes of interpreting this descriptive report, the following definitions will generally apply and, where appropriate, terms used in the singular will also include the plural and vice versa.

[087] Throughout this descriptive report, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps, or group of compositions of matter shall be taken as encompassing one and a plurality (i.e., one or more) of these steps, compositions of matter, groups of steps, or groups of compositions of matter. Thus, as used in the present invention, the singular forms one, an, and the, a include the plural aspects and vice versa, unless the context clearly determines otherwise. For example, Petition 870250078796, dated 03 / 09 / 2025, pages 255 / 342 38 / 99 reference to “a” includes a single one, as well as two or more; reference to “an” includes a single one, as well as two or more; reference to “the” includes a single one, as well as two or more, and so on.

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

[089] Any example or embodiment of the present invention in the present invention shall be taken as applying mutatis mutandis to any other example or embodiment of the invention unless specifically mentioned otherwise.

[090] Unless specifically defined otherwise, all technical and scientific terms used in the present invention shall be taken to have the same meaning as commonly understood by a person skilled in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry and biochemistry).

[091] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques used in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DM Glover and BD Hames (editors), Petition 870250078796, dated 03 / 09 / 2025, pp. 256 / 342 39 / 99 DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FM Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to the present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and JE Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to the present).

[092] The terms and / or, for example, X and / or Y, will be understood as meaning X and Y or X or Y and will be taken to provide explicit support for both meanings or for either meaning.

[093] As used in the present invention, the term derived from will be taken to indicate that a specified integer can be obtained from a particular source although not necessarily directly from that source.

[094] When comparing amino acid sequences, the sequences must be compared relative to a comparison window that is determined by the length of the polypeptide. The comparison window may include additions or deletions (i.e., gaps) of about 20% or less compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences to align a comparison window can be conducted by computerized implementations of algorithms such as the BLAST family of programs, as, for example, 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. Petition 870250078796, dated 03 / 09 / 2025, pages 257 / 342 40 / 99 (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). These two 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 be found in Unit 19.3 of Ausubel et al (Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998).

[095] “Purinergic receptor” generally refers to a receptor that uses a purine (such as ATP) as a ligand.

[096] “P2X7 receptor” generally refers to a purinergic receptor formed from three protein subunits or monomers, with at least one of the monomers having an amino acid sequence substantially as shown in SEQ ID NO: 141 below:

[097] SEQ ID NO: 141 MPACCSCSDVFQYETNKVTRIQSMNYGTIKWFFHVIIFSYVCFALVSDKLYQRKEPV IS SVHTKVKGIAEVKEEIVENGVKKLVHSVFDTADYTFPLQGNSFFVMTNFLKTEGQEQRL CPEYPTRTLCSSDRGCKKGWMDPQSKGIQTGRCVVYEGNQKTCEVSAWCPIEAVE EAPRPALLNSAENFTVLIKNNIDFPGHNYTTRNILPGLNITCTFHKTQNPQCPIFRLGDIF RETGDNFSDVAIQGGIMGIEIYWDCNLDRWFHHCRPKYSFRRLDDKTTNVSLYPGYNF RYAKYYKENNVEKRTLIKVFGIRFDILVFGTGGKFDIIQLVVYIGSTLSYFGLAAVFIDFLID TYSSNCCRSHIYPWCKCCQPCVVNEYYYRKKCESIVEPKPTLKYVSFVDESHIRMVNQ QLLGRSLQDVKGQEVPRPAMDFTDLSRLPLALHDTPPIPGQPEEIQLLRKEATPRSRD SPVWCQCGSCLPSQLPESHRCLEELCCRKKPGACITTSELFRKLVLSRHVLQFLLLYQ EPLLALDVDSTNSRLRHCAYRCYATWRFGSQDMADFAILPSCCRWRIRKEFPKSEG Petition 870250078796, dated 03 / 09 / 2025, pages 258 / 342 41 / 99 Q YSGFKSPY.

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

[099] Functional P2X7 receptor generally refers to a form of the P2X7 receptor having three intact binding sites or clefts for ATP binding. When bound to ATP, the functional receptor forms a non-selective sodium / calcium channel that converts into a pore-like structure allowing the influx of calcium ions and molecules up to 900 Da into the cytosol, which can induce programmed cell death. In normal homeostasis, the expression of functional P2X7 receptors is generally limited to cells that undergo programmed cell death such as thymocytes, dendritic cells, lymphocytes, macrophages, and monocytes. There may also be some expression of functional P2X7 receptors in erythrocytes and other Petition 870250078796, dated 03 / 09 / 2025, pages 259 / 342 42 / 99 types of cells.

[100] “Dysfunctional P2X7 receptor” (also referred to as “non-functional” or (nf)P2X7) is a P2X7 receptor that has an impaired response to ATP such that it is unable to form an apoptotic pore under normal physiological conditions. A dysfunctional P2X7 receptor (or nfP2X7 receptor) generally refers to a form of a P2X7 receptor having a conformation, distinct from functional P2X7, whereby the receptor is unable to form an apoptotic pore, but is still able to operate as a non-selective channel by maintaining a single functional ATP binding site situated between adjacent monomers. An example arises where one or more of the monomers has a cis isomerization at Pro210 (according to the sequence established in SEQ ID NO: 141). Isomerization can originate from any molecular event that leads to improper monomer folding, including, for example, primary monomer sequence mutation or abnormal post-translational processing.One consequence of isomerization is that the receptor is unable to bind ATP at one, or more particularly two, ATP-binding sites on the trimer and, as a consequence, is unable to extend the channel opening. Under these circumstances, the receptor cannot form a pore, and this limits the extent to which calcium ions can enter the cytosol. Dysfunctional P2X7 receptors are expressed in a wide range of epithelial and hematopoietic cancers. As used in the present invention, the term “dysfunctional P2X7 receptors” can be used interchangeably with the term “non-functional P2X7 receptors” or “nfP2X7 receptors”.

[101] “Cancer-associated P2X7 receptors” are generally P2X7 receptors that are found in cancer cells (including preneoplastic, neoplastic, malignant, benign or metastatic cells), but not in non-cancerous or normal cells. Petition 870250078796, dated 03 / 09 / 2025, pages 260 / 342 43 / 99

[102] “Epitope E200 generally refers to an epitope having the sequence GHNYTTNILPGLNITC (SEQ ID NO: 95) and variants thereof (e.g., SEQ ID NOs: 96 to 119). The antigen recognition domains of the CARs of the invention are those that are preferentially capable of binding to E200 or a variant thereof.

[103] “Epitope E300 generally refers to an epitope having the sequence KYYKENNVEKRTLIK and variants thereof (e.g., SEQ ID Nos: 120 and 121).

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

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

[106] The terms “binds to,” “specifically binds to,” or “specific to,” with respect to the antigen-binding domain of a CAR, 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 not Petition 870250078796, dated 03 / 09 / 2025, pp. 261 / 342 44 / 99 contradicts the definition that the antigen-binding domain is specific. An antigen-binding domain that binds specifically to an antigen may also bind to different allelic forms of the antigen (allelic variants, splice variants, isoforms, etc.) or homologous variants of that antigen from the same genetic family. This cross-reactivity is typical of many antibodies and, therefore, does not contradict the definition that the antigen-binding domain is specific.

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

[108] The terms “immune cell” or “immune effector cell” refer to a cell that may be part of the immune system, the adaptive (i.e., cellular or humoral) or innate immune system and performs an effector function. Petition 870250078796, dated 03 / 09 / 2025, pages 262 / 342 45 / 99 particularly such as alpha-beta T cells, NK cells, NKT cells, B cells, Breg cells, Treg cells, innate lymphoid cells (ILCs), cytokine-induced killer cells (CIKs), lymphokine-activated killer cells (LAKs), gamma-delta T cells, mesenchymal stem cells or mesenchymal stromal cells (MSCs), monocytes or macrophages or any hematopoietic progenitor cells such as pluripotent stem cells and early progenitor subsets that can mature or differentiate into somatic cells. Cells can be naturally occurring or generated by cytokine exposure, genetically modified / artificial cells (such as iPSCs and other types of artificial cells). The immune cell can be a subset of artificial cells including induced pluripotent stem cells and cells matured from them.Preferred immune cells are cells with cytotoxic effector function such as alpha-beta T cells, NK cells, NKT cells, ILC, CIK cells, LAK cells, or gamma-delta T cells. "Effector function" means a specialized function of a cell; for example, in a T cell, an effector function might be cytolytic activity or helper cell activity, including cytokine secretion.

[109] The term “treat” (treatment of) a disorder, as used in the present invention, means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by an individual.

[110] The term “expression”, as used in the present invention, is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter in a cell.

[111] “Antibodies” or “immunoglobulins” or “Igs” are gamma globulin proteins that are found in the blood or other body fluids of vertebrates that function in the immune system to bind antigen, consequently identifying and / or neutralizing foreign objects.

[112] Antibodies are generally a heterotetrameric glycoprotein Petition 870250078796, dated 03 / 09 / 2025, pp. 263 / 342 46 / 99 is composed of two identical light chains (L) and two identical heavy chains (H). Each L chain is linked to an H chain by a covalent disulfide bond. The two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges.

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

[114] Antibodies can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated α, δ, ε, γ, and μ, respectively. The α and γ 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, IgAI, and IgA2. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, referred to as kappa and lambda, based on the amino acid sequences of their constant domains.

[115] The constant domain includes the Fc portion comprising the carboxy-terminal portions of both H chains held together by disulfide bonds. The effector functions of antibodies such as ADCC are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain cell types.

[116] The pairing of a Vh and Vl together forms a “region Petition 870250078796, dated 03 / 09 / 2025, pages 264 / 342 The variable domain, or "variable domain," includes the amino-terminal domains of the antibody's heavy or light chain. The variable domain of the heavy chain may be referred to as "VH." The variable domain of the light chain may be referred to as "VL." The V domain contains an "antigen-binding site" that affects antigen binding and defines the specificity of a particular antibody for its particular antigen. The V regions span approximately 110 amino acid residues and consist of relatively invariant stretches referred to as framework regions (FRs) (usually about 4) of 15-30 amino acids separated by shorter regions of extreme variability referred to as "hypervariable regions" (usually about 3) that are each generally 9-12 amino acids long. The FRs largely adopt a β-sheet conformation, and the hypervariable regions form loops that connect to, and in some cases are part of, the β-sheet structure.

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

[118] The sequences of complementarity-determining regions (CDRs) of an antigen-binding protein can be defined according to any of a number of different numbering systems, including the IMGT numbering system, Kabat or Chotia systems. It is well understood within the scope of the skilled in the art to be able to identify and determine the CDRs after knowledge of the total variable heavy and light sequences.

