Chimeric antigen receptors for binding to dysfunctional P2X7 receptors

Abstract

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

Description

TECHNICAL FIELD

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

[0002] RELATED APPLICATIONS This application claims priority from Australian provisional application AU 2023900626, the entire contents of which are hereby incorporated by reference. BACKGROUND

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

[0004] While CARs have been designed to bind a variety of tumour specific antigens, the architecture and binding affinity of CARs often need to be optimised to ensure that an appropriate level of binding is achieved where a T cell binds a cancer cell. In other words, antigen binding domains that are derived directly from therapeutic antibodies can have an inappropriate affinity in the context of a CAR and can result in manufactured CAR T cells producing toxicity in a patient.

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

[0006] The reference in this specification to any prior publication (or information derived from it) is not, and should not be taken as an acknowledgment or admission that the prior publication (or information derived from it) forms part of the common general knowledge in any jurisdiction, or that the prior publication is known or must be reasonably expected to be known to the skilled person in any jurisdiction, or that the prior publication (or information derived from it) and / or combinations thereof are known or must be reasonably expected to be known to be relevant to the subject matter of the application. SUMMARY

[0007] The present invention is based on the inventors’ surprising finding that specific antigen recognition domain sequences provide improved functionality and cell killing in the context of a chimeric antigen receptor.

[0008] In a first aspect, the present invention provides a chimeric antigen receptor (CAR) comprising: i) an antigen recognition domain that recognises a dysfunctional P2X7 receptor (nfP2X7 receptor) or an epitope derived therefrom; ii) a transmembrane domain; and iii) Intracellular domains, The antigen recognition domain comprises a complementarity-determining region (CDR) from VH and a CDR from VL, wherein VH comprises a sequence as shown in SEQ ID NO: 4 and VL comprises a sequence as shown in SEQ ID NO: 12.

[0009] In a second aspect, the present invention provides a chimeric antigen receptor (CAR) comprising: i) An antigen recognition domain that recognizes a dysfunctional P2X7 receptor (nfP2X7 receptor) or an epitope derived therefrom. ii) Transmembrane domains; and iii) Intracellular domains, The antigen recognition domain includes a complementarity-determining region (CDR) from a VH, which contains a sequence as shown in any of SEQ ID NO: 135, 136 or 137.

[0010] According to the first aspect, the antigen recognition domain of CAR includes: FR1 – CDR1 – FR2 – CDR2 – FR3 – CDR3 – FR4, and FR1a – CDR1a – FR2a – CDR2a – FR3a – CDR3a – FR4a, in: FR1, FR2, FR3 and FR4 are each a frame region; CDR1, CDR2 and CDR3 are each complementary determinant regions; FR1a, FR2a, FR3a and FR4a are each a frame region; CDR1a, CDR2a, and CDR3a are each complementary determinant regions. The complementarity-determining region contains the amino acid sequence described in Table 1 below.

[0011] According to a second aspect of the invention, the antigen recognition domain of the CAR includes: FR1 – CDR1 – FR2 – CDR2 – FR3 – CDR3 – FR4 in: FR1, FR2, FR3 and FR4 are each a frame region; CDR1, CDR2 and CDR3 are each complementary determinant regions; The complementarity-determining region contains the amino acid sequence described in Table 3 below.

[0012] Preferably, and according to a first aspect of the invention, a chimeric antigen receptor (CAR) is provided, comprising: an antigen recognition domain, a transmembrane domain, and an intracellular domain. The antigen recognition domain includes: (i) VH, which includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR 1 contains 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% of the sequence shown in SEQ ID NO: 1, 29, 36, or 43; CDR 2 contains 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% of the sequence shown in SEQ ID NO: 2, 30, 37, or 44; and CDR 3 ...43; and CDR 3 contains at least 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 The sequences shown in NO:3, 31, 38 or 45 are 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 sequences; (ii) VH, which contains at least about 95% or 96% or 97% or 98% or 99% of the sequence shown in SEQ ID NO: 4; (iii) VL, comprising CDR1, CDR2, and CDR3, wherein CDR1 comprises 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% of the sequence shown in SEQ ID NO: 9, 50, 57, or 64; CDR2 comprises 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% of the sequence shown in SEQ ID NO: 10, 51, 58, or 65; and CDR3 .... The sequences shown in 11, 52, 59 or 66 are 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 sequences; (iv) VL, which contains at least about 95% or 96% or 97% or 98% or 99% of the sequence shown in SEQ ID NO: 12; (v)VH, which contains CDR1, CDR2 and CDR3, wherein CDR1 contains the sequence shown in SEQ ID NO: 1, 29, 36 or 43, CDR2 contains the sequence shown in SEQ ID NO: 2, 30, 37 or 44, and CDR3 contains the sequence shown in SEQ ID NO: 3, 31, 38 or 45; (vi) VH, which contains the sequence shown in SEQ ID NO: 4; (vii) VL, which includes CDR1, CDR2 and CDR3, wherein CDR1 includes the sequence shown in SEQ ID NO: 9, 50, 57 or 64, CDR2 includes the sequence shown in SEQ ID NO: 10, 51, 58 or 65, and CDR3 includes the sequence shown in SEQ ID NO: 11, 52, 59 or 66; (viii) VL, which contains the sequence shown in SEQ ID NO: 12; (ix) VH and VL, wherein VH comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO: 1, 29, 36, or 43, CDR2 comprises the sequence shown in SEQ ID NO: 2, 30, 37, or 44, and CDR3 comprises the sequence shown in SEQ ID NO: 3, 31, 38, or 45; and VL comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO: 9, 50, 57, or 64, CDR2 comprises the sequence shown in SEQ ID NO: 10, 51, 58, or 65, and CDR3 comprises the sequence shown in SEQ ID NO: 11, 52, 59, or 66; or (x)VH and VL, wherein VH contains the sequence shown in SEQ ID NO: 4 and VL contains the sequence shown in SEQ ID NO: 12.

[0013] In any embodiment of the first aspect of the invention, the antigen recognition domain further comprises at least one of the following: (i) VH, which comprises frame regions (FR) 1, FR2, FR3, and FR4, wherein FR1 comprises 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% of the sequence shown in SEQ ID NO: 5, 32, 39, or 46; FR2 comprises 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% of the sequence shown in SEQ ID NO: 6, 33, 40, or 47; and FR3 comprises 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% of the sequence shown in SEQ ID NO: 49, 30, 41, 42, 43, 44, 45, 46, FR4, FR5, FR6, FR7, FR8, FR9 ... The FR4 contains 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% of the sequence shown in SEQ ID NO: 8, 35, 42, or 49. (ii) VL, comprising FR1, FR2, FR3, and FR4, wherein FR1 comprises 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% of the sequence shown in SEQ ID NO: 13, 53, 60, or 67; FR2 comprises 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% of the sequence shown in SEQ ID NO: 14, 54, 61, or 68; and FR3 comprises 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% of the sequence shown in SEQ ID NO: 14, 54, 61, or 68; and FR4 ... The FR4 contains 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% of the sequence shown in SEQ ID NO: 16, 56, 63, or 70. (iii) VH, which comprises FR1, FR2, FR3 and FR4, wherein FR1 comprises the sequence shown in SEQ ID NO: 5, 32, 39 or 46, FR2 comprises the sequence shown in SEQ ID NO: 6, 33, 40 or 47, FR3 comprises the sequence shown in SEQ ID NO: 7, 34, 41 or 48, and FR4 comprises the sequence shown in SEQ ID NO: 8, 35, 42 or 49; (iv) VL, comprising FR1, FR2, FR3, and FR4, wherein FR1 comprises the sequence shown in SEQ ID NO: 13, 53, 60, or 67, FR2 comprises the sequence shown in SEQ ID NO: 14, 54, 61, or 68, FR3 comprises the sequence shown in SEQ ID NO: 15, 55, 62, or 69, and FR4 comprises the sequence shown in SEQ ID NO: 16, 56, 63, or 70; or (v) VH and VL, wherein VH comprises FR1, FR2, FR3 and FR4, wherein FR1 comprises the sequence shown in SEQ ID NO: 5, 32, 39 or 46, FR2 comprises the sequence shown in SEQ ID NO: 6, 33, 40 or 47, FR3 comprises the sequence shown in SEQ ID NO: 7, 34, 41 or 48, and FR4 comprises the sequence shown in SEQ ID NO: 8, 35, 42 or 49; and VL comprises FR1, FR2, FR3 and FR4, wherein FR1 comprises the sequence shown in SEQ ID NO: 13, 53, 60 or 67, FR2 comprises the sequence shown in SEQ ID NO: 14, 54, 61 or 68, FR3 comprises the sequence shown in SEQ ID NO: 15, 55, 62 or 69, and FR4 comprises the sequence shown in SEQ ID NO: 16, 56, 63 or 70.

[0014] In any embodiment of the first aspect, the antigen recognition domain comprises, is substantially composed of, or is composed of, the amino acid sequences of SEQ ID NO: 4 and 12 (in the order of N-terminus to C-terminus or C-terminus to N-terminus).