[119] The description and definitions of variable regions and parts thereof, immunoglobulins, antibodies and fragments thereof in the present invention Petition 870250078796, dated 03 / 09 / 2025, pages 265 / 342 48 / 99 can be further clarified by the discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991, Bork et al., J Mol. Biol. 242, 309-320, 1994, Chothia and Lesk J. Mol Biol. 196:901-917, 1987, Chothia et al. Nature 342, 877-883, 1989, Martin (“enhancedChothia; Mol Immunol. (2008) 45:3832-9; and / or or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

[120] As used in the present invention, the term “complementarity-determining regions” (syn. CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of a variable region of antibody whose presence is the main contributor to specific antigen binding. Each variable region domain (Vh or Vl) typically has three CDRs identified as CDR1, CDR2, and CDR3. The Vh CDRs are also referred to in the present invention as CDR Hi, CDR H2, and CDR H3, respectively, where CDR H1 corresponds to CDR 1 of Vh, CDR H2 corresponds to CDR 2 of Vh, and CDR H3 corresponds to CDR 3 of Vh.In one example, the amino acid positions assigned to CDRs and FRs are defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to in the present invention as “the Kabat numbering system”). In another example, the amino acid positions assigned to CDRs and FRs are defined according to the Enhanced Chothia Numbering Scheme (http: / / www.bioinfo.org.uk / mdex.html). The present invention is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including the canonical or Chothia and Lesk J. Mol. Biol. 196: 901-917, 1987; Chothia et al., Nature 342: 877-. Petition 870250078796, dated 03 / 09 / 2025, pages 266 / 342 49 / 99 883, 1989; and / or Al-Lazikani et al., J. Mol. Biol. 273: 927-948, 1997; the numbering system of Honnegher and Plükthun J. Mol. Biol. 309: 657-670, 2001; or the IMGT system discussed in Giudicelli et al., Nucleic Acids Res. 25: 206-211 1997.

[121] “Structure regions” (FRs) are those variable region residues different from CDR residues. The VH FRs are also referred to in the present invention as FR H1, FR H2, FR H3 and FR H4, respectively, wherein FR H1 corresponds to VH FR 1, FR H2 corresponds to VH FR 2, FR H3 corresponds to VH FR 3 and FR H4 corresponds to VH FR 4. Similarly, the VL FRs are also referred to in the present invention as FR L1, FR L2, FR L3 and FR L4, respectively, wherein FR L1 corresponds to VL FR 1, FR L2 corresponds to VL FR 2, FR L3 corresponds to VL FR 3 and FR L4 corresponds to VL FR 4.

[122] An “antigen-binding site” generally refers to a molecule that includes at least the structure and hypervariable regions that are necessary to impart antigen-binding function to a V domain. An antigen-binding site may be in the form of an antibody or an antibody fragment (such as an mAb, single domain (SD)-mAb, dAb, Fab, SD-Fv, Fd, SD-Fv, Fv, F(ab')2 or scFv) in a method described in the present invention.

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

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

[125] The “Fab fragment” consists of an entire L chain together Petition 870250078796, dated 03 / 09 / 2025, pages 267 / 342 50 / 99 with the variable region domain of the H chain (VH) and the first constant domain of a heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, that is, it has a single antigen-binding site.

[126] A “Fab’ fragment differs from Fab fragments by having a few additional residues at the carboxy-terminal end of the CH1 domain including one or more cysteines from the antibody hinge region. Fab’-SH is the designation in the present invention for Fab’ in which the cysteine ​​residue(s) of the constant domains contain a free thiol group.

[127] An “F(ab')2 fragment” roughly corresponds to two Fab fragments linked by disulfide having divalent antigen-binding activity and is also capable of cross-linking antigen.

[128] A “Fv” is the minimal antibody fragment that contains a complete antigen-binding and recognition site. This fragment consists of a dimer of a heavy chain variable region domain and a light chain variable region domain in tight, non-covalent association.

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

[130] “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments comprising the VH and VL antibody domains connected to form a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker. Petition 870250078796, dated 03 / 09 / 2025, pages 268 / 342 51 / 99 between the VHe and VL domains allow scFv to form the desired structure for antigen binding.

[131] A “single variable domain” is half of an Fv (comprising only three CDRs specific to an antigen) that has the ability to recognize and bind antigen, although generally at a lower affinity than the whole binding site.

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

[133] Diabodies can be bivalent or bispecific. Bispecific diabodies are heterodimers of two sFv “crossover” fragments in which the VH and VL domains of the two antibodies are present in different polypeptide chains. Triabodies and tetrabodies are also commonly known in the art.

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

[135] A “human antibody” refers to an antibody that has an amino acid sequence that corresponds to that of an antibody produced Petition 870250078796, dated 03 / 09 / 2025, pages 269 / 342 52 / 99 by a human. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous sites have been disabled.

[136] “Humanized” forms of non-human antibodies (e.g., rodents) are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (receptor antibody) in which residues from a hypervariable region of the receptor are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as a mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and antibody capacity. In some cases, residues from the structure region (FR) of the human immunoglobulin are replaced by corresponding non-human residues. Additionally, humanized antibodies may comprise residues that are not found in the receptor antibody or the donor antibody. These modifications are made to further refine antibody performance.In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulin and all or substantially all of the RFs are those of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[137] “Monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the Petition 870250078796, dated 03 / 09 / 2025, pages 270 / 342 53 / 99 individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor quantities. Monoclonal antibodies are highly specific, being directed against a single antigenic site or determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized without contamination by other antibodies. Monoclonal antibodies can be prepared by hybridoma methodology. Monoclonal antibodies can also be isolated from phage antibody libraries using molecular manipulation techniques.

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

[139] As used in the present invention, the term “antigen” is intended to include substances that bind to or evoke the production of one or more antibodies and may include, but are not limited to, proteins, peptides, Petition 870250078796, dated 03 / 09 / 2025, pages 271 / 342 54 / 99 polypeptides, oligopeptides, lipids, carbohydrates and combinations thereof, for example, a glycosylated protein or a glycolipid. The term antigen as used in the present invention refers to a molecular entity that can be expressed on a target cell and that can be 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 perform high-affinity binding to a structure.

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

[141] The term target cell as used in the present invention refers to a cell that expresses nfP2X7 receptor. The target cell can be a cancerous cell or any other diseased cell.

[142] The term disorder or condition means a functional alteration or abnormality in an individual such as cancer, an autoimmune disorder, or an infection by a virus, bacteria, parasite, or others.

[143] For example, a nucleic acid or peptide naturally present in a living animal is not isolated, but the same nucleic acid or peptide is partially or totally separated from materials Petition 870250078796, dated 03 / 09 / 2025, pages 272 / 342 55 / 99 coexisting in its natural state is "isolated". An isolated nucleic acid or protein can also exist in a non-native environment, such as in a host cell.

[144] The term “autologous”, as used in the present invention, refers to any material derived from the same individual, into whom it is subsequently reintroduced.

[145] The term “allogeneic”, as used in the present invention, refers to any material derived from a different individual of the same species as the individual into whom the material is subsequently reintroduced.

[146] The terms “therapeutically effective quantity” or “therapeutically effective population” mean a quantity, for example, of a population of cells that provides a therapeutic benefit to an individual. Chimeric antigen receptors

[147] Generally, an “antigen-binding domain” or “antigen recognition domain” refers to the region of the CAR that specifically binds to an antigen (and is thus able to target a cell containing the antigen). The CARs of the invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is for binding to 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, full-length heavy chain, Fab fragments, single-chain Fv fragments (scFv), antibodies, or divalent single-chain diabodies. Any molecule that specifically binds to a given antigen such as affibodies or ligand-binding domains from naturally occurring receptors may be used as an antigen-binding domain. Frequently the antigen-binding domain is an scFv.Normally, in. Petition 870250078796, dated 03 / 09 / 2025, pages 273 / 342 56 / 99 In an scFv, the variable regions of an immunoglobulin heavy chain and light chain are fused by a flexible ligand to form an scFv. Such a ligand can be, for example, the (G4 / Si)3-ligand and variations thereof, but those skilled in the art will recognize that various sequences and formats of ligand can be used.

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

[149] A “signal peptide” refers to a sequence of peptides that directs the transport and localization of the protein in a cell, for example, in a certain cell organelle (such as the endoplasmic reticulum) and / or the cell surface.

[150] “Spacer” or “hinge” as used in the present invention refers to the hydrophilic region that lies between the antigen-binding domain and / or transmembrane domain. The CARs of the invention may comprise an extracellular spacer domain, but it is also possible to omit such a spacer. The spacer may include, for example, Fc fragments of antibodies or fragments thereof, hinge regions of antibodies or fragments thereof, CH2 or CH3 regions of antibodies, accessory proteins, artificial spacer sequences, or combinations thereof. A prominent example of a spacer is the CD8alpha hinge.

[151] The transmembrane domain of the CAR can be derived from Petition 870250078796, dated 03 / 09 / 2025, pages 274 / 342 57 / 99 any desired natural or synthetic source for such a domain. When the source is natural, the domain can be derived from any transmembrane or membrane-bound protein. The transmembrane domain can be derived, for example, from CD8α CD28. When the antigen recognition and key signaling modules (domains) are in two (or even more) polypeptides, then the CAR can have two (or more) transmembrane domains. The splitting of two antigen recognition and key signaling modules allows for small molecule-dependent, titratable, and reversible control over CAR cell expression (Wu et al., 2015, Science 350: 293-303) due to small molecule-dependent heterodimerization domains in each CAR polypeptide.

[152] The cytoplasmic domain (or intracellular signaling domain) of CAR is responsible for activating at least one of the normal effector functions of the immune cell in which CAR is expressed. “Effector function” means a specialized function of a cell, for example, in a T cell an effector function might be cytolytic activity or helper cell activity including cytokine secretion. The intracellular signaling domain refers to the part of a protein that transduces the effector function signal and directs the cell expressing CAR to perform a specialized function. The intracellular signaling domain may include any complete, mutated, or truncated part of the intracellular signaling domain of a given protein sufficient to transduce a signal that initiates or blocks immune cell effector functions.

[153] The CAR signaling domain can be any suitable domain that is capable of inducing, or participating in the induction of, an intracellular signaling cascade after CAR activation as a result of antigen recognition by the CAR antigen recognition domain. The signaling domain of a CAR will be specifically chosen Petition 870250078796, dated 03 / 09 / 2025, pages 275 / 342 58 / 99 depending on the desired cellular outcome after CAR activation. Although there are many possible signaling domains, when used in immunotherapy and cancer therapy, signaling domains can be grouped into two general categories based on the receptor from which they are derived, namely activating receptors and co-stimulatory receptors. Therefore, in some modalities, the CAR signaling domain includes a portion derived from an activating receptor. In some modalities, the signaling domain includes a portion derived from a co-stimulatory receptor.

[154] As used throughout the descriptive report, the term “activation receptor” refers to receptors, or coreceptors, that form a component of, or are involved in the formation of, the T cell receptor (TCR) complex or receptors involved in the specific activation of immune cells as a result of the recognition of an antigenic stimulus or other immunogenic stimuli.

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

[156] Some examples of intracellular signaling domains for use in CARs include T cell receptor (CR) cytoplasmic signaling sequences and coreceptors that initiate signal transduction after antigen receptor engagement.

[157] Generally, T cell activation can be mediated by two distinct classes of cytoplasmic signaling sequences, firstly those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and secondly those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences, co-stimulatory signaling domain). Therefore, a domain of Petition 870250078796, dated 03 / 09 / 2025, pages 276 / 342 59 / 99 Intracellular signaling of a CAR may comprise one or more primary cytoplasmic signaling domains and / or one or more secondary cytoplasmic signaling domains.