[0015] In any embodiment of the first aspect, the antigen recognition domain comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 4 or a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or constitutes thereof, and the light chain variable domain comprises, SEQ ID NO: The amino acid sequence shown in SEQ ID NO: 4 or 12, or a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or constitutes the amino acid sequence shown in SEQ ID NO: 4 or 12; wherein the heavy chain and light chain do not contain any sequence changes in the CDR compared to the sequence shown in SEQ ID NO: 4 or 12, and / or wherein the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

[0016] In any embodiment of the first aspect, the antigen-recognizing domain comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 4, and the light chain variable domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 12, wherein, compared to the amino acid sequence shown in SEQ ID NO: 4 or 12, the heavy chain variable domain and the light chain variable domain of the antigen-binding domain each comprise no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 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 residues substituted, deleted, or added; preferably, wherein the amino acid substitution, deletion, or addition is not in the CDR, and / or wherein the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

[0017] Preferably, the antigen recognition domain comprises, is substantially composed of, or is composed of SEQ ID NO: 12 and SEQ ID NO: 4 (i.e., VL to VH) in the order from N-terminus to C-terminus. Optionally, the antigen-binding protein comprises SEQ ID NO: 12 (VL) – linker – SEQ ID NO: 4 (VH).

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

[0019] Optionally, the variable heavy and variable light regions of the antigen recognition domain are connected via a linker. In any embodiment, the antigen recognition domain may include: FR1 – CDR1 – FR2 – CDR2 – FR3 – CDR3 – FR4 – Linker – FR1a – CDR1a –FR2a – CDR2a – FR3a – CDR3a – FR4a.

[0020] As defined herein, a linker can be a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

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

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

[0023] Preferably, and according to a second aspect of the invention, a chimeric antigen receptor (CAR) is provided, comprising: an antigen recognition domain, a transmembrane domain, and an intracellular domain. The antigen recognition domain includes: (i) VH, which includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR 1 contains 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% of the sequence shown in SEQ ID NO: 124; CDR 2 contains 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% of the sequence shown in SEQ ID NO: 124; and CDR 3 ... The sequences shown in NO:128, 129 or 130 are 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 sequences; (ii) VH, which contains at least about 95% or 96% or 97% or 98% or 99% identical sequences to those shown in SEQ ID NO: 135, 136 or 137; (iii) VH, comprising CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO: 124, CDR2 comprises the sequence shown in SEQ ID NO: 125, 126, or 127, and CDR3 comprises the sequence shown in SEQ ID NO: 128, 129, or 130; preferably, CDR1 comprises the sequence shown in SEQ ID NO: 124, CDR2 comprises the sequence shown in SEQ ID NO: 125, and CDR3 comprises the sequence shown in SEQ ID NO: 128; or preferably, CDR1 comprises the sequence shown in SEQ ID NO: 124, CDR2 comprises the sequence shown in SEQ ID NO: 126, and CDR3 comprises the sequence shown in SEQ ID NO: 129; or preferably, CDR1 comprises the sequence shown in SEQ ID NO: 124, CDR2 comprises the sequence shown in SEQ ID NO: 127, and CDR3 comprises the sequence shown in SEQ ID NO: 130; or (iv) VH, which contains the sequence shown in SEQ ID NO: 135, 136 or 137.

[0024] In any embodiment of the second aspect of the invention, the antigen recognition domain further comprises at least one of the following: (i) VH, which comprises frame regions (FR) 1, FR2, FR3, and FR4, wherein FR1 comprises 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% of the sequence shown in SEQ ID NO: 131; FR2 comprises 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% of the sequence shown in SEQ ID NO: 132; FR3 comprises 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% of the sequence shown in SEQ ID NO: 133; and FR4 .... The sequence shown in 134 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. (ii) VH, which includes FR1, FR2, FR3 and FR4, wherein FR1 includes the sequence shown in SEQ ID NO: 131, FR2 includes the sequence shown in SEQ ID NO: 132, FR3 includes the sequence shown in SEQ ID NO: 133 and FR4 includes the sequence shown in SEQ ID NO: 134.

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

[0026] In any embodiment, the antigen-recognizing domain comprises a heavy chain variable domain comprising an amino acid sequence as shown in SEQ ID NO: 135, or a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or constitutes thereof; wherein the heavy chain does not contain any sequence changes in the CDR compared to the sequence of SEQ ID NO: 135, and / or wherein the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

[0027] In any embodiment, the antigen-recognizing domain comprises a heavy chain variable domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 135, wherein, compared to the amino acid sequence shown in SEQ ID NO: 135, the heavy chain variable domain of the antigen-binding domain comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residues substituted, deleted, or added; preferably, wherein the amino acid substitution, deletion, or addition is not in the CDR, and / or wherein the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

[0028] In any embodiment, the antigen-recognizing domain comprises a heavy chain variable domain comprising, or being composed of, an amino acid sequence as shown in SEQ ID NO: 136, or a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or constitutes thereof; wherein, compared with the sequence of SEQ ID NO: 136, the heavy chain does not contain any sequence changes in the CDR, and / or wherein the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

[0029] In any embodiment, the antigen-recognizing domain comprises a heavy chain variable domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 136, wherein, compared to the amino acid sequence shown in SEQ ID NO: 136, the heavy chain variable domain of the antigen-binding domain comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residues substituted, deleted, or added; preferably, the amino acid substitution, deletion, or addition is not in the CDR, and / or the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

[0030] In any embodiment, the antigen-recognizing domain comprises a heavy chain variable domain comprising, or being composed of, an amino acid sequence as shown in SEQ ID NO: 137, or a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or constitutes thereof; wherein, compared with the sequence of SEQ ID NO: 137, the heavy chain does not contain any sequence changes in the CDR, and / or wherein the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

[0031] In any embodiment, the antigen-recognizing domain comprises a heavy chain variable domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 137, wherein, compared to the amino acid sequence shown in SEQ ID NO: 137, the heavy chain variable domain of the antigen-binding domain comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residues substituted, deleted, or added; preferably, the amino acid substitution, deletion, or addition is not in the CDR, and / or the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

[0032] In any embodiment of any aspect, the CAR also includes a hinge region (also referred to herein as a spacer region). The hinge region may be derived from CD28, CD8a, or IgG4. Preferably, the hinge region is derived from CD8a. Typically, the hinge is located at the C-terminus of the antigen recognition domain. In one embodiment, the hinge region is located between the antigen recognition domain and the transmembrane domain. In a preferred embodiment, the hinge region comprises or consists of the amino acid sequence shown in SEQ ID NO: 88.

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

[0034] In any embodiment of any aspect, the signal transduction domain of the CAR includes a portion derived from the activating receptor. In some embodiments, the activating receptor is a member of the CD3 co-receptor complex. In some embodiments, the portion derived from the CD3 co-receptor complex is CD3-ζ (CD3-zeta) and optionally includes the amino acid sequence shown in SEQ ID NO: 92.

[0035] In any embodiment of any aspect, the signal transduction domain of the CAR 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 shown in SEQ ID NO: 90 and / or 91.

[0036] The signal transduction domain may comprise a portion derived from an activating receptor and a portion derived from a co-stimulatory receptor. In some embodiments, the activating receptor is a member of the CD3 co-receptor complex, and the co-stimulatory receptor is selected from CD28, ICOS, CD27, OX40, and / or 4-1BB. In some embodiments, the activating receptor is a member of the CD3 co-receptor complex, and the co-stimulatory receptor is CD28 and 4-1BB (CD137). Preferably, the portion derived from the CD3 co-receptor complex is CD3-ζ (CD3-zeta). In some embodiments, the signal transduction domain comprises an amino acid sequence containing the sequences shown in SEQ ID NO: 90, 91, and / or 92, and combinations thereof.

[0037] In any embodiment, the CAR includes (from N-terminus to C-terminus) an antigen-binding domain, a linker, a CD8a hinge, a CD28 transmembrane domain, a CD28 signaling domain, a 4-1BB signaling domain, and a CD3ζ (zeta) signaling domain as described herein. Optionally, the CAR may also include an epitope or affinity tag.

[0038] In a preferred embodiment of the first aspect, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 94 or a functional variant thereof, the functional variant comprising a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown herein; wherein the functional variant comprises an antigen recognition domain as defined herein, preferably comprising VH and VL, the VH comprising the amino acid sequence of SEQ ID NO: 4; and the VL comprising the amino acid sequence of SEQ ID NO: 12.

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

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

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

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

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

[0044] In a preferred embodiment of the second aspect, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 140 or a functional variant thereof, the functional variant comprising a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 137; wherein the functional variant comprises an antigen recognition domain as defined herein, preferably comprising a VH, the VH comprising the amino acid sequence shown in SEQ ID NO: 137.

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

[0046] In one respect, any changes described herein (e.g., identity % of the amino acid sequence or substitution, deletion or addition of the amino acid sequence) are variations in the frame region or constant region of the variable domain.

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

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

[0049] In some implementations, the nucleic acid construct also includes an internal ribosome entry site (IRES) that enables translation initiation after the mRNA is expressed by the nucleic acid construct.

[0050] In some implementations, the nucleic acid construct is a vector (e.g., a viral vector) that can be used to transform immune cells (e.g., T cells) to induce CAR expression.

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

[0052] In another aspect, the present invention provides genetically modified cells comprising the nucleic acid molecules described herein according to the invention, or the nucleic acid constructs described herein, or constructs in a genome-integrated form.

[0053] In another aspect, the present invention provides a method for generating genetically modified cells, the method comprising transducing cells (preferably immune cells) with a nucleic acid construct encoding a CAR of the present invention as described herein, such that the transduced cells express the CAR, thereby generating genetically modified cells.

[0054] In some implementations, the cell is an immune cell, such as a leukocyte. In some implementations, the cell is a peripheral blood mononuclear cell (PBMC), lymphocyte, T cell (including CD4+ T cells or CD8+ T cells), natural killer (NK) cell, natural killer T cell, or tumor-infiltrating lymphocyte (TIL).

[0055] In a preferred embodiment, the immune cells expressing CAR are T cells. Illustrative examples of suitable T cells include helper T cells (HTL; CD4+). + T cells, cytotoxic T cells (CTL; ​​CD8) + T cells, CD4 + CD8 + T cells, CD4 - CD8 - T cells or any other subset of T cells. Other illustrative examples of suitable T cells include T cells expressing one or more of the following markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, CD197, and HLA-DR.

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

[0057] In another aspect, the present invention provides a method for killing cells expressing the nfP2X7 receptor, the method comprising exposing cells expressing the nfP2X7 receptor to genetically modified cells of the present invention as described herein, thereby killing the cells expressing the nfP2X7 receptor.

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

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

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

[0061] In another aspect, the present invention provides a method for in vitro expansion of the genetically modified cells of the present invention, the method comprising the step of exposing the cells to an antigen of CAR. In some embodiments, the method includes a further step of exposing the cells to cytokines.

[0062] In another aspect, the present invention provides a method for in vitro expansion of the genetically modified cells of the present invention, the method comprising the steps of: exposing the cells to an antigen of CAR (e.g., nfP2X7 receptor) and simultaneously exposing the cells to cytokines.

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

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

[0065] In another aspect, the present invention provides a method for in vitro expansion of the genetically modified cells of the present invention, the method comprising: - Expose the cells to fixed CD3 and CD28 agonists; and -The cells (preferably T cells, especially human T cells) are brought into contact with the culture medium under conditions that allow cell (preferably T cells, preferably human T cells) to proliferate.

[0066] In some embodiments, the agonist is immobilized on a bead-like substrate. In one embodiment, the agonist may be immobilized on “human activators” Dynabeads™. In another preferred example, the agonist is immobilized on a colloidal polymer nanomatrix bead-like substrate conjugated with recombinant humanized CD3 and CD28 agonists (e.g., in “MACS GMP” TransAct). TM (On the bead-like base).

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

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

[0069] In some implementations, antibodies are immobilized on the surface of a tissue culture container, such as a culture flask, culture plate, or bioreactor.

[0070] In another aspect, the present invention provides a pharmaceutical composition comprising the genetically modified cells and pharmaceutically acceptable carriers of the present invention as described herein. In some embodiments, the pharmaceutical composition comprises a suitable adjuvant, which may be composed of cytokines. In some embodiments, the pharmaceutical composition may also comprise intermediates as described herein.

[0071] In another aspect, the present invention provides a method, which: To treat, prevent, or minimize cancer progression in subjects. To minimize, reduce, or prevent tumor growth in the subject. To minimize, reduce, or prevent cancer metastasis in the subject, or Increase the survival rate of subjects with cancer. Optionally, the cancer or tumor is characterized by the expression of the nfP2X7 receptor; the method comprises administering to a subject a CAR of the present invention as described herein, a nucleic acid construct of the present invention as described herein, a genetically modified cell of the present invention as described herein, or a pharmaceutical composition of the present invention as described herein, thereby: To treat, prevent, or minimize cancer progression in subjects. To minimize, reduce, or prevent tumor growth in the subject. Minimize, reduce, or prevent subject transfer, or Increase the survival rate of subjects with cancer.

[0072] In another aspect, the present invention provides the use of the CAR of the present invention as described herein, the nucleic acid construct of the present invention as described herein, the genetically modified cell of the present invention as described herein, or the pharmaceutical composition of the present invention as described herein in the preparation of a medicament for: To treat, prevent, or minimize cancer progression in subjects. To minimize, reduce, or prevent tumor growth in the subject. To minimize, reduce, or prevent cancer metastasis in the subject, or Increase the survival rate of subjects with cancer. Optionally, the cancer or tumor is characterized by the expression of the nfP2X7 receptor.

[0073] In another aspect, the present invention provides the CAR of the present invention as described herein, the nucleic acid construct of the present invention as described herein, the genetically modified cell of the present invention as described herein, or the pharmaceutical composition of the present invention as described herein, for use in: To treat, prevent, or minimize cancer progression in subjects. To minimize, reduce, or prevent tumor growth in the subject. To minimize, reduce, or prevent cancer metastasis in the subject, or Increase the survival rate of subjects with cancer. Optionally, cancer or tumor is characterized by the expression of the nfP2X7 receptor.

[0074] As used herein, unless the context otherwise requires, the term “comprising” or variations thereof, such as “including” and “containing”, is not intended to exclude additional additives, components, whole or steps.

[0075] Other aspects of the invention and other embodiments of the various aspects described in the foregoing paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings. Attached Figure Description

[0076] Figure 1 : MOLM-13 cell count after contact with untransduced T cells (UTD) or T cells transduced with CAR12AV1 or CAR10A. NB = No bridging molecule (indicating direct cell killing). "With BRiDGE" = Co-incubated with 200 ng / mL anti-CD33BRiDGE molecule, which contains the E200 sequence for binding the CAR antigen recognition domain.

[0077] Figure 2JeKo-1 cell viability was measured after contact with untransduced T cells (UTD) or T cells transduced with CAR10A, CAR12A, 3a-B9-CAR, or 4A7-CAR supplemented with anti-CD19 BRiDGE. BRiDGE concentrations ranged from 0 ng / mL to 100 ng / mL. "BRiDGE" = fusion protein containing an E200 sequence for binding a CAR antigen recognition domain and an anti-CD19 antigen binding domain. Effector-target cell ratio = 2.77 / 1. Cell viability was read 21 hours after co-incubation.

[0078] Figure 3 MOLM-13 cell viability after contact with untransduced T cells (UTD) or T cells transduced with 3a-B9-CAR or 4A7-CAR. The CAR-positive cell to cancer cell ratio was 10:1. "BRiDGE" = co-incubated with 100 ng / mL BRiDGE molecules containing an E200 sequence for binding to the CAR antigen recognition domain and either the anti-CD33 binding domain or the anti-CD19 binding domain. Cell viability was read 24 hours after co-incubation.

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

[0080] Sequence information Table 1: Sequence Information

[0081] Table 2: Further sequence information

[0082] Table 3: Sequence information of sdAb-based CAR of the present invention Detailed Implementation

[0083] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with embodiments, it should be understood that it is not intended to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the invention as defined by the claims.

[0084] Those skilled in the art will recognize that many methods and materials are similar to or equivalent to those described and materials herein, and that can be used in the practice of this invention. This invention is by no means limited to the methods and materials described.

[0085] It should be understood that the invention disclosed and defined in this specification includes all alternative combinations of two or more individual features mentioned in or clearly apparent from the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0086] All patents and publications mentioned in this article are incorporated in full by way of citation.

[0087] This invention seeks to address one or more deficiencies in the prior art and is based on the inventors' understanding that a specific antigen recognition domain provides improved anti-nfP2X7 receptor CAR function. More specifically, the preferred CAR of this invention is believed to provide a higher binding affinity to cancer cells expressing the nfP2X7 receptor, thereby exhibiting stronger cell-killing ability (potency). The inventors have discovered that specific single-domain (sdAb-based) CARs and specific scFv-based CARs offer superior efficacy compared to prior art sdAb-based CARs.

[0088] Definition - Overview Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0089] For the purposes of interpreting this specification, the following definitions will generally apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0090] Throughout this specification, unless otherwise specified or required by context, references to a single step, a composition of substances, a group of steps, or a group of compositions of substances shall encompass one or more of those steps, compositions of substances, groups of steps, or groups of compositions of substances (i.e., one or more). Therefore, as used herein, the singular forms “an,” “an,” and “the” include the plural aspect, and vice versa, unless the context clearly indicates otherwise. For example, reference to “an” includes a single and two or more; reference to “an” includes a single and two or more; reference to “the” includes a single and two or more, and so on.

[0091] Those skilled in the art will understand that variations and modifications can be made to the present invention beyond those specifically described. It should be understood that the present invention includes all such variations and modifications. The present invention also includes all steps, features, compositions, and compounds individually or collectively mentioned or indicated in this specification, as well as any and all combinations of said steps or features, or any two or more thereof.