[158] Primary cytoplasmic signaling sequences that act in a stimulatory mode may contain ITAMs (immunoreceptor tyrosine-based activation motifs) signaling motifs.

[159] Examples of ITAMs containing primary cytoplasmic signaling sequences frequently 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. The most prominent is the sequence derived from CD3 zeta.

[160] The cytoplasmic domain of the CAR can be designed to comprise the CD3-zeta signaling domain alone or combined with any other desired cytoplasmic domain(s). The cytoplasmic domain of the CAR may comprise a CD3 zeta chain portion and a co-stimulatory signaling region. The co-stimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient lymphocyte response to an antigen. Examples of a co-stimulatory molecule 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.

[161] Cytoplasmic signaling sequences in the cytoplasmic signaling portion of the CAR can be linked together with or without a linker in a random or specified order. A short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length, can form Petition 870250078796, dated 03 / 09 / 2025, pages 277 / 342 The 60 / 99 linkage. A prominent linker is the glycine-serine doublet.

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

[163] The extracellular portion or transmembrane domain or cytoplasmic domain of a CAR may comprise a heterodimerization domain with the aim of splitting the key antigen recognition and signaling modules of the CAR.

[164] A CAR for use in accordance with the present invention, that is, a CAR comprising an E200 nfP2X7 binding domain, may be designed to comprise any portion or part of the aforementioned domains as described in the present invention in any order and / or combination resulting in a functional CAR.

[165] The CARs as disclosed in the present invention, or polypeptide(s) derived therefrom, nucleic acid molecule(s) or recombinant expression vector cells encoding said CARs, or cell populations expressing said CARs, may be isolated and / or purified. The term “isolated” means altered or removed from the natural state. For example, an isolated cell population means an enrichment of such cells and separation from other cells that are normally associated in their natural occurrence state with said isolated cells. An isolated cell population means a substantially purified cell population that is a more homogeneous population Petition 870250078796, dated 03 / 09 / 2025, pages 278 / 342 61 / 99 of cells compared to those found in nature. Preferably, the enriched cell population comprises at least about 90% of the selected cell type. In particular aspects, the cell population comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% of the selected cell type.

[166] The affinity at which the CAR antigen recognition domain binds to the nfP2X7E200 recognition site on a cell (or the E200 sequence comprised in a binding molecule as described elsewhere in the present invention) may vary, but generally the binding affinity may be in the range of 100 μM, 1 nM, 10 nM, or 100 nM, preferably at least about 1 pM or 10 pM, even more preferably at least about 100 pM. Binders

[167] A linker may be a peptide having a length of up to 20 amino acids. The term “linked to” or “fused with” refers to a covalent bond, for example, a peptide bond formed between two moieties. Therefore, in the context of the present invention, the linker may 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 in the present invention may comprise a linker between the VH and VL of the antigen-binding domain and / or the antigen-binding domain and the hinge. Such linkers have the advantage that they can make it more likely that the different polypeptides of the fusion protein will fold independently and commit as expected.

[168] A person skilled in the art will be familiar with the design and use of various peptide ligands comprising various amino acids and of various lengths, which would be suitable for use as ligands according to the Petition 870250078796, dated 03 / 09 / 2025, pages 279 / 342 62 / 99 present invention. The linker may comprise various combinations of repeating amino acid sequences. The linker may be a flexible linker (such as those comprising repeats of glycine and serine residues), a rigid linker (such as those comprising lysine and glutamic acid residues flanking alanine repeats) and / or a cleavable linker (such as sequences that are susceptible to protease cleavage).

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

[170] In any embodiment, the peptide linker may include the amino acid sequence GGGGGS (a linker 6 amino acids in length, SEQ ID NO: 19) or even longer. The linker may be a series of repeating glycine and serine (GS) residues of different lengths, i.e., (Gs)n where n is any number from 1 to 15 or more. For example, the linker may be (GS)3 (i.e., GSGSGS) (SEQ ID NO: 20) or longer (Gs)11 (SEQ ID NO: 142) or longer. It will be recognized that n may be any number including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more. Nucleic acids

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

[172] In some embodiments of the invention, the nucleic acid molecule includes a nucleotide sequence that encodes the amino acid sequence set forth in SEQ ID NO: 4 and / or 12, such as SEQ ID NO: 94, or encodes a Petition 870250078796, dated 03 / 09 / 2025, pp. 280 / 342 63 / 99 functional variant thereof, in which the functional variant retains the ability to bind to the nfP2X7 receptor. In some embodiments, the nucleic acid molecule encodes a functional variant that includes an amino acid sequence that is at least 80% identical to SEQ ID NO: 94 and retains the ability to bind to the nfP2X7 receptor. In some embodiments, the nucleic acid molecule encodes a functional variant that includes an amino acid sequence that is at least 80% identical to SEQ ID NO: 94, retains the ability to bind to nfP2X7, and comprises an antigen recognition domain comprising the amino acid sequences of SEQ ID NOs 4 and 12.

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

[174] In some embodiments of the invention, the nucleic acid molecule includes a nucleotide sequence set out at SEQ ID NO: 74 and / or SEQ ID NO: 82. Petition 870250078796, dated 03 / 09 / 2025, pp. 281 / 342 64 / 99

[175] It would be understood by a person skilled in the art that any nucleotide sequence encoding a chimeric antigen receptor comprising the amino acid sequence set forth in SEQ ID NOs: 4 and / or 12, for example, SEQ ID NO: 94, or a functional variant thereof, is considered to be included in the present invention. For example, variants of SEQ ID NO: 94 are considered to comprise one or more different nucleic acids for SEQ ID NOs: 74 and / or 82, but which still encode identical amino acid sequences. 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.Therefore, every nucleotide sequence of the present invention that encodes a chimeric antigen receptor having the amino acid sequence set forth in SEQ ID NO: 94, or a functional variant thereof, also describes every possible silent variation of the nucleotide sequence. A person skilled in the art will recognize that every codon in a nucleic acid (except AUG, which is commonly the single codon for methionine, and TGG, which is commonly the single codon for tryptophan) can be modified to provide a functionally identical molecule. Consequently, every silent variation of a nucleotide sequence encoding a polypeptide is implied in every sequence described.

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

[177] As used in the present invention, the term “marker gene” Petition 870250078796, dated 03 / 09 / 2025, pp. 282 / 342 "65 / 99 selectable" includes any gene that confers a phenotype in a cell in which it is expressed, to facilitate the identification and / or selection of cells that are transfected or transformed with the construct.

[178] Selectable marker genes include any nucleotide sequences that, when expressed by a cell transformed with the construct, confer a phenotype on the cell that facilitates the identification and / or selection of these transformed cells. A range of nucleotide sequences encoding suitable selectable markers are known in the art (e.g., Mortesen, RM. and Kingston RE. Curr Protoc Mol Biol, 2009; Unit 9.5).Exemplary nucleotide sequences encoding selectable markers include: Adenosine deaminase (ADA) gene; cytokine 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 genes, puromycin resistance genes, bleomycin resistance genes, igromycin resistance genes, kanamycin resistance genes and ampicillin resistance gene; fluorescent reporter genes such as genes encoding green, red, yellow or blue fluorescent protein; and luminescence-based reporter genes such as the luciferase gene, among others, that allow for the optical selection of cells using techniques such as Fluorescence-Activated Cell Sorting (FACS).

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

[180] As established above, the nucleic acid construct can Petition 870250078796, dated 03 / 09 / 2025, pages 283 / 342 66 / 99 also includes one or more transcriptional control sequences. The term “transcriptional control sequence” should be understood as including any nucleic acid sequence that effects the transcription of an operationally connected nucleic acid. A transcriptional control sequence may include, for example, a leader, polyadenylation sequence, promoter, enhancer or upstream activation sequence, and transcription terminator. Typically, a transcriptional control sequence includes at least one promoter. The term “promoter” as used in the present invention describes any nucleic acid that confers, activates, or enhances the expression of a nucleic acid in a cell.

[181] In some embodiments, at least one transcriptional control sequence is operationally connected to the nucleic acid molecule of the invention. For the purposes of this descriptive report, a transcriptional control sequence is considered to be “operationally connected” to a given nucleic acid molecule when the transcriptional control sequence is capable of promoting, inhibiting, or otherwise modulating the transcription of the nucleic acid molecule. Therefore, in some embodiments, the nucleic acid molecule is under the control of a transcriptional control sequence, such as a constitutive promoter or an inducible promoter.

[182] The “nucleic acid construct” may be in any suitable form, such as a plasmid, phage, transposon, cosmid, chromosome, vector, etc., which is capable of replication when associated with appropriate control elements and which can transfer gene sequences, contained in 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.

[183] ​​A promoter can regulate the expression of an acid molecule. Petition 870250078796, dated 03 / 09 / 2025, pages 284 / 342 67 / 99 nucleic acid operationally connected constitutively, or differentially, with respect to the cell, tissue, or organ in which expression occurs. As such, the promoter may include, for example, a constitutive promoter, or an inducible promoter. A “constitutive promoter” is a promoter that is active under most environmental and physiological conditions. An “inducible promoter” is a promoter that is active under most environmental and physiological conditions. The present invention considers the use of any promoter that is active in a cell of interest. As such, a wide range of promoters would be readily determined by a person skilled in the art.

[184] 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 / chicken early enhancer β-actin (CAGG).

[185] 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 alcohols, antibiotics, steroids, metal ions, or other compounds. Examples of chemically inducible promoters include: tetracycline-regulated promoters (e.g., see U.S. Patent 5,851,794 and U.S. Patent 5,464,758); steroid-responsive promoters such as glucocorticoid receptor promoters (e.g., see U.S. Patent 5,512,483), ecdysone receptor promoters (e.g., see U.S. Patent 6,379,945), and the like; and metal-responsive promoters such as metallothionein promoters (e.g., see U.S. Patent 4,940,661, U.S. Patent 4,579,821 and U.S. Patent 4,601,978) among others.

[186] As mentioned above, control sequences may also include a terminator. The term “terminator” refers to a sequence of Petition 870250078796, dated 03 / 09 / 2025, pages 285 / 342 68 / 99 DNA at the end of a transcription unit that signals transcriptional termination. Terminators are untranslated 3' DNA sequences generally containing a polyadenylation signal, which facilitate the addition of polyadenylate sequences to the 3' end of a primary transcript. As with promoter sequences, the terminator can be any terminator sequence that is operable in the cells, tissues, or organs in which it is intended to be used. Suitable terminators would be known to a person skilled in the art.

[187] As will be understood, the nucleic acid construct of the invention may additionally include additional sequences, for example, sequences that allow enhanced expression, membrane or cytoplasmic transport, and localization signals. Non-limiting specific examples include an Internal Ribosome Entry Site (IRES).

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

[189] Methods are known in the art for the deliberate introduction (transfection / transduction) of exogenous genetic material, such as the nucleic acid construct of the present invention, into eukaryotic cells. As will be understood, the most suitable method for introducing the nucleic acid construct into the desired host cell depends 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 desired, or required, final viability of the transfected / transduced cells. Non-limiting examples of such methods include: chemical transfection with chemicals such as Petition 870250078796, dated 03 / 09 / 2025, pages 286 / 342 69 / 99 cationic polymers, calcium phosphate or structures such as liposomes and dendrimers; non-chemical methods such as electroporation, sonoporation, optical or thermal shock transfection; particle-based methods such as 'genetic weapon' distribution, magnetofection, or viral impalafection or transduction.