[0092] Unless otherwise stated, any instance or embodiment of the present invention herein should be applied with the necessary modifications to any other instance or embodiment of the invention.

[0093] Unless otherwise specifically defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0094] Unless otherwise stated, the recombinant proteins, cell cultures, and immunotherapies used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained throughout the literature, for example, in the following sources: J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al.Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989); TA Brown (editor), Essential Molecular Biology: APractical Approach, Volumes 1 and 2, IRL Press (1991); DM Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996); FMAusubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date); Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988); and JEColigan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0095] The term “and / or”, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y”, and should be considered to provide clear support for both meanings or either meaning.

[0096] As used in this article, the term “derived from” should be understood to mean that the specified whole can be obtained from a particular source, although not necessarily directly from that source.

[0097] When comparing amino acid sequences, the sequences should be compared within a comparison window determined by the polypeptide length. For optimal alignment of two sequences, the comparison window can contain approximately 20% or less of additions or deletions (i.e., vacancies) compared to the reference sequence (excluding additions or deletions). Optimal alignment of the sequences used for the comparison window can be achieved through computerized implementations of algorithms (e.g., the BLAST family of programs), such as those developed by Altschul. et al ., 1997,Nucl.Acids Res. Published at 25:3389-3402. Global alignment programs can also be used to align similar sequences of approximately equal size. Examples of global alignment programs include NEEDLE (available at www.ebi.ac.uk / Tools / psa / emboss_needle / ), which is part of the EMBOSS package (Rice P...). et al ., 2000, Trends Genet ., 16: 276-277) and the GGSEARCH program as part of the FASTA package (available at fasta.bioch.virginia.edu / fasta_www2 / fasta_www.cgi?rm=compare&pgm=gnw) (Pearson W and Lipman D, 1988, Proc.Natl.Acad.Sci.USA Both programs are based on the Needleman-Wunsch algorithm, which is used to find the best alignment of two sequences along their entire length (including gaps). A detailed discussion of sequence analysis can also be found in Ausubel (85:2444-2448). et al Unit 19.3 of "Current Protocols in Molecular Biology" John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998.

[0098] "Purine receptor" usually refers to a receptor that uses purine (such as ATP) as a ligand.

[0099] "P2X7 receptor" generally refers to a purine receptor formed by three protein subunits or monomers, wherein at least one of the monomers has an amino acid sequence substantially as shown in SEQ ID NO: 141 below: SEQ ID NO: 141 MPACCSCSDVFQYETNKVTRIQSMNYGTIKWFFHVIIFSYVCFALVSDKLYQRKEPVISSVHTKVKGIAEVKEEIVENGVKKLVHSVFDTADYTFPLQGNSFFVMTNFLKTEGQEQRLCPEYPTRRTLCSSDRGCKKGWMDPQSKGIQ TGRCVVYEGNQKTCEVSAWCPIEAVEEAPRPALLNSAENFTVLIKNNIDFPGHNYTTRNILPGLNITCTFHKTQNPQCPIFRLGDIFRETGDNFSDVAIQGGIMGIEIYWDCNLDRWFHHCRPKYSFRRLDDKTTNVSLYPGYNFRYAK YYKENNVEKRTLIKVFGIRFDILVFGTGGKFDIIQLVVYIGSTLSYFGLAAVFIDFLIDTYSSNCRSHIYPWCKCCQPCVVNEYYYRKKCESIVEPKPTLKYVSFVDESHIRMVNQQLLGRSLQDVKGQEVPRPAMDFTDLSRLPLAL HDTPPIPGQPEEIQLLRKEATPRSRDSPVWCQCGSCLPSQLPESHRCLEELCCRKKPGACITTSELFRKLVLSRHVLQFLLLYQEPLLALDVDSTNSRLRHCAYRCYATWRFGSQDMADFAILPSCCRWRIRKEFPKSEGQYSGFKSPY The term "P2X7 receptor" is "trimer" or "trimeric" in relation to the fact that the P2X7 receptor is formed from three monomers. "P2X7 receptor" encompasses naturally occurring variants of the P2X7 receptor, such as splice variants, allele variants, SNPs, and isotypes, including naturally occurring truncated or secreted forms of the monomers forming the P2X7 receptor (e.g., forms consisting of extracellular domain sequences or truncated forms thereof), naturally occurring variant forms (e.g., alternative splice forms), and naturally occurring allele variants. In some embodiments of the invention, the naturally occurring sequence P2X7 monomeric polypeptide disclosed herein is a mature or full-length naturally occurring sequence polypeptide comprising the full-length amino acid sequence shown in SEQ ID NO: 141. In some embodiments, the P2X7 receptor may have a modified amino acid sequence; for example, various amino acids in the sequence shown in SEQ ID NO: 141 may be substituted, deleted, or have residues inserted.

[0100] A “functional P2X7 receptor” typically refers to a form of P2X7 receptor with three complete binding sites or grooves for ATP binding. When bound to ATP, the functional receptor forms a non-selective sodium / calcium channel, which transforms into a porous structure capable of allowing calcium ions and molecules up to 900 Da into the cytosol, potentially inducing programmed cell death. In normal homeostasis, expression of the functional P2X7 receptor is generally limited to cells undergoing programmed cell death, such as thymocytes, dendritic cells, lymphocytes, macrophages, and monocytes. Partial expression of the functional P2X7 receptor may also be present on erythrocytes and other cell types.

[0101] "Dysfunctional P2X7 receptors" (also known as "nonfunctional" or (nf)P2X7) are P2X7 receptors with impaired ATP response, preventing them from forming apoptotic pores under normal physiological conditions. Dysfunctional P2X7 receptors (or nfP2X7 receptors) generally refer to a form of P2X7 receptor with a conformation different from that of a functional P2X7, where the receptor cannot form apoptotic pores but can still function as a non-selective channel by maintaining a single functional ATP-binding site located between adjacent monomers. One example is when one or more of these monomers have cis isomerization at Pro210 (as shown in SEQ ID NO: 141). Isomerization can be caused by any molecular event leading to monomer misfolding, including, for example, mutations in the monomer's primary sequence or aberrant post-translational processing. One consequence of isomerization is that the receptor cannot bind ATP at one or more specifically two ATP-binding sites on the trimer, thus preventing the channel opening from extending. In these cases, the receptor cannot form a pore, which limits the extent to which calcium ions can enter the cytosol. Dysfunctional P2X7 receptors are expressed in a wide range of epithelial and hematopoietic carcinomas. As used herein, the term “dysfunctional P2X7 receptor” may be used interchangeably with the terms “nonfunctional P2X7 receptor” or “nfP2X7 receptor”.

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

[0103] "E200 epitope" generally refers to an epitope having the sequence GHNYTTNILPGLNITC (SEQ ID NO: 95) and its variants (e.g., SEQ ID NO: 96 to 119). The antigen recognition domain of the CAR of the present invention is preferably an antigen recognition domain capable of binding to E200 or its variants.

[0104] "E300 epitope" generally refers to an epitope with the sequence KYYKENNVEKRTLIK and its variants (such as SEQ ID NO: 120 and 121).

[0105] A “composite epitope” typically refers to an epitope formed by juxtaposing E200 and E300 epitopes or portions thereof. An example of a composite epitope containing E200 and E300 epitopes is GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO:122).

[0106] As used herein, the term "chimeric antigen receptor" or CAR refers to an artificially constructed protein intended for expression on the surface of immune cells. This protein comprises an extracellular domain (extracellular portion) containing an antigen-binding domain (also known as an antigen recognition domain), a transmembrane domain, and an intracellular signal transduction domain. The extracellular domain can connect to the transmembrane domain via a linker. The extracellular domain may also contain a signal peptide.

[0107] The terms "binding," "specific binding," or "specific to" in relation to the antigen-binding domain of a CAR refer to a domain that recognizes and binds to a specific antigen and substantially does not recognize or bind to other molecules in the sample. An antigen-binding domain that specifically binds to an antigen from one species can also bind to that antigen from another species. This cross-species reactivity is characteristic of many antibodies and therefore does not contradict the definition that an antigen-binding domain is specific. An antigen-binding domain that specifically binds to an antigen can also bind to different allelic forms of the antigen (allelic variants, splice variants, isotypes, etc.) or homologous variants of the antigen from the same gene family. This cross-reactivity is characteristic of many antibodies and therefore does not contradict the definition that an antigen-binding domain is specific.

[0108] As used herein, the terms “engineered cell” and “genetically modified cell” are used interchangeably. These terms refer to cells containing and / or expressing exogenous gene or nucleic acid sequences that subsequently modify the genotype or phenotype of the cell or its progeny. In particular, these 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 their natural state. For example, immune cells are engineered to express artificial constructs, such as chimeric antigen receptors, on their cell surface. For example, nucleic acid sequences encoding CARs can be delivered to cells using adenoviruses, adeno-associated virus (AAV)-based vectors, retroviral or lentiviral vectors, or any other pseudotype variants thereof, or any other gene delivery mechanism (e.g., electroporation or lipid transfection using CRISPR / Cas9, transposons (e.g., Sleeping Beauty) or variants thereof). Gene delivery can be in the form of mRNA (transient) or DNA (transient or permanent).