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

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

[192] The cell may be a “manipulated cell”, “genetically modified cell”, “immune cell” or “immune effector cell”, as described in the present invention. Additionally, the cell may be capable of differentiating into an immune cell. A cell that is capable of differentiating into an immune cell Petition 870250078796, dated 03 / 09 / 2025, pages 287 / 342 70 / 99 (for example, a T cell that will express the dysfunctional CAR P2X7) can be a stem cell, a multilineage progenitor cell, or an induced pluripotent stem cell.

[193] In any embodiment, the cell may be a T cell, in which optionally said T cell does not express TcRap, PD1, CD3 or CD96 (for example, by deletion or knockout of one of these genes at a genetic or functional level).

[194] In any modality, the cell can be an immune cell, in which optionally said cell does not express accessory molecules that can be checkpoint, exhaustion or apoptosis signaling receptors as well as ligands such as PD-1, LAG-3, TIGIT, CTLA-4, FAS-L and FAS-R, (for example, by knocking out one of these genes at a genetic or functional level).

[195] In some embodiments, the genetically modified cell includes two or more different CARs. For example, the genetically modified cell may include CARs of different architectures (e.g., with different signaling domains) but comprising the same antigen recognition sequence. Alternatively, the genetically modified cell may include CARs that have different antigen recognition domains but bind to the nfpX2 receptor. The CARs may bind to the same or different epitopes of the nfP2X7 receptor. In alternative embodiments, the genetically modified cell may comprise CARs for binding to different antigens (e.g., nP2X7 receptor and a different antigen on a cancer cell).

[196] In some embodiments of the invention, the genetically modified cell includes a nucleic acid molecule, or a nucleic acid construct that codes for two or more different CARs. In some Petition 870250078796, dated 03 / 09 / 2025, pages 288 / 342 71 / 99 embodiments of the invention, the genetically modified cell includes two or more nucleic acid molecules, or two or more nucleic acid constructs, each of which codes for a different CAR.

[197] As mentioned in the present invention, a “genetically modified cell” includes any cell comprising a non-naturally occurring and / or introduced nucleic acid construct or nucleic acid molecule covered by the present invention. The introduced nucleic acid molecule or nucleic acid construct may be maintained in the cell as a discrete DNA molecule or may be integrated into the cell's genomic DNA.

[198] The genomic DNA of a cell should be understood in its broadest context to include any and all endogenous DNA that comprises the genetic complement of a cell. As such, the genomic DNA of a cell should be understood as including chromosomes, mitochondrial DNA, and the like. As such, the term “genomically integrated” considers chromosome integration, mitochondrial DNA integration, and the like. The “genomically integrated form” of the construct may be all or part of the construct. However, in some embodiments, the genomically integrated form of the construct at least includes the nucleic acid molecule of the second aspect of the invention.

[199] As used in the present invention, the term “different CARs” or “different chimeric antigen receptors” refers to any two or more CARs that have non-identical antigen recognition and / or non-identical signaling domains. In one example, “different CARs” include two CARs with the same antigen recognition domains (for example, both CARs can recognize a dysfunctional P2X7 receptor) but have different signaling domains, such as one CAR having a different domain. Petition 870250078796, dated 03 / 09 / 2025, pages 289 / 342 72 / 99 signaling with a portion of an activating receptor and the other CAR having a signaling domain with a portion of a co-stimulatory receptor. As will be understood, at least one of two or more CARs in this mode will have an antigen recognition domain that recognizes the dysfunctional P2X7 receptor and the other CAR(s) may assume any suitable form and may be directed against any suitable antigen.

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

[201] In some modalities, the activation receptor (from which a signaling domain portion is derived) is the CD3 coreceptor complex or is an Fc receptor.

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

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

[204] In some embodiments, the genetically modified cell is further modified to constitutively express costimulatory receptors. Petition 870250078796, dated 03 / 09 / 2025, pp. 290 / 342 73 / 99

[205] As described above, a cellular immune response is typically induced only when an activation signal (typically in response to an antigen) and a co-stimulation signal are simultaneously experienced. Therefore, having a genetically modified cell according to some of the above modalities, which includes two or more CARs that in combination provide both an intracellular activation signal and an intracellular co-stimulation signal, ensures that a sufficient immune response can be induced in response to recognition by the CAR(s) of its cognate antigen. Alternatively, the genetically modified cell may include only one CAR, which has an antigen recognition domain that recognizes a dysfunctional P2X7 receptor, and may constitutively express co-stimulatory receptors, thereby increasing the likelihood of co-stimulation being provided simultaneously when the CAR is activated.Alternatively, the genetically modified cell can be further modified to constitutively express both costimulatory receptor(s) and its ligand(s). In this way, the cell is continuously experiencing costimulation and only needs the activation of one CAR, with a signaling domain including a portion from an activating receptor, for immune activation of the cell.

[206] Therefore, in some embodiments, the genetically modified cell expressing CAR is further modified to constitutively express costimulatory receptors. In further embodiments, the genetically modified cell is further modified to express ligands for the costimulatory receptors, thereby facilitating self-stimulation of the cell. Examples of CAR-expressing T cells that also express both costimulatory receptors and their cognate ligands (so as to induce self-stimulation) are known in the art and Petition 870250078796, dated 03 / 09 / 2025, pages 291 / 342 74 / 99 include, inter alia, those revealed in Stephen MT. et al. Nat Med, 2007; 13: 1440-9.

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

[208] Although any one or more cytokines may 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 a combination thereof.

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

[210] The immune cell may be a T cell, where optionally said T cell does not express TcRae, PD1, CD3 or CD96 (for example, by means of deletion or knockout of one of these genes at a genetic or functional level). Petition 870250078796, dated 03 / 09 / 2025, pages 292 / 342 75 / 99

[211] The immune cell may not express accessory molecules that may be signaling receptors associated with checkpoint, exhaustion or apoptosis as well as ligands such as PD-1, LAG-3, TIGIT, CTLA-4, FAS-L and FAS-R, (for example, by knockout or elimination, one of these genes at a genetic or functional level). Treatment and administration methods

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

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

[214] In preferred embodiments, the target cell is a cancer cell and the cell surface molecule of the cancer cell is an antigen that is associated with cancer. The antigen may be a tumor-specific antigen or a tumor-associated antigen. The antigen may be one that is associated with a particular type of cancer. For example, overexpression of the antigen may be associated with a specific cancer or a specific class of cancer. For example, where the cancer is breast cancer, the antigen may be associated with breast cancer but not with another form of cancer. Alternatively, the antigen may be associated with a class of cancers such as solid tumors but not associated with hematologic (i.e., liquid) tumors, or vice versa. The antigen may be associated with cancers of a particular lineage but not with others. For example, the antigen may be associated with sarcomas but not Petition 870250078796, dated 03 / 09 / 2025, pages 293 / 342 76 / 99 lymphomas or carcinoma. As used in the present invention, the term “associated with” in relation to cancer will be understood to mean that the expression of the antigen (whether increased or decreased) is considered a marker of cancer. It will be recognized that there may be low levels of expression of an antigen, but this does not equate to the antigen being “associated” with a given cancer.

[215] In preferred embodiments, the present invention provides a method of killing a target cell expressing nfP2X7 receptor, the method including exposing the cell expressing an nfP2X7 receptor to a genetically modified cell of the invention, thereby killing the target cell.

[216] According to preferred embodiments of the invention, the antigen recognition domain of the CAR of the invention is for directly recognizing the nfP2X7 receptor. As used in the present invention, the term “directly recognizes” includes direct binding of the CAR antigen recognition domain to the nfP2X7 receptor, or an epitope thereof. In another non-limiting example, the antigen recognition domain may directly bind to a processed form of the nfP2X7 receptor, which may be presented by antigen-presenting molecules such as the major histocompatibility complex (MHC).

[217] It will be recognized that, under certain circumstances, the CAR of the invention may indirectly recognize the nfP2X7 receptor or other antigen on a target cell. In such embodiments, the CAR may bind to a target cell via an intermediate. As such, the present invention also provides a method of killing a target cell, the method including exposing the target cell to a genetically modified cell of the invention, thereby killing the target cell. In some embodiments of the invention, the method of killing a target cell further includes the step of exposing the target cell to an intermediate. Petition 870250078796, dated 03 / 09 / 2025, pp. 294 / 342 77 / 99

[218] An intermediate can be a molecule such as a probe, polypeptide, or fusion protein that binds to or interacts directly with the CAR (such as through the antigen-binding domain) and that also binds to or interacts directly with an antigen on a target cell. Non-limiting examples of such intermediates include peptides, antibodies or fragments thereof, a Fab of an antibody, an scFv, a soluble engineered TCR, or an aptamer. The target cell antigen may comprise nfP2X7 receptor, including an nfP2X7 receptor epitope that is different from the epitope recognized by the CAR antigen-recognition domain.The antigen on the target cell may comprise an antigen that is not an nfP2X7 receptor (such as any other tumor-associated or tumor-specific antigen present on the surface of a cancer cell, such as, but not limited to, CD33 (Siglec-3), CD123 (IL3RA), CD135 (FLT-3), CD44 (HCAM), CD44V6, CD47, CD184 (CXCR4), CLEC12A (CLL1), LeY, FRp, MICA / B, CD305 (LAIR-1), CD366 (TIM-3), CD96 (TACTILE), CD133, CD56, CD29 (ITGB1), CD44 (HCAM), CD47 (IAP), CD66 (CEA), CD112 (Nectin2), CD117 (c-Kit), CD133, CD146 (MCAM), CD155 (PVR), CD171 (LICAM), CD200 (OX-2), CD221 (IGF1), CD227 (MUC1), CD243 (MRD1), CD246 (ALK), CD271 (LNGFR), CD19, CD20, GD2, and EGFRvll).

[219] The CAR may be able to directly recognize the intermediate or the intermediate may have a tag that is recognized by the CAR. In either case, the intermediate provides the specificity for the target cell while the genetically modified cell having the CAR provides the efficacy and directs an immune response against the target cell. Examples of a CAR that recognizes cells via an intermediate are known in the art, for example, European patent application EP 2651442. Such intermediates may also be referred to in the art as “binding molecules”. Additional examples of binding molecules (also referred to as BRiDGE molecules) Petition 870250078796, dated 03 / 09 / 2025, pp. 295 / 342 Suitable 78 / 99 molecules for directing an nfP2X7 receptor-binding CAR or a CAR of the present invention to a target cell are described in WO 2022 / 187906, incorporated herein by reference. Such binding molecules typically comprise a polypeptide comprising an E200 sequence, or derivative thereof, which can be recognized by the antigen recognition domain of the CARs of the present invention.

[220] The term aptamer, as used throughout the descriptive report, refers to any oligonucleic acid, polynucleic acid, peptide or polypeptide that specifically binds to, or preferentially forms a complex with, a target (specifically mesothelin).

[221] In some embodiments of the invention, the target cell is inside an individual’s body. In some embodiments, the individual is a human being. In some embodiments, the method further includes exposing the target cell to a genetically modified cytokine along with an exogenous cytokine.

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

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

[224] The terms treat, treating or treatment, as used in the present invention, should be understood as including within their scope one or more of the following results: (I) inhibiting to some extent the growth of a primary tumor in an individual, including decreasing and completely halting Petition 870250078796, dated 03 / 09 / 2025, pages 296 / 342 79 / 99 growth and including reducing the growth of the primary tumor after resection; (ii) inhibiting to some extent the growth and formation of one or more secondary tumors in an individual; (iii) reducing the number of tumor cells in an individual; (iv) reducing the size of a tumor in an individual; (v) inhibiting (i.e., reducing, decreasing, or completely stopping) tumor cell infiltration into peripheral organs; (vi) inhibiting (i.e., reducing, decreasing, or completely stopping) metastasis; (vii) improving an individual's life expectancy compared to the untreated state; (viii) improving an individual's quality of life compared to the untreated state; (ix) alleviating, abateing, or improving at least one symptom of cancer in an individual; (x) causing regression or remission of cancer in an individual; (xi) alleviating a condition in an individual that is caused by cancer; and (xii) stopping symptoms in an individual that are associated with cancer.

[225] The terms “prevent” or “prevention”, as used in the present invention, should be understood as including within their scope inhibiting the formation of a primary tumor in an individual, inhibiting the formation of one or more secondary tumors in an individual, or reducing or eliminating cancer recurrence in an individual in remission.

[226] The term “inhibition”, as used in the present invention, is intended to mean a decrease or reduction in the growth of a cancer, cancerous cell or tumor when compared with the growth in a control, such as an untreated cell or individual. In some embodiments, the growth may be decreased or reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to an untreated control.

[227] The inhibition of the growth of a cancer, tumor or cancer cell can be assessed by a range of methods known in the art. For example, for a cancer cell in vitro, cell growth can be Petition 870250078796, dated 03 / 09 / 2025, pages 297 / 342 The 80 / 99 ratio is determined by a suitable proliferation assay, or by a method that assesses the extent of incorporation of tritiated thymidine into cellular DNA over a given period of time. For a tumor or cancer cell present in vivo, tumor or cell growth can be determined, for example, by a suitable imaging method known in the art.

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

[229] A reference to “providing to an individual” refers to administering the genetically modified cell to the individual. Alternatively, the genetically modified cell may be generated in the individual. For example, the genetically modified cell may be generated in vivo so that the individual 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 an individual.

[230] The provision of a genetically modified cell expressing a CAR directed against a target cell expressing or displaying nfP2X7 receptor may be sufficient to provide effective immunotherapy against precancerous or cancerous cells; the provision of adjuvants along with the genetically modified cells may further enhance the induction of the immune response and may augment immunotherapy. Cytokines, preferably pro-inflammatory cytokines, are particularly suitable adjuvants for provision to an individual along with genetically modified cells. Petition 870250078796, dated 03 / 09 / 2025, pages 298 / 342 81 / 99 having CARs.

[231] Therefore, in some embodiments of the invention, the genetically modified cell is administered to the individual together with a cytokine. It should be understood that, as used throughout the descriptive report, the term “together with” includes the genetically modified cell being administered simultaneously with a cytokine or administered in combination with a cytokine. Consequently, when administered in combination with a cytokine, this can be considered as including a combination therapy whereby the immunotherapy of an individual includes both treatment with a cytokine and treatment with a genetically modified cell having a CAR directed against a target cell that expresses or displays nfP2X7 receptor, or an epitope thereof. In some embodiments, the cytokine is administered on a different day (>24 h) from the administration of the genetically modified cells. In other embodiments, the cytokine is administered on the same day (within 24 h) as the genetically modified cells.In additional forms, the cytokine(s) and the genetically modified cell are administered 18h, 12h, 6h, 4h, 2h, 1h, 45 min, 30 min, 15 min, 10 min, 5 min, 2 min, or 1 min apart.

[232] Suitable cytokine(s) for administration together with the genetically modified cell include(s) IL-2, IL-4, IL-6, IL-7, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, IFNa, IFNβ, IFNγ, GM-CSF, TGFβ and TNFα. Preferred cytokines include IL-7 and IL-15. Additionally, cytokines can be administered as recombinant forms, natural forms, or via delivery systems such as protein fusions, delivered as a nucleic acid sequence that is expressed in the genetically modified cell, or coupled with a polymer such as polyethylene glycol (PEG).

[233] The cell to be genetically modified can be obtained from Petition 870250078796, dated 03 / 09 / 2025, pages 299 / 342 82 / 99 any suitable source. In some embodiments of the invention, the cell to be genetically modified is an autologous cell, an autologous cell being a cell that expresses or displays the nfP2X7 receptor, or an epitope thereof. Advantageously, an autologous cell would not be recognized as 'foreign to the organism' by the individual's immune system and would therefore be tolerated by the individual. However, in some forms of cancer, suitable autologous cells may not be readily available. Therefore, in some embodiments of the invention, the cell to be genetically modified is an allogeneic or heterologous cell.

[234] It will be clearly understood that, although this descriptive report refers specifically to applications in humans, the invention is also useful for veterinary purposes. Thus, in all respects the invention is useful for domestic animals such as cattle, sheep, horses and poultry; for companion animals such as cats and dogs; and for zoo animals. Therefore, the general term individual or “individual to be / being treated” is understood as including all animals (such as humans, monkeys, dogs, cats, horses and cows).

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

[236] Individuals requiring treatment include those already having a Petition 870250078796, dated 03 / 09 / 2025, pages 300 / 342 83 / 99 benign, precancerous, or non-metastatic tumors, as well as those in which the occurrence or recurrence of cancer should be prevented. Individuals may have metastatic cells, including metastatic cells present in ascitic fluid and / or lymph nodes.

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

[238] Treatment effectiveness can be measured by assessing survival duration, time to disease progression, response rates (RR), duration of response and / or quality of life.

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

[240] The method is particularly useful for extending human survival, including general survival as well as survival without progression.

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

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

[243] The goal or outcome of treatment may be any one or more of the following: Petition 870250078796, dated 03 / 09 / 2025, pages 301 / 342 84 / 99 - to reduce the number of cancer cells; - to reduce the size of the primary tumor; - to inhibit (that is, to reduce to a certain extent and preferably stop) the infiltration of cancer cells into peripheral organs; - to inhibit (that is, to reduce to a certain extent and preferably stop) tumor metastasis; - to inhibit, to some extent, the growth of the tumor; - to alleviate, to some extent, one or more of the symptoms associated with the disorder.

[244] In one modality, individuals requiring treatment include those having a benign, precancerous, non-metastatic tumor.

[245] In one modality, the cancer is pre-cancerous or pre-neoplastic.

[246] In one modality, the cancer is a secondary cancer or metastasis. Secondary cancer can be located in any organ or tissue, and particularly those organs or tissues having relatively higher hemodynamic pressures, such as the lung, liver, kidney, pancreas, intestine, and brain. Secondary cancer can be detected in ascites fluid and / or lymph nodes.

[247] In one form, the cancer may be substantially undetectable.

[248] “Precancerous or “preneoplastic” generally refers to a condition or growth that precedes or typically develops into cancer. A “precancerous” growth may have cells that are characterized by abnormal cell cycle regulation, proliferation, or differentiation, which can be determined by cell cycle markers.

[249] Cancer can be a solid tumor or a “liquid” one. In other words, cancer can be a growth in a tissue (carcinoma, sarcoma, Petition 870250078796, dated 03 / 09 / 2025, pages 302 / 342 85 / 99 adenomas etc.) or it may be a cancer present in bodily fluids such as blood or bone marrow (e.g., lymphomas and leukemias).

[250] The term cancer will be understood to include benign, precancerous, preneoplastic or non-metastatic tumors or metastatic tumors. In some forms, cancer is metastatic, such as stage III or stage IV cancer.

[251] In some modalities, the type of cancer to be treated includes those having a benign, precancerous, pre-neoplastic, or non-metastatic tumor. A benign tumor will be understood as not being a malignant tumor and not invading nearby tissue or spreading to other parts of the body. Similarly, non-metastatic cancer will be understood as not invading nearby tissue or spreading to other parts of the body. Precancerous or pre-neoplastic generally refers to a condition or growth that precedes or develops into cancer. A precancerous growth may have cells that are characterized by abnormal cell cycle regulation, proliferation, or differentiation, which can be determined by cell cycle markers.

[252] In one modality, the cancer is a secondary cancer or metastasis. Secondary cancer can be located in any organ or tissue, and particularly those organs or tissues having relatively higher hemodynamic pressures, such as the lung, liver, kidney, pancreas, intestine, and brain. Secondary cancer can be detected in ascites fluid and / or lymph nodes.

[253] In certain modalities, the cancer that requires treatment may be a cancer characterized by low levels of dysfunctional P2X7 receptor expression.

[254] Cancers particularly suitable for prevention or Petition 870250078796, dated 03 / 09 / 2025, pages 303 / 342 86 / 99 treatment refers to those that express or overexpress the nfP2X7 receptor. The expression or overexpression of the nfP2X7 receptor in a cancer or a specific tumor can be determined by detecting the nfP2X7 receptor protein present in or on a cancer cell.

[255] Pre-neoplastic, neoplastic and metastatic cancers are particular examples to which the methods of the invention can be applied. Broad examples include breast tumors, colorectal tumors, adenocarcinomas, mesothelioma, bladder tumors, prostate tumors, germ cell tumor, hepatoma / colongiocarcinoma, carcinomas, neuroendocrine tumors, pituitary neoplasm, small round cell tumors, squamous cell carcinoma, melanoma, atypical fibroxanthoma, seminomas, non-seminomas, Leydig stromal 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 Wilms' tumor.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.