[0109] The term "immune cell" or "immune effector cell" refers to a cell that can be part of the immune system—that is, the adaptive (i.e., cellular or humoral) or the innate immune system—and performs specific effector functions, such as α-β T cells, NK cells, NKT cells, B cells, Breg cells, Treg cells, innate lymphocytes (ILCs), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, γ-δ 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 cell subsets that can mature or differentiate into somatic cells. Cells can be naturally occurring or produced through cytokine exposure, artificially / genetically modified cells (e.g., iPSCs and other artificial cell types). Immune cells can be artificial cell subsets, including induced pluripotent stem cells and cells matured from them. Preferred immune cells are cells with cytotoxic effector functions, such as α-β T cells, NK cells, NKT cells, ILCs, CIK cells, LAK cells, or γ-δ T cells. "Effective function" refers to the specialized function of a cell. For example, in T cells, effector function can be cytolytic activity or helper cell activity, including the secretion of cytokines.

[0110] As used herein, the term “treat or treatment of” means reducing the frequency or severity of at least one sign or symptom of a disease or condition experienced by the subject.

[0111] As used herein, the term “expression” is defined as the transcription and / or translation of a specific nucleotide sequence in a cell driven by its promoter.

[0112] Antibodies, or immunoglobulins, or Ig are gamma globulins found in the blood or other bodily fluids of vertebrates. They function in the immune system to bind to antigens, thereby identifying and / or neutralizing foreign substances.

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

[0114] The H and L chains define specific Ig domains. More specifically, each H chain has a variable domain (VN) at its N-terminus. H Following this are three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isoforms. Each L chain has a variable domain (V) at the N-terminus. L ), followed by a constant structural domain (CL) at its other end. V L With V H Alignment, and C L Aligned with the first constant structural domain (CH1) of the heavy chain.

[0115] Antibodies can be designated as different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains named α, δ, ε, γ, and μ, respectively. The γ and α classes are based on... The relatively subtle differences in sequence and function are further subdivided into subclasses; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The L-chain from any vertebrate species can be designated as one of two distinct types (called κ and λ) based on the amino acid sequence of its constant domain.

[0116] The constant domain contains the Fc region, which includes the carboxyl-terminal portions of two H chains held together by disulfide bonds. The effector function of an antibody (such as ADCC) is determined by the sequence in the Fc region, which is also the part recognized by the Fc receptor (FcR) found on certain cell types.

[0117] V H and V L Together they form "variable regions" or "variable domains," including the amino-terminal domains of either the heavy or light chain of the antibody. The variable domain of the heavy chain can be called a "V..." H The variable structural domain of a light chain can be called a "V". LThe V domain contains an antigen-binding site that influences antigen binding and defines the specificity of a particular antibody for its specific antigen. The V region spans approximately 110 amino acid residues and consists of relatively invariant segments called framework regions (FRs) (typically about four, each consisting of 15 to 30 amino acids). These are separated by shorter, highly variable regions called hypervariable regions (typically about three, each typically 9 to 12 amino acids long). FRs primarily adopt a β-sheet configuration, and the hypervariable regions form loops connecting the β-sheet structures and, in some cases, form part of the β-sheet structure.

[0118] "Hypervariant regions" refer to the regions within the variable domains of an antibody where the sequence exhibits hypervariability and / or forms structurally defined loops. Typically, antibodies contain six hypervariant regions (also known as complementarity-determining regions or CDRs); three are located in the V... H In (H1, H2, H3), and three of them are in V L (L1, L2, L3)

[0119] The complementarity-determining region (CDR) sequence of an antigen-binding protein can be defined according to any of a variety of numbering systems, including the IMGT numbering system, the Kabat system, or the Chothia system. Technicians are fully capable of identifying and determining the CDR once they have the complete heavy and light chain variable sequences.

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

[0121] As used herein, the term "complementarity-determining region" (synonymous CDR; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of the antibody variable region, the presence of which is the primary reason for specific antigen binding. Each variable region domain (V H or V L It typically has three CDRs, which are designated as CDR1, CDR2, and CDR3. H The CDRs in this paper are also referred to as CDR H1, CDR H2, and CDR H3, respectively, where CDR H1 corresponds to V. H CDR 1 and CDR H2 correspond to V H CDR 2, and CDR H3 corresponds to V H CDR 3. Similarly, V L The CDRs in this paper are referred to as CDR L1, CDR L2, and CDR L3, respectively, where CDR L1 corresponds to V. L CDR1 and CDR L2 correspond to V L CDR 2, and CDR L3 corresponds to V LCDR 3. In one instance, the amino acid positions assigned to CDR and FR are defined according to the Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as the "Kabat numbering system"). In another instance, the amino acid positions assigned to CDR and FR are defined according to the Enhanced Chothia Numbering Scheme (http: / / www.bioinfo.org.uk / mdex.html). This invention is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including normalized numbering systems or the following numbering systems: the numbering systems of Chothia and Lesk J. Mol.Biol.196: 901-917, 1987; Chothia et al., Nature 342: 877-883, 1989; and / or Al-Lazikani et al., J. Mol.Biol.273: 927-948, 1997; 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.

[0122] The "frame region" (FR) consists of the variable region residues other than the CDR residues. The FRs of VH are referred to in this paper as FR H1, FR H2, FR H3, and FR H4, where FR H1 corresponds to VH. H FR1, FRH2 correspond to V H FR2, FRH3 correspond to V H FR 3, and FR H4 corresponds to V H FR 4. Similarly, V L The FRs in this paper are referred to as FR L1, FR L2, FR L3, and FR L4, respectively, where FR L1 corresponds to V. L FR1, FRL2 correspond to V L FR2, FRL3 correspond to V L FR3, and FRL4 corresponds to V. L FR 4.

[0123] "Antigen binding site" generally refers to a molecule that contains at least the hypervariable region and frame region required to confer antigen-binding function to the V domain. In the methods described herein, the antigen binding site may be in the form of an antibody or antibody fragment (e.g., mAb, single-domain (SD)-mAb, dAb, Fab, SD-Fab, Fd, SD-Fv, Fv, F(ab')2, or scFv). A "complete" or "whole" antibody is an antibody that contains an antigen-binding site and a C-cell antibody. L And at least heavy chain constant domains CH1, CH2, and CH3. The constant domains can be natural sequence constant domains (e.g., human natural sequence constant domains) or amino acid sequence variants thereof.

[0124] "Complete antibody fragments containing variable domains" include SD-mAb, Fab, Fab', F(ab')2 and Fv fragments; biantibodies; linear antibodies, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0125] The “Fab fragment” consists of the entire L chain and the variable region (V) of the H chain. H It consists of a first constant domain (CH1) of a heavy chain. Each Fab fragment is monovalent in terms of antigen binding, that is, the Fab fragment has a single antigen binding site.

[0126] The difference between a “Fab’ fragment” and a Fab fragment is that the Fab fragment has a few additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. Fab’-SH is the name given in this paper to Fab’ fragments in which the cysteine ​​residues of the constant domain have free thiol groups.

[0127] The “F(ab')2 fragment” roughly corresponds to two disulfide-linked Fab fragments with bivalent antigen-binding activity and can still crosslink antigens.

[0128] "Fv" is the smallest antibody fragment containing complete antigen recognition and binding sites. This fragment consists of a dimer of a tightly non-covalently bound heavy chain variable region domain and a light chain variable region domain.

[0129] In single-chain Fv (scFv) types, a heavy chain variable domain and a light chain variable domain are covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate in a "dimeric" structure similar to that of double-chain Fv types. The folding of these two domains generates six hypervariable rings (three rings each in the H chain and L chain), which provide amino acid residues for antigen binding and confer specificity for antibody-antigen binding.

[0130] A "single-chain Fv" (also abbreviated as "sFv" or "scFv") is an antibody fragment containing V-shaped molecules linked together to form a single polypeptide chain. H and V L Antibody domain. Preferably, the scFv polypeptide also contains V H and V L A polypeptide linker between the domains enables scFv to form the desired structure for antigen binding.

[0131] The "single variable domain" is half of the Fv (containing only three antigen-specific CDRs), which has the ability to recognize and bind antigens, although its affinity is usually lower than that of the full binding site.

[0132] "Dual antibody" refers to an antibody fragment with two antigen-binding sites, which are contained within the same polypeptide chain and bound to a light chain variable domain (V). L ) connected heavy chain variable structural domain (V H (V) H -V L By building on V H and V L Small antibody fragments are prepared by using sFv fragments (see the preceding paragraph) with short linkers (about 5 to 10 residues) between the domains, which enables inter-chain rather than intra-chain pairing of the V domains, thereby producing bivalent fragments, i.e. fragments with two antigen-binding sites.

[0133] Biantibodies can be divalent or bispecific. A bispecific biantibody is a heterodimer of two "crossover" sFv fragments, where the V of the two antibodies... H and V L The domains are located on different polypeptide chains. Triantibodies and tetraantibodies are also commonly known in this field.

[0134] "Isolated antibody" is an antibody that has been identified, isolated, and / or recovered from components of its pre-existing environment. Contaminant components are substances that will interfere with the therapeutic use of the antibody and may include enzymes, hormones, and other protein or non-protein solutes.

[0135] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of antibodies produced by humans. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Human antibodies can also be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen attack, but whose endogenous loci have been deactivated.

[0136] The “humanized” form of non-human (e.g., rodent) antibodies is a chimeric antibody containing a minimal sequence derived from a non-human antibody. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies) where residues from the receptor hypervariable region are replaced by residues from the hypervariable region of a non-human species (donor antibody) (e.g., mice, rats, rabbits, or non-human primates) that possess the desired antibody specificity, affinity, and potency. In some cases, frame region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may include residues not found in the receptor or donor antibody. These modifications are made to further improve antibody performance. Typically, humanized antibodies will contain substantially all, usually both, variable domains, where all or substantially all hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all FRs are FRs of the human immunoglobulin sequence. Humanized antibodies may optionally also include at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the constant region of human immunoglobulins.

[0137] Monoclonal antibodies are antibodies obtained from a substantially homogeneous group of antibodies; that is, the individual antibodies constituting the group are identical, except for the possibility of a small number of natural mutations. Monoclonal antibodies are highly specific, targeting a single antigenic site or determinant on an antigen. In addition to their specificity, another advantage of monoclonal antibodies is that they can be synthesized without contamination from other antibodies. Monoclonal antibodies can be prepared using hybridoma methods. They can also be isolated from phage antibody libraries using molecular engineering techniques.

[0138] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies typically bind antigens slowly and tend to dissociate readily, while high-affinity antibodies typically bind antigens more quickly and tend to remain bound for longer periods. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this invention.

[0139] As used herein, the term "antigen" is intended to include substances that bind to or cause the production of one or more antibodies, and may include, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates, and combinations thereof, such as glycosylated proteins or glycolipids. As used herein, the term "antigen" refers to a molecular entity that can be expressed on target cells and recognized by an 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 bind to a structure with high affinity.

[0140] An epitope typically refers to the portion of an antigen that is bound to an antibody's antigen-binding site. An epitope can be "linear," meaning that the hypervariable loop of the antibody's CDR, which forms the antigen-binding site, binds to an amino acid sequence in the primary protein structure. In some embodiments, an epitope is a "conformational epitope," where the hypervariable loop of the CDR binds to residues present in the tertiary or quaternary protein structure.

[0141] As used in this article, the term "target cell" refers to a cell that expresses the nfP2X7 receptor. Target cells can be cancer cells or any other diseased cells.

[0142] The term “symptom” or “condition” refers to a functional abnormality or disorder in a subject, such as cancer, an autoimmune disease, or an infection by a virus, bacteria, parasite, or other substance.

[0143] For example, nucleic acids or peptides that are naturally present in living animals are not "isolated," but the same nucleic acids or peptides that are partially or completely isolated from their native coexisting material are "isolated." Isolated nucleic acids or proteins can also exist in non-natural environments, such as host cells.

[0144] As used in this article, the term “autologous” refers to any material derived from the same subject that will later be reintroduced into that subject.

[0145] As used in this article, the term "alien" refers to any material derived from different subjects of the same species that will be reintroduced into that subject.

[0146] The terms “therapeutic effective amount” or “therapeutic effective population” refer to, for example, the amount of cell population that provides therapeutic benefit in a subject.

[0147] Chimeric antigen receptor Generally, an "antigen-binding domain" or "antigen-recognition domain" refers to the region of a CAR that specifically binds to an antigen (thereby enabling it to target cells containing that antigen). The CAR of this invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is used to bind to the nfP2X7 receptor. The antigen-binding domain may comprise an antibody or an antibody-binding fragment thereof. The antigen-binding domain may comprise, for example, a full-length heavy chain, a Fab fragment, a single-chain Fv (scFv) fragment, a bivalent single-chain antibody, or a biantibody. Any molecule that specifically binds to a given antigen, such as an affinity or a ligand-binding domain from a naturally occurring receptor, can be used as an antigen-binding domain. Typically, the antigen-binding domain is an scFv. Typically, in an scFv, variable regions of the immunoglobulin heavy and light chains are fused together by a flexible linker to form the scFv. Such a linker may be, for example, a "(G4 / S1)3 linker" and variants thereof, but those skilled in the art will understand that various linker sequences and forms can be used.

[0148] In some cases, it is advantageous to derive the antigen-binding domain from the same species from which the CAR will be used. For example, when therapeutic applications in humans are planned, it may be advantageous for the antigen-binding domain of the CAR to contain a human or humanized antibody or its antigen-binding fragment. Human or humanized antibodies or their antigen-binding fragments can be prepared by a variety of methods well known in the art.

[0149] "Signal peptide" refers to a peptide sequence that guides the transport and localization of proteins within cells, such as to specific organelles (e.g., endoplasmic reticulum) and / or the cell surface.

[0150] As used herein, a "spacer region" or "hinge" refers to a hydrophilic region between an antigen-binding domain and a transmembrane domain. The CAR of this invention may include an extracellular spacer region, but such a spacer region may be omitted. The spacer region may include, for example, an Fc fragment of an antibody or a fragment thereof, a hinge region of an antibody or a fragment thereof, a CH2 or CH3 region of an antibody, an accessory protein, an artificial spacer region sequence, or a combination thereof. A prominent example of a spacer region is the CD8α hinge.

[0151] The transmembrane domain of a CAR can be derived from any desired natural or synthetic source of such a domain. When the source is natural, the domain can be derived from any membrane-binding or transmembrane protein. The transmembrane domain can be derived from, for example, CD8α or CD28. A CAR can have two (or more) transmembrane domains when the key signaling and antigen recognition modules (domains) are located on two (or more) peptides. Due to the small molecule-dependent heterodimerization of the domains in each peptide of the CAR, the splitting of the key signaling and antigen recognition modules enables small molecule-dependent, titratable, and reversible control of CAR cellular expression (Wu et al, 2015, Science 350: 293-303).

[0152] The cytoplasmic domains (or intracellular signaling domains) of a CAR are responsible for activating at least one of the normal effector functions of CAR-expressing immune cells. "Effective function" refers to a cell's specialized function; for example, in T cells, effector functions may be cytolytic activity or helper cell activity, including cytokine secretion. An intracellular signaling domain is the portion of a protein that transduces effector signals and directs CAR-expressing cells to perform their specialized functions. Intracellular signaling domains may include any complete, mutated, or truncated portion of an intracellular signaling domain of a given protein that is sufficient to transduce signals that initiate or block effector functions of immune cells.

[0153] The signal transduction domain of a CAR can be any suitable domain that, upon recognition of an antigen by the CAR's antigen recognition domain and activation of the CAR, induces or participates in inducing an intracellular signaling cascade. The specific selection of the CAR's signal transduction domain will depend on the desired cellular outcome after CAR activation. While many possible signal transduction domains exist, when used in immunotherapy and cancer therapy, they can be categorized into two general classes based on the receptors from which they originate: activating receptors and co-stimulatory receptors. Therefore, in some embodiments, the CAR's signal transduction domain includes a portion derived from an activating receptor. In some embodiments, the signal transduction domain includes a portion derived from a co-stimulatory receptor.

[0154] As used throughout this specification, the term "activating receptor" refers to the following receptors or co-receptors that form or participate in the formation of the T-cell receptor (TCR) complex, or the following receptors that participate in the specific activation of immune cells due to recognition of antigens or other immunogenic stimuli.

[0155] The function of intracellular domains can be pro-inflammatory, anti-inflammatory, and / or immunomodulatory, or a combination thereof.

[0156] Some examples of intracellular signaling domains used in CARs include cytoplasmic signaling sequences of T-cell receptors (TCRs) and cytoplasmic signaling sequences of co-receptors that initiate signal transduction upon antigen-receptor binding.

[0157] Typically, T cell activation can be mediated by two different classes of cytoplasmic signaling sequences: the first class consists of sequences that induce antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and the second class consists of sequences that act in a non-antigen-dependent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences, co-stimulatory signaling domains). Therefore, the intracellular signaling domains of a CAR may include one or more primary cytoplasmic signaling domains and / or one or more secondary cytoplasmic signaling domains.

[0158] Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain ITAM (immunoreceptor tyrosine-based activation motif) signaling motifs.

[0159] Examples of primary cytoplasmic signaling sequences containing ITM commonly used in CARs are those derived from TCRζ (CD3ζ), FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. The most prominent are sequences derived from CD3ζ.

[0160] The cytoplasmic domain of a CAR can be designed to contain the CD3-ζ signaling domain itself or in combination with any other desired cytoplasmic domain. The cytoplasmic domain of a CAR may contain a portion of the CD3ζ chain and a co-stimulatory signaling region. The co-stimulatory signaling region refers to the portion of the intracellular domain of the CAR that contains the co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for an effective lymphocyte response to an antigen. Examples of co-stimulatory molecules are CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0161] The cytoplasmic signaling sequences within the cytoplasmic signaling moiety of CARs can be linked together randomly or in a specified order, with or without linkers. Short oligopeptides or polypeptides of preferably 2 to 10 amino acids in length can form bonds with linkers. A prominent linker is the glycine-serine duplex.