[256] Examples of particular cancers include, but are not limited to, adenocarcinoma, adenoma, adenofibroma, adenolymphoma, odontoma, AIDS-related cancers, acoustic neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenocystic carcinoma, adrenocortical cancer, agnogenic myeloid metaplasia, alopecia, alveolar soft tissue sarcoma, ameloblastoma, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, sclerosing angioma, angiomatosis, apudoma, anal cancer, angiosarcoma, Petition 870250078796, dated 03 / 09 / 2025, pages 304 / 342 87 / 99 aplastic anemia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancers, bowel cancer, brainstem glioma, CNS and brain tumors, breast cancer (preferably triple-negative breast cancer), bronchoma, CNS tumors, carcinoid tumors, cervical cancer, brain tumors in children, cancer in children, leukemia in children, soft tissue sarcoma in children, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancers, cutaneous T-cell lymphoma, carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, Merkel cell, mucinous, non-small cell lung, sand-grain cell, papillary, cirrhosis, bronchiolar, bronchogenic, squamous cell and cell transition), carcinosarcoma, cervical dysplasia, phyllodes tumors, cementoma, chordoma, choristoma, chondrosarcoma, chondroblastoma, craniopharyngioma, cholangioma,cholangiocarcinoma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, dermatofibrosarcoma protuberans, desmoplastic small round cell tumor, ductal carcinoma, dysgerminoma, endocrine cancers, endometrial cancer, ependymoma, somatic cancer, Ewing's sarcoma, extrahepatic bile duct cancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tube cancer, Fanconi anemia, fibroma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancers, gastrointestinal carcinoid tumor, genitourinary cancers, germ cell tumors, gestational trophoblastic disease, glioma, gynecological cancers, giant cell tumors, ganglioneuroma, glioma, glomangioma, granulosa cell tumor, gynandroblastoma, hematological malignancies, hairy cell leukemia, Head and neck cancer, hepatocellular carcinoma, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole.hypercalcemia, cancer of, Petition 870250078796, dated 03 / 09 / 2025, pages 305 / 342 88 / 99 hypopharynx, hamartoma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, hemangiosarcoma, histiocytic disorders, malignant histiocytosis, histiocytoma, hepatoma, hidradenoma, small immunoproliferative, opoma, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, renal 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, leiomyosarcoma, leukemia (e.g., B-cell, mixed-cell, null-cell, T-cell, chronic T-cell, HTLV-IL-associated, lymphangiosarcoma, acute lymphocytic, lymphocytic chronic, mast cell and myeloid), leukosarcoma, Leydig cell tumor, liposarcoma, leiomyoma, leiomyosarcoma, lymphangioma, lymphangiocytoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma, male breast cancer, malignant rhabdoid tumor of the kidney,medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, oral cancer, multiple endocrine neoplasia, fundoides mycosis, myelodysplastic syndromes, myeloma, myeloproliferative disorders; malignant carcinoid syndrome, carcinoid heart disease, medulloblastoma, meningioma, melanoma, mesenchymal stem cell carcinoma, mesonephroma, mesothelioma, myoblastoma, fibroid, myosacroma, myxoma, myxosacroma, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen rupture syndrome, non-melanoma skin cancer, non-small cell lung cancer (NSCLC), neurilemmoma, neuroblastoma, neuroepithelioma, neurofibromatosis, neurofibroma, neuroma, neoplasms (e.g., of bone, breast, digestive system, colorectal, liver), eye cancers, cancer, esophageal cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer with stoma formation, pancreatic cancer, paranasal cancer, parathyroid cancer,parotid gland cancer Petition 870250078796, dated 03 / 09 / 2025, pages 306 / 342 89 / 99 penile cancer, peripheral neuroectodermal tumors, pituitary cancer, polycythemia vera, prostate cancer, osteoma, osteosarcoma, ovarian carcinoma, papilloma, paraganglioma, non-chromaffin paraganglioma, pinealoma, plasmacytoma, proto-oncogene, rare cancers and associated disorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, reticuloendotheliosis, rhabdomyoma, salivary gland cancer, sarcoma, schwa noma, Sézary syndrome, skin cancer, small cell lung cancer (SCLC), small bowel cancer, soft tissue sarcoma, spinal cord tumors, squamous cell carcinoma (skin), stomach cancer, synovial sarcoma, sarcoma (e.g., experimental Ewing's sarcoma, Kaposi's sarcomas, and mast cell sarcomas), Sertoli cell tumor, Synovioma, testicular cancer, thymus cancer, thyroid cancer, transitional cell carcinoma (bladder), transitional cell carcinoma (renal-pelvis- / ureter),Trophoblastic cancer, teratoma, theca cell tumor, thymoma, trophoblastic tumor, urethral cancer, urinary tract cancer, uroplakins, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom's tumor-macroglobulinemia and Wilms' tumor. Preferred particular examples include pleural or peritoneal mesothelioma, gastric cancer, endometrial cancers, colorectal cancer, non-small cell lung adenocarcinoma, cholangiocarcinoma, ovarian carcinoma, esophageal cancer, pancreatic ductal adenocarcinoma, triple-negative breast cancer, and any other mesothelin-positive cancers.

[257] In some embodiments of the invention, the method is used for the prevention or treatment of a cancer selected from one or more of: brain cancer, esophageal cancer, mouth cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cancer Petition 870250078796, dated 03 / 09 / 2025, pages 307 / 342 90 / 99 cervical cancer, epithelial cell carcinomas, skin cancer, leukemia, lymphoma, myeloma, breast cancer, ovarian cancer, endometrial cancer, and testicular cancer. Preferably, the cancer is selected from one or more of the following: lung cancer, esophageal cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer, cervical cancer, vaginal cancers, epithelial cell carcinomas, skin cancer, blood-related cancers, breast cancer, endometrial cancer, uterine cancer, and testicular cancer.

[258] The existence of, improvement in, treatment of, or minimization of cancer progression can be determined by any clinically or biochemically relevant method as described in the present invention or known in the art. A positive response to treatment or a minimization of cancer progression can be determined by any method known in the art and may include the determination of: - a reduction in the number of cancer cells; - a reduction in tumor size; - an inhibition (that is, reducing to a certain extent and preferably stopping) of the infiltration of cancer cells into peripheral organs; - an inhibition (that is, slowing down to a certain extent and preferably stopping) of tumor metastasis; - a reduction or complete prevention of tumor metastasis after removal of the primary tumor; - an inhibition, to some extent, of tumor growth; - relief, to some extent, of one or more of the symptoms associated with cancer; and / or - increased survival of the individual.

[259] The determination of any of the above may be considered as a positive response to treatment as described in Petition 870250078796, dated 03 / 09 / 2025, pages 308 / 342 91 / 99 present invention.

[260] The individual who has received cancer treatment may be in partial or total remission. In other words, the individual, having received cancer treatment as described above, may have a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in measurable tumor growth parameters as can be found on physical examination, radiological study or by biomarker levels from a blood or urine test. Alternatively, where the individual is in total remission, there is a complete disappearance of all detectable manifestations of disease, so that the individual has no detectable signs of cancer. The individual may have substantially undetectable signs of cancer."A cancer that is substantially undetectable" generally refers to a circumstance where therapy has depleted the size, volume, or other physical measure of a cancer, such that, using relevant standard detection techniques such as in vivo imaging, the cancer, as a consequence of the therapy, is not clearly detectable.

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

[262] Treatment effectiveness can be measured by assessing survival duration, time to disease progression, response rates (RR), duration of response and / or quality of life.

[263] In one embodiment, the method is particularly useful for delaying Petition 870250078796, dated 03 / 09 / 2025, pages 309 / 342 92 / 99 cancer progression. In one embodiment, the method is particularly useful for extending an individual's survival, including overall survival as well as progression-free survival. Overall survival will be understood as the length of time from the date of diagnosis or the start of treatment of a cancer that patients diagnosed with cancer are still alive. Progression-free survival will be understood as the length of time during and after cancer treatment that a patient lives with the disease but it does not worsen.

[264] Survival analysis can be performed using well-known techniques in the field, including the Kaplan-Meier method. The Kaplan-Meier method estimates the survival function from lifetime data. In medical research, it can be used to measure the fraction of patients who live for a certain period of time after treatment. A Kaplan-Meier method plot of the survival function is a series of horizontal steps of declining magnitude that, when a large enough sample is taken, approximates the true survival function for that population. The value of the survival function between successive distinct sampled observations (“cliques”) is assumed to be constant.

[265] An important advantage of the Kaplan-Meier curve is that the method can account for “censored” data – losses from the sample before the final result is observed (for example, if a patient withdraws from a study). In the graph, small vertical scale marks indicate losses, where the patient’s data has been censored. When no truncation or censoring occurs, the Kaplan-Meier curve is equivalent to the empirical distribution.

[266] In one modality, the method is particularly useful for providing a complete response to therapy, whereby all signs of cancer in response to treatment have disappeared. This does not always mean that the cancer has been Petition 870250078796, dated 03 / 09 / 2025, pages 310 / 342 93 / 99 cured. In one modality, the method is particularly useful for providing a partial response to therapy, whereby there has been a decrease in the size of one or more tumors or lesions, or in the extent of cancer in the body, in response to treatment. Compositions and administration

[267] The distribution or administration of the genetically modified cell according to the invention may be distribution or administration of the cell alone, or distribution or administration of the cell formulated in a suitable pharmaceutical composition. Therefore, the present invention provides a pharmaceutical composition including a genetically modified cell of the invention and a pharmaceutically acceptable carrier.

[268] Methods are known in the art for providing CAR-containing cells for immunotherapy (see, for example, Kershaw, MH et al. Clin Cancer Res. 2006;12(20): 6106-15; Parker LL. et al. Hum Gene Ther 2000; 11:2337- 87). Additionally, protocols and methods are known in the art for the preparation, expansion, and evaluation of mammalian CAR-expressing cells (see, for example, Cheadle, EJ, et al. Antibody Engineering: Methods and Protocols, second edition, Methods in Molecular Biology, vol. 907: 645-66) and are summarized in the Examples below.

[269] 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 particular physical and chemical characteristics of the cell to be administered. In some embodiments, the pharmaceutical composition includes a suspension of genetically modified cells of the invention in a suitable medium, such as isotonic saline solution. In some embodiments, the pharmaceutical composition may Petition 870250078796, dated 03 / 09 / 2025, pages 311 / 342 94 / 99 may include suitable adjuvants, such as one or more cytokines as described above. In some embodiments, the pharmaceutical composition may also include an intermediate as described above.

[270] The pharmaceutical composition may also be administered via parenteral means, which include intravenous, intraventricular, intraperitoneal, intramuscular, intrapleural or intracranial injection, or local injections at the site of a tumor or cancerous mass.

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

[272] Single-domain CARs for binding to nfPX27 are known, including CARs comprising the sdAb designated “BIL03s”.

[273] The inventors sought to identify alternative sdAb-based CARs to target cancer cells. A number of different anti-nfP2X7 sdAb sequences were tested in the context of a CAR.

[274] Three suitable sdAb sequences (4A7, 3aB9 and 3aD9, as defined in the present invention in Table 3) have been identified as providing efficacy in the context of a CAR including in the context of a BRiDGE / CAR T-cell system (as disclosed in WO 2022 / 187906), preferably wherein the CAR has the hinge region of CD8A, the transmembrane domain of CD28 and the co-stimulatory / signaling domains of CD28, 41BB (CD137) and CD3zeta. Petition 870250078796, dated 03 / 09 / 2025, pages 312 / 342 95 / 99

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

[276] Although single-domain antigen-binding domains for inclusion in anti-nfP2X7 receptor CARs are known, it is not clear whether the binding affinity could be improved by the addition of a paired variable light chain. As indicated in Example 1, in some cases, light chain pairings with heavy chains in the context of a CAR did not result in improved CAR function. Furthermore, the selection of an appropriate light chain for pairing with a known heavy chain is not evident from the state of the art.

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

[278] More than 10 different light chain pairings were tested, including the variable light chain defined as G B1 in Table 1 of the present invention.