[0162] As an example, the cytoplasmic domain may contain a CD3-ζ signal transduction domain and a CD28 signal transduction domain. In another example, the cytoplasmic domain may contain a CD3-ζ signal transduction domain and a CD27 signal transduction domain. In yet another example, the cytoplasmic domain may contain a CD3-ζ signal transduction domain, a CD28 signal transduction domain, and a CD27 signal transduction domain.

[0163] The extracellular portion, transmembrane domain, or cytoplasmic domain of a CAR may also contain heterodimerization domains for the cleavage of key signal transduction and antigen recognition modules of the CAR.

[0164] The CAR used according to the present invention, namely the CAR containing the nfP2X7E200 combined structural domain, can be designed to include any part or portion of the structural domains described herein in any order and / or combination, thereby producing a functional CAR.

[0165] The CAR or its derived polypeptides, nucleic acid molecules, or recombinant expression vector cells encoding the CAR, or cell populations expressing the CAR, as disclosed herein, can be isolated and / or purified. The term "isolated" means altered or removed from its natural state. For example, an isolated cell population means an enrichment of such cells that is separate from other cells typically associated with the isolated cells in their natural state. An isolated cell population means a substantially purified cell population that is more homogeneous than those found in nature. Preferably, the enriched cell population contains at least about 90% of the selected cell types. In certain aspects, the cell population contains at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% of the selected cell types.

[0166] The binding affinity of the antigen recognition domain of the CAR to the nfP2X7 recognition site E200 on the cell (or the E200 sequence contained in the “bridging molecule” described elsewhere in this document) may vary, but typically the binding affinity can be in the range of 100 μM, 1 nM, 10 nM or 100 nM, preferably at least about 1 pM or 10 pM, and even more preferably at least about 100 pM.

[0167] connector Linkers can be peptides with a length of up to 20 amino acids. The terms "link" or "fusion" refer to a covalent bond, such as a peptide bond, formed between two parts. Therefore, in the context of this invention, linkers can have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids. For example, the CAR provided herein can comprise linkers between the VH and VL of the antigen-binding domain and / or between the antigen-binding domain and the hinge. Such linkers have the advantage of allowing different polypeptides of the fusion protein to fold more independently and function as intended.

[0168] Those skilled in the art are familiar with the design and use of various peptide linkers composed of a variety of amino acids and of varying lengths, which would be suitable as linkers according to the present invention. Linkers can comprise various combinations of repeating amino acid sequences. Linkers can be flexible linkers (e.g., those containing repeating sequences of glycine and serine residues), rigid linkers (e.g., those containing repeating sequences of glutamic acid and lysine residues, flanked by alanine repeating sequences), and / or cleavable linkers (e.g., sequences susceptible to protease cleavage).

[0169] The peptide linker can be any one or more repetitive sequences of Gly-Ser (GS), Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS) (SEQ ID NO: 17), or Gly-Gly-Gly-Gly-Ser (GGGGS) (SEQ ID NO: 18) or variants thereof. In any embodiment, the linker may comprise or consist of the sequence GGGGSGGGGSGGGGS, i.e., (G4S)3 (SEQ ID NO: 93).

[0170] In any embodiment, the peptide linker may comprise the amino acid sequence GGGGGS (a linker of 6 amino acids in length, SEQ ID NO: 19) or even longer. The linker may be a series of repeating glycine and serine residues of varying lengths (GS), i.e., (GS)n, where n is any value from 1 to 15 or greater. 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 should be understood that n can be any value, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or greater.

[0171] Nucleic acid In another aspect, the present invention provides a nucleic acid molecule (e.g., a recombinant nucleic acid molecule) comprising a nucleotide sequence encoding a chimeric antigen receptor according to the present invention.

[0172] In some embodiments of the invention, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4 and / or 12 (e.g., SEQ ID NO: 94) or encoding a functional variant thereof, wherein the functional variant retains the ability to bind to the nfP2X7 receptor. In some embodiments, the nucleic acid molecule encodes a functional variant comprising at least 80% of the same amino acid sequence as 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 comprising at least 80% of the same amino acid sequence as 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 NO: 4 and 12.

[0173] Nucleic acid molecules can contain any polynucleotide or polydeoxynucleotide, which can be unmodified or modified RNA or DNA. For example, nucleic acid molecules can include single-stranded and / or double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA as a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA, which can be single-stranded, or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. Furthermore, nucleic acid molecules can contain triple-stranded regions containing RNA or DNA, or both. Nucleic acid molecules can also contain one or more modified bases or DNA or RNA backbones that are modified for stability or other reasons. DNA and RNA can be modified in a variety of ways; therefore, the term "nucleic acid molecule" encompasses forms of chemical, enzymatic, or metabolic modification.

[0174] In some embodiments of the present invention, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 74 and / or SEQ ID NO: 82.

[0175] Those skilled in the art will understand that this invention covers any of the following nucleotide sequences encoding a chimeric antigen receptor comprising the amino acid sequence shown in SEQ ID NO: 4 and / or 12 (e.g., SEQ ID NO: 94) or a functional variant thereof. For example, variants of SEQ ID NO: 94 are considered, which comprise one or more nucleic acids different from SEQ ID NO: 74 and / or 82, but still encode the same amino acid sequence. 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, each nucleotide sequence encoding the following chimeric antigen receptors herein also describes each possible silent variant of the nucleotide sequence having the amino acid sequence shown in SEQ ID NO: 94 or a functional variant thereof. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG (which is typically the only codon for methionine) and TGG (which is typically the only codon for tryptophan)) can be modified to produce a functionally identical molecule. Therefore, each silent variant of the nucleotide sequence encoding a polypeptide is implicit in each of the said sequences.

[0176] In another aspect, the present invention provides a nucleic acid construct comprising a nucleic acid molecule according to the invention. The nucleic acid construct may also comprise one or more of the following: a replication origin of one or more hosts; a selection marker gene active in one or more hosts; and / or one or more transcriptional control sequences.

[0177] As used herein, the term "selectable marker gene" includes any gene that can confer a phenotype in cells in which it is expressed, so as to facilitate identification and / or selection of cells transfected or transformed with the construct.

[0178] "Optional marker genes" include any of the following nucleotide sequences that, when expressed in cells transformed with the construct, confer a phenotype that facilitates identification and / or selection of these transformed cells. The range of nucleotide sequences encoding suitable optional markers is 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) genes; cytosine deaminase (CDA) genes; dihydrofolate reductase (DHFR) genes; histidine dehydrogenase (hisD) genes; puromycin-N-acetyltransferase (PAC) genes; thymidine kinase (TK) genes; xanthine-guanine phosphoribosyltransferase (XGPRT) genes or antibiotic resistance genes (e.g., ampicillin resistance genes, puromycin resistance genes, bleomycin resistance genes, hygromycin resistance genes, kanamycin resistance genes, and ampicillin resistance genes); fluorescence reporter genes (e.g., genes encoding green, red, yellow, or blue fluorescent proteins); and luminescence-based reporter genes (e.g., luciferase genes), as well as other genes that allow optical selection of cells using techniques such as fluorescence-activated cell sorting (FACS).

[0179] In addition, it should be noted that the optional marker gene can be a different open reading frame in the construct, or it can be expressed as a fusion protein with another polypeptide (e.g., CAR).

[0180] As described above, nucleic acid constructs may also contain one or more transcriptional control sequences. The term "transcriptional control sequence" should be understood to include any nucleic acid sequence that affects the transcription of operatively linked nucleic acids. Transcriptional control sequences may include, for example, leader sequences, polyadenylated sequences, promoters, enhancers or upstream activating sequences and transcription terminators. Typically, transcriptional control sequences include at least a promoter. As used herein, the term "promoter" describes any nucleic acid that confers, activates or enhances nucleic acid expression in a cell.

[0181] In some embodiments, at least one transcriptional control sequence is operatively linked to the nucleic acid molecule of the present invention. For the purposes of this specification, a transcriptional control sequence is considered "operatively linked" to a given nucleic acid molecule when it is capable of promoting, inhibiting, or otherwise regulating the transcription of the nucleic acid molecule. Thus, in some embodiments, the nucleic acid molecule is under the control of a transcriptional control sequence (e.g., a constitutive or inducible promoter).

[0182] A “nucleic acid construct” can be in any suitable form (e.g., plasmid, bacteriophage, transposon, granulosome, chromosome, vector, etc.) that is capable of replication when bound to appropriate control elements and can transfer the gene sequence contained within the construct between cells. Therefore, the term includes cloning and expression vectors, as well as viral vectors. In some embodiments, the nucleic acid construct is a vector. In some embodiments, the vector is a viral vector.