[279] The inventors discovered that specific light chain pairing with WT B1 (defined in Table 1) provided stable expression and reduced aggregation. Surprisingly, this light-heavy chain pairing also significantly improved the affinity for binding to the target antigen, as demonstrated in the table below. Table 2: Affinity for BILO3 heavy chain binding compared to affinity when paired with various variable light chain domains. Petition 870250078796, dated 03 / 09 / 2025, pages 313 / 342 96 / 99 Description KD (M) ka kd affinity (nM) (E200) BIL03 VH only 1.27E-07 2.0E+05 2.5E-02 127 BIL03-WT B1 (scFv format) 2.42E-08 5.41E+04 1.31E-03 24 BIL03-Germline B1 8.27E-08 3.14E+04 2.60E-03 83 BIL03-WT F5 2.03E-07 7.06E+04 1.43E-02 203 BIL03-WT B4 4.22E-07 3.33E+04 1.41E-02 422 BIL03-Germline G3 6.36E-07 1.88E+04 1.19E-02 636

[280] Binding affinity was evaluated with respect to two different antigens derived from the dysfunctional P2X7 receptor: E200 peptide (GHNYTTRNILPGLNITC) (SEQ ID NO: 143) and Ext 17 peptide (GHNYTTRNILPGLNITSTFHKTSGSGK) (SEQ ID NO: 102). Example 3: design and production of CAR scFv CAR Construction:

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

[282] 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 co-stimulatory / signaling domains of CD28, 41BB and CD3zeta.

[283] The two CARs comprise a tEGFR C terminal to facilitate CAR detection.

[284] Nucleic acids encoding CARs were designed in silica, synthesized, cloned into the transfer vector, and packaged into self-inactivating lentiviral particles (SIN) (3rd generation LV system) in cells Petition 870250078796, dated 03 / 09 / 2025, pages 314 / 342 97 / 99 HEK293T. The supernatant was concentrated by ultracentrifugation according to standard protocols. CAR T cell production

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

[286] Activated non-transduced T cells (aUT) do not express any receptor that can engage with EGFR.

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

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

[289] The results indicate that cells transduced with CAR12AV1 were capable of greater direct cell killing compared with cells transduced with CAR10A, although both CARs allowed cells to kill cells indirectly when combined with BRiDGE molecule.

[290] Figure 1 shows a direct comparison of the cell-killing ability of T cells expressing CAR10A compared to cells expressing CAR12AV1. Target cells used were the AML MOLM cell line. Petition 870250078796, dated 03 / 09 / 2025, pages 315 / 342 98 / 99 13. ET 5:1, CAR to target ratio of 2.1 to 1. Cells were deprived of cytokines for 3 days prior to exposure to CAR T cells.

[291] The results also show significantly greater direct cell killing (as shown by reduced MOL-13 cell count) by CAR12AV1 T cells compared to CAR10A T cells. Co-incubation with 200 ng / mL of Fab-based anti-CD33 BRiDGE molecule (as disclosed in WO 2022 / 187906) allowed CAR-expressing T cells to kill MOLM-13 cells more effectively. Example 4: Cell death by T cells expressing CAR molecule of the invention.

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

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

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

[295] Figure 3 shows that T cells expressing 3a-B9-CAR and 4A7-CAR were also capable of direct and indirect killing of MOLM-13 cells when co-incubated with an anti-CD33 BRiDGE molecule. Similar results were Petition 870250078796, dated 03 / 09 / 2025, pages 316 / 342 99 / 99 achieved with CAR-positive cell and cancer cell ratios of 5:1 and 2.5:1 (data not shown).

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

[297] It will be understood that the invention disclosed and defined in this descriptive report extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All such different combinations constitute various alternative aspects of the invention. Petition 870250078796, dated 03 / 09 / 2025, pages 317 / 342

Claims

1 / 19 CLAIMS 1. Chimeric antigen receptor (CAR), characterized in that it comprises: i) an antigen recognition domain that recognizes dysfunctional P2X7 receptor or epitope derived therefrom; ii) a transmembrane domain; and iii) an intracellular domain, wherein the antigen recognition domain comprises complementarity-determining regions (CDRs) from a VH comprising a sequence as set forth in SEQ ID NO: 4 and CDRs from a VL comprising a sequence as set forth in SEQ ID NO:

12.

2. CAR, according to claim 1, characterized in that the antigen recognition domain comprises: (i) a VH comprising a complementarity-determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to a sequence established in SEQ ID NO: 1, 29, 36 or 43, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to a sequence established in SEQ ID NO: 2, 30, 37 or 44 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to a sequence established in SEQ ID NO: 3, 31, 38, or 45;(ii) a VH comprising a sequence that is at least about 95% or 96% or 97% or 98% or 99% identical to a sequence established in SEQ ID Petition 870250078796, dated 03 / 09 / 2025, page 323 / 342 2 / 19 NO: 4; (iii) a VL comprising a CDR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence established in SEQ ID NO: 9, 50, 57 or 64, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence established in SEQ ID NO: 10, 51, 58 or 65 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence established in SEQ ID NO: 11, 52, 59 or 66;(iv) a VL comprising a sequence at least about 95% identical to a sequence established in SEQ ID NO: 12; (v) a VH comprising a CDR1 comprising a sequence established in SEQ ID NO: 1, 29, 36 or 43, a CDR2 comprising a sequence established in SEQ ID NO: 2, 30, 37 or 44 and a CDR3 comprising a sequence established in SEQ ID NO: 3, 31, 38 or 45; (vi) a VH comprising a sequence established in SEQ ID NO: 4; (vii) a VL comprising a CDR1 comprising a sequence established in SEQ ID NO: 9, 50, 57 or 64, a CDR2 comprising a sequence established in SEQ ID NO: 10, 51, 58 or 65 and a CDR3 comprising a sequence established in SEQ ID NO: 11, 52, 59 or 66; (viii) a VL comprising a sequence established in SEQ ID NO: 12;(ix) a VH comprising a CDR1 comprising a sequence Petition 870250078796, dated 03 / 09 / 2025, page 324 / 342 3 / 19 established in SEQ ID NO: 1, 29, 36 or 43, a CDR2 comprising a sequence established in SEQ ID NO: 2, 30, 37 or 44 and a CDR3 comprising a sequence established in SEQ ID NO: 3, 31, 38 or 45; and a VL comprising a CDR1 comprising a sequence established in SEQ ID NO: 9, 50, 57 or 64, a CDR2 comprising a sequence established in SEQ ID NO: 10, 51, 58 or 65 and a CDR3 comprising a sequence established in SEQ ID NO: 11, 52, 59 or 66; or (x) a VH comprising a sequence established in SEQ ID NO: 4 and a VL comprising a sequence established in SEQ ID NO: 12.; 3. CAR, according to claim 2, characterized in that the antigen recognition domain further comprises at least one of: (i) a VH comprising a structure region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence established 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%, at least 99% identical to a sequence established 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%, at least 99% identical to a sequence established in SEQ ID NO: 7, 34, 41 or 48 and an FR4 comprising a sequence at least approximately 80%, at least 85%, at least 90%, at least 92%,at least 95%, at least 97%, at least 99% identical to a sequence established in SEQ ID NO: 8, 35, 42 or 49; (ii) a VL comprising an FR1 comprising a sequence Petition 870250078796, dated 03 / 09 / 2025, page. 325 / 342 4 / 19 at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence established in SEQ ID NO: 13, 53, 60 or 67, an FR2 comprising a sequence at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence established in SEQ ID NO: 14, 54, 61 or 68, an FR3 comprising a sequence at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence established in SEQ ID NO: 15, 55, 62 or 69 and an FR4 comprising a sequence of at least approximately 80%, at least 85%, at least 90%, at least 92%,(iii) a VH comprising an FR1 comprising a sequence established in SEQ ID NO: 5, 32, 39 or 46, an FR2 comprising a sequence established in SEQ ID NO: 6, 33, 40 or 47, an FR3 comprising a sequence established in SEQ ID NO: 7, 34, 41 or 48 and an FR4 comprising a sequence established in SEQ ID NO: 8, 35, 42 or 49; (iv) a VL comprising an FR1 comprising a sequence established in SEQ ID NO: 13, 53, 60 or 67, an FR2 comprising a sequence established in SEQ ID NO: 14, 54, 61 or 68, an FR3 comprising a sequence established in SEQ ID NO: 15, 55, 62 or 69 and an FR4 comprising a sequence established in SEQ ID NO: 16, 56, 63 or 70; or (v) a VH comprising an FR1 comprising a sequence established in SEQ ID NO: 5, 32, 39 or 46, an FR2 comprising a sequence established in SEQ ID NO: 6, 33,40 or 47, an FR3 comprising a sequence established in SEQ ID NO: 7, 34, 41 or 48 and an FR4 Petition 870250078796, dated 03 / 09 / 2025, page 326 / 342 5 / 19 comprising a sequence established in SEQ ID NO: 8, 35, 42 or 49; and a VL comprising an FR1 comprising a sequence established in SEQ ID NO: 13, 53, 60 or 67, an FR2 comprising a sequence established between SEQ ID NO: 14, 54, 61 or 68, an FR3 comprising a sequence established in SEQ ID NO: 15, 55, 62 or 69 and an FR4 comprising a sequence established in SEQ ID NO: 16, 56, 63 or 70., 4. CAR, according to any one of claims 1 to 3, characterized in that the antigen recognition domain comprises a variable heavy chain domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 4, or a functional variant thereof comprising a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto;and a variable light chain domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 12, or a functional variant thereof comprising a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; and wherein the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

5. CAR, according to any one of claims 1 to 3, Petition 870250078796, dated 03 / 09 / 2025, pp. 327 / 342 6 / 19 characterized in that the antigen recognition domain comprises a variable heavy chain domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 4 and a variable light chain domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 12;wherein the variable heavy and / or light chain domain comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residue substitutions, deletions, or additions, compared to the amino acid sequence as set forth in SEQ ID NO: 4 or 12, respectively, and wherein the antigen recognition domain retains the ability to bind to the nfP2X7 receptor.

6. CAR, according to claim 4, characterized in that the functional variant does not comprise amino acid substitutions, deletions or additions in the CDRs or in the CAR defined in claim 5, wherein the amino acid substitutions, deletions or additions are not in the CDRs.

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

8. CAR, according to any one of claims 1 to 7, characterized in that the antigen recognition domain comprises, consists essentially of, or consists of, in order of N to C terminal, SEQ ID NO: 12 and SEQ ID NO: 4 (i.e., VL to VH).

9. CAR, according to claim 7, characterized in that the antigen-binding protein comprises SEQ ID NO: 12 (VL) - ligand - SEQ ID NO: 4 (VH).

10. CAR, according to any one of claims 1 to 9, characterized in that the antigen recognition domain is a single-strand variable fragment (scFv).

11. Chimeric antigen receptor (CAR), characterized in that it comprises: i) an antigen recognition domain that recognizes a dysfunctional P2X7 receptor or an epitope derived therefrom; ii) a transmembrane domain; and iii) an intracellular domain, wherein the antigen recognition domain comprises complementarity-determining regions (CDRs) from a VH comprising a sequence as set out in SEQ ID NO: 135, 136 or 137.

12. CAR, according to claim 11, characterized in that the antigen recognition domain comprises: (i) a VH comprising a complementarity-determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence established in SEQ ID NO: 124, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to Petition 870250078796, dated 03 / 09 / 2025, page.329 / 342 8 / 19 sequence established in SEQ ID NO: 125, 126 or 127 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 a sequence established in SEQ ID NO: 128, 129 or 130; (ii) a VH comprising a sequence that is at least about 95% or 96% or 97% or 98% or 99% identical to a sequence established in SEQ ID NO: 135, 136 or 137; (iii) a VH comprising a CDR1 comprising a sequence established in SEQ ID NO: 124, a CDR2 comprising a sequence established in SEQ ID NO: 125, 126, or 127 and a CDR3 comprising a sequence established in SEQ ID NO: 128, 129 or 130; or (iv) a VH comprising a sequence established in SEQ ID NO: 135, 136 or 137.

13. CAR, according to claim 11 or 12, characterized in that the antigen recognition domain further comprises at least one of: (i) a VH comprising a structure region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence established in SEQ ID NO: 131, or 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 a sequence established in SEQ Petition 870250078796, dated 03 / 09 / 2025, page.330 / 342 9 / 19 ID NO: 132, an FR3 comprising a sequence at least approximately 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence established in SEQ ID NO: 133 and an FR4 comprising a sequence at least approximately 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence established in SEQ ID NO: 134; (ii) a VH comprising an FR1 comprising a sequence established in SEQ ID NO: 131, an FR2 comprising a sequence established in SEQ ID NO: 132, an FR3 comprising a sequence established in SEQ ID NO: 133 and an FR4 comprising a sequence established in SEQ ID NO:

134.

14. CAR, according to any one of claims 11 to 13, characterized in that the antigen recognition domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 135, 136 or 137.

15. CAR, according to any one of claims 11 to 13, characterized in that the antigen recognition domain comprises a variable heavy chain domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 135, or a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to Petition 870250078796, dated 03 / 09 / 2025, p. 331 / 342 10 / 19 same; wherein the heavy chain does not comprise any sequence variation in the CDRs compared with the sequence of SEQ ID NO: 135, and / or wherein the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

16. CAR, according to any one of claims 11 to 13, characterized in that the antigen recognition domain comprises a variable heavy chain domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 135, wherein the variable heavy chain domain of the antigen-binding domain comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions or additions, compared to the amino acid sequence as set forth in SEQ ID NO: 135, preferably where replacements,Deletions or additions of amino acids are not present in CDRs and / or where the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

17. CAR, according to any one of claims 11 to 13, characterized in that the antigen recognition domain comprises a variable heavy chain domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 136, or a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, as per Petition 870250078796, dated 03 / 09 / 2025, p. 332 / 342 11 / 19 less than 96%, at least 97%, at least 98%, at least 99% identical to the same; wherein the heavy chain does not comprise any sequence variation in the CDRs compared to the sequence of SEQ ID NO: 136, and / or wherein the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

18. CAR, according to any one of claims 11 to 13, characterized in that the antigen recognition domain comprises a variable heavy chain domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 136, wherein the variable heavy chain domain of the antigen-binding domain comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions or additions, compared to the amino acid sequence as set forth in SEQ ID NO: 136, preferably where replacements,Deletions or additions of amino acids are not present in CDRs and / or where the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

19. CAR, according to any one of claims 11 to 13, characterized in that the antigen recognition domain comprises a variable heavy chain domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 137, or a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least Petition 870250078796, dated 03 / 09 / 2025, p. 333 / 342 12 / 19 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 to the same; wherein the heavy chain does not comprise any sequence variation in the CDRs compared with the sequence of SEQ ID NO: 137, and / or wherein the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

20. CAR, according to any one of claims 11 to 13, characterized in that the antigen recognition domain comprises a variable heavy chain domain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 137, wherein the variable heavy chain domain of the antigen-binding domain comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions or additions, compared to the amino acid sequence as set forth in SEQ ID NO: 137, preferably where replacements,Deletions or additions of amino acids are not present in CDRs and / or the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

21. CAR, according to any one of claims 1 to 20, characterized in that the CAR additionally comprises a hinge region.

22. CAR, according to claim 21, characterized in that the hinge region is derived from CD8a, CD28, or an IgG4 hinge region. Petition 870250078796, dated 03 / 09 / 2025, pp. 334 / 342 13 / 19 23. CAR, according to claim 22, characterized in that the hinge region comprises or consists of the amino acid sequence set forth in SEQ ID NO:

88.

24. CAR, according to any one of claims 21 to 23, characterized in that the hinge is C-terminal to the antigen recognition domain.

25. CAR, according to claim 24, characterized in that the hinge is situated between the antigen recognition domain and the transmembrane domain of the CAR.

26. CAR, according to any one of claims 1 to 26, characterized in that the transmembrane domain of the CAR comprises a portion originating from CD8a or CD28.

27. CAR, according to claim 26, characterized in that the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:

89.

28. CAR, according to any one of claims 1 to 27, characterized in that the signaling domain of the CAR comprises a portion derived from an activation receptor.

29. CAR, according to claim 28, characterized in that the activation receptor is a member of the CD3 coreceptor complex.

30. CAR, according to claim 29, characterized in that the portion derived from the CD3 coreceptor complex is CD3-Z (CD3-zeta).

31. CAR, according to claim 30, characterized in that the signaling domain comprises an amino acid sequence as set forth in SEQ ID NO:

92.

32. CAR, according to any one of claims 1 to 31, characterized in that the signaling domain of the CAR comprises Petition 870250078796, dated 03 / 09 / 2025, pp. 335 / 342 14 / 19 a portion derived from a co-stimulatory receptor.

33. CAR, according to claim 32, characterized in that the co-stimulatory receptor is CD28 and / or 4-1BB (CD137).

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

35. CAR, according to any one of claims 1 to 34, characterized in that the signaling domain comprises a portion derived from an activating receptor and a portion derived from a co-stimulatory receptor.

36. CAR, according to claim 35, characterized in that the activating receptor is a member of the CD3 coreceptor complex and the co-stimulating receptor is selected from CD28 and / or 4-1BB.

37. CAR, according to claim 36, characterized in that the signaling domain comprises an amino acid sequence comprising the sequences set forth in SEQ ID NOs: 90, 91 and / or 92 and combinations thereof.

38. CAR, according to any one of claims 1 to 37, characterized in that the CAR comprises (from the N to the C terminal) an antigen recognition domain defined in 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 CD3z (zeta) signaling domain.

39. CAR, according to claim 38, characterized in that the CAR comprises an amino acid sequence as set forth in SEQ ID NO: 94, or a functional variant thereof comprising a sequence at least about 80%, at least 81%, at least 82%, at least 83%, Petition 870250078796, dated 03 / 09 / 2025, page 336 / 342 15 / 19 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 in the present invention, 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. CAR, according to claim 38, characterized in that the CAR comprises an amino acid sequence as set forth in SEQ ID NO: 138, or a functional variant thereof comprising a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; wherein the functional variant comprises an antigen recognition domain as defined in the present invention, preferably comprising a VH comprising the amino acid sequence of SEQ ID NO:

135.

41. CAR, according to claim 38, characterized in that the CAR comprises an amino acid sequence as set forth in SEQ ID NO: 139, or a functional variant thereof comprising a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto; wherein Petition 870250078796, dated 03 / 09 / 2025, p. 337 / 342 16 / 19 functional variant comprises an antigen recognition domain as defined in the present invention, preferably comprising a VH comprising the amino acid sequence of SEQ ID NO:

136.

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

137.

43. Nucleic acid, characterized in that it encodes the chimeric antigen receptor defined in any one of claims 1 to 42.

44. Nucleic acid construct, characterized in that it includes a nucleic acid molecule defined in claim 43.

45. Genetically modified cell, characterized in that it comprises a CAR defined in any one of claims 1 to 42.

46. ​​Genetically modified cell, characterized in that it comprises a nucleic acid molecule defined in claim 43 or a construct defined in claim 44.

47. Genetically modified cell, according to claim 45 or 46, characterized in that the cell is an immune cell.

48. Genetically modified cell, according to claim 47, characterized in that the immune cell is a leukocyte. Petition 870250078796, dated 03 / 09 / 2025, pp. 338 / 342 17 / 19 49. Genetically modified cell, according to claim 48, characterized in that 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. Genetically modified cell, according to claim 49, characterized in that the T cell is either a CD4+ T cell or a CD8+ T cell.

51. A method for generating a genetically modified cell, said method characterized in that it comprises transducing the cell, preferably an immune cell, with a nucleic acid construct defined in claim 44, such that the transduced cell expresses the CAR, thereby generating a genetically modified cell.

52. Method according to claim 51, characterized in that the immune cell is a T cell.

53. A method for killing a cell expressing nfP2X7 receptor, characterized in that it comprises exposing a cell expressing nfP2X7 receptor to a genetically modified cell defined in any one of claims 45 to 50, thereby killing a cell expressing an nfP2X7 receptor.

54. Method according to claim 53, characterized in that the cell expressing nfP2X7 receptor is a cancer cell.

55. Method, according to claim 54, characterized in that the cancer is selected from the group consisting of: brain cancer, esophageal cancer, mouth cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cervical cancer, epithelial cell cancers, skin cancer, leukemia, lymphoma, myeloma, Petition 870250078796, dated 03 / 09 / 2025, pp. 339 / 342 18 / 19 breast cancer, ovarian cancer, endometrial cancer, thymic cancer and testicular cancer.

56. Method according to claim 55, characterized in that the cancer is selected from the group consisting of: lung cancer, esophageal cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer, cervical cancer, vaginal cancers, epithelial cell cancers, skin cancer, blood-related cancers, breast cancer, endometrial cancer, uterine cancer and testicular cancer.

57. Pharmaceutical composition, characterized in that it includes a genetically modified cell defined in any one of claims 45 to 50 and a pharmaceutically acceptable carrier.

58. Method for: • treating, preventing or minimizing the progression of cancer in an individual, • minimizing, reducing or preventing the growth of a tumor in an individual, • minimizing, reducing or preventing metastasis in an individual, or • increasing the survival of an individual, the method characterized in that it comprises administering to the individual a CAR defined in any one of claims 1 to 42, a nucleic acid defined in claim 43, a nucleic acid construct defined in claim 44, a genetically modified cell defined in any one of claims 45 to 50, or a pharmaceutical composition defined in claim 57, so as to: • treat, prevent or minimize the progression of cancer in an individual, • minimize, reduce or prevent the growth of a tumor in an individual, Petition 870250078796, dated 03 / 09 / 2025, pp. 340 / 342 19 / 19 • minimize, reduce or prevent metastasis in an individual, or • increase the survival of an individual.

59. Use of a CAR defined in any one of claims 1 to 42, of a nucleic acid defined in claim 43, of a nucleic acid construct defined in claim 44, of a genetically modified cell defined in any one of claims 45 to 50, or of a pharmaceutical composition defined in claim 57, characterized in that it is for the manufacture of a medicament to: • treat, prevent or minimize the progression of cancer in an individual, • minimize, reduce or prevent the growth of a tumor in an individual, • minimize, reduce or prevent metastasis in an individual, or • increase the survival of an individual.

60. CAR, defined in any one of claims 1 to 42, a nucleic acid defined in claim 43, a nucleic acid construct defined in claim 44, a genetically modified cell defined in any one of claims 45 to 50, or a pharmaceutical composition defined in claim 57, characterized in that they are for use in: • treating, preventing or minimizing the progression of cancer in an individual, • minimizing, reducing or preventing the growth of a tumor in an individual, • minimizing, reducing or preventing metastasis in an individual, or • increasing the survival of an individual. Petition 870250078796, dated 03 / 09 / 2025, pp. 341 / 342

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