[0183] Promoters regulate the expression of nucleic acid molecules operably linked to them; this regulation can be constitutive or differential, depending on the cell, tissue, or organ in which expression occurs. Therefore, promoters can include, for example, constitutive promoters or inducible promoters. A “constitutive promoter” is a promoter that is active under most environmental and physiological conditions. An “inducible promoter” is a promoter that is active under specific environmental or physiological conditions. The present invention contemplates the use of any promoter that is active in the cells of interest. Therefore, those skilled in the art will readily identify a wide variety of promoters.

[0184] Constitutive promoters in mammals may include, but are not limited to, simian virus 40 (SV40). Cytomegalovirus (CMV) CMV, P-actin, ubiquitin C (UBC), elongation factor-1α (EF1A), phosphoglycerate kinase (PGK), and early enhancer of CMV / chicken β-actin (CAGG).

[0185] Inducible promoters can include, but are not limited to, chemically inducible promoters and physically inducible promoters. Chemically inducible promoters include promoters that have activity modulated by a chemical compound (e.g., alcohols, antibiotics, steroids, metal ions, or other compounds). Examples of chemically inducible promoters include: tetracycline-regulated promoters (e.g., see U.S. Patent 5,851,796 and 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), etc.; and metal-responsive promoters, such as metallothionein promoters (e.g., see U.S. Patent 4,940,661, 4,579,821, and 4,601,978), etc.

[0186] As described above, the control sequence may also include a terminator. The term "terminator" refers to a DNA sequence at the end of a transcription unit that signals the termination of transcription. A terminator is typically a 3' untranslated DNA sequence containing a polyadenylation signal that facilitates the addition of a polyadenylation sequence to the 3' end of the primary transcript. Like promoter sequences, terminators can be any terminator sequence that is operable in the cell, tissue, or organ in which it is intended to be used. Suitable terminators are known to those skilled in the art.

[0187] As will be understood, the nucleic acid constructs of the present invention may also include additional sequences, such as sequences capable of enhancing expression, cytoplasmic or membrane transport sequences, and position signaling sequences. Specific, non-limiting examples include internal ribosome entry sites (IRES).

[0188] This invention extends to virtually all genetic constructs as described herein. These constructs may also contain nucleotide sequences designed to maintain and / or replicate the genetic construct in eukaryotes, and / or integrate the genetic construct or a portion thereof into the eukaryotic cell genome.

[0189] Methods known in the art can be used to intentionally introduce (transfect / transduct) exogenous genetic material (such as the nucleic acid constructs of the present invention) into eukaryotic cells. It should be understood that the optimal method for introducing the nucleic acid construct into the desired host cell depends on a variety of factors, such as the size of the nucleic acid construct, the type of host cell, the desired transfection / transduction efficiency, and the final desired or required viability of the cells after transfection / transduction. Non-limiting examples of such methods include: chemical transfection using chemicals (e.g., cationic polymers, calcium phosphate, or structures (e.g., liposomes and dendritic polymers)); non-chemical methods (e.g., electroporation, acoustic transfection, heat shock, or optical transfection); and particle-based methods (e.g., gene gun delivery, magnetic transfection, puncture transfection, or viral transduction).

[0190] The nucleic acid construct will be selected based on the desired transfection / transduction method. In some embodiments of the invention, the nucleic acid construct is a viral vector, and the method for introducing the nucleic acid construct into host cells is viral transduction. Methods for inducing CAR expression in PBMCs using viral transduction are known in the art (Parker, LL). et al(Hum GeneTher. 2000;11: 2377-87), and more generally utilize retroviral or lentiviral systems to transduce mammalian cells (Barde et al, “Production and Titration of Lentiviral Vectors,” Current Protocols in Neuroscience, Volume 53, Issue 1 (2010) (also cited in Current Protocols in Neuroscience 4.21.1-4.21.23 (October 2010) and Cepko, C. and Pear, W. CurrProtoc Mol Biol. 2001, Unit 9.9). In other embodiments, the nucleic acid construct may be a plasmid, viscera, artificial chromosome, etc., and may be transfected into cells by any suitable method known in the art.

[0191] Genetically modified cells As described herein, in some embodiments, the present invention includes a treatment method involving the use of cells expressing the chimeric antigen receptor (CAR) of the present invention.

[0192] The cell can be an “engineered cell,” a “genetically modified cell,” an “immune cell,” or an “immune effector cell,” as described herein. Furthermore, the cell is capable of differentiating into an immune cell. Cells capable of differentiating into immune cells (e.g., T cells expressing dysfunctional P2X7CAR) can be stem cells, multilineage progenitor cells, or induced pluripotent stem cells.

[0193] In any embodiment, the cell may be a T cell, wherein the T cell optionally does not express TcRαβ, PD1, CD3 or CD96 (e.g., by knocking down or knocking out one of these genes at the genetic or functional level).

[0194] In any embodiment, the cell may be an immune cell, wherein the cell optionally does not express accessory molecules, which may be checkpoint, exhaustion or apoptosis-related signaling receptors and ligands, such as PD-1, LAG-3, TIGIT, CTLA-4, FAS-L and FAS-R (e.g., by knocking out one of these genes at the genetic or functional level).

[0195] In some embodiments, the genetically modified cells contain two or more different CARs. For example, genetically modified cells may contain CARs with different architectures (e.g., different signal transduction domains) but containing the same antigen recognition sequence. Alternatively, genetically modified cells may contain CARs with different antigen recognition domains but binding to the nfP2X7 receptor. The CARs may bind to the same or different epitopes of the nfP2X7 receptor. In another embodiment, genetically modified cells may contain CARs for binding to different antigens (e.g., the nfP2X7 receptor and different antigens on cancer cells).

[0196] In some embodiments of the present invention, the genetically modified cells comprise nucleic acid molecules or nucleic acid constructs encoding two or more different CARs.

[0197] As mentioned herein, "genetically modified cells" include any cells containing non-naturally occurring and / or introduced nucleic acid molecules or nucleic acid constructs covered by this invention. The introduced nucleic acid molecules or nucleic acid constructs may be maintained in the cell as discrete DNA molecules, or they may be integrated into the cell's genomic DNA.

[0198] The genomic DNA of a cell should be understood in its broadest context as any and all endogenous DNA containing the genetic complements that constitute the cell. Therefore, the genomic DNA of a cell should be understood to include chromosomal DNA, mitochondrial DNA, etc. Thus, the term "genome integration" encompasses chromosomal integration, mitochondrial DNA integration, etc. The "genome-integrated form" of a construct can be all or part of the construct. However, in some embodiments, the genome-integrated form of the construct contains at least the nucleic acid molecule of the second aspect of the invention.

[0199] As used herein, the terms "different CAR" or "different chimeric antigen receptor" refer to any two or more CARs having different antigen recognition domains and / or different signal transduction domains. In one instance, "different CAR" includes two CARs having the same antigen recognition domain (e.g., both CARs recognize a dysfunctional P2X7 receptor) but different signal transduction domains (e.g., one CAR has a signal transduction domain with a portion of the activating receptor, and the other CAR has a signal transduction domain with a portion of the co-stimulatory receptor). As will be understood, at least one of the two or more CARs in this embodiment will have an antigen recognition domain that recognizes a dysfunctional P2X7 receptor, and the other CARs may take any suitable form and may target any suitable antigen.

[0200] Therefore, in some embodiments of the present invention, two or more different CARs have different signal transduction domains and may have the same or different antigen recognition domains. Specifically, the genetically modified cells of the present invention may comprise: a first chimeric antigen receptor having a signal transduction domain comprising a portion derived from an activating receptor; and a second chimeric antigen receptor having a signal transduction domain comprising a portion derived from a co-stimulatory receptor.

[0201] In some implementations, the activating receptor (from which a portion of the signal transduction domain is derived) is a CD3 co-receptor complex or an Fc receptor.

[0202] In some implementations, the co-stimulatory receptor (from which a portion of the signal transduction domain is derived) is selected from the group consisting of CD27, CD28, CD-30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS.

[0203] In some implementations, the co-stimulatory receptor (from which a portion of the signal transduction domain is derived) is selected from the group consisting of CD28, OX40, or 4-1BB.

[0204] In some implementations, the genetically modified cells are further modified to constitutively express co-stimulatory receptors.

[0205] As described above, a cellular immune response is typically induced only when both an activation signal (usually in response to an antigen) and a co-stimulatory signal are simultaneously received. Therefore, by using genetically modified cells according to some of the embodiments described above, the cells contain two or more CARs that can provide both intracellular activation and intracellular co-stimulatory signals, thereby ensuring that a sufficient immune response is induced when the CAR recognizes its homologous antigen. Alternatively, the genetically modified cells may contain only one CAR having an antigen-recognizing domain that recognizes a dysfunctional P2X7 receptor and constitutively expressing a co-stimulatory receptor, thereby increasing the likelihood of simultaneous co-stimulation when the CAR is activated. Alternatively, the genetically modified cells may be further modified to constitutively express both the co-stimulatory receptor and its ligand. In this way, the cells continuously experience co-stimulation and only require activation of the CAR, which has a signal transduction domain containing a portion of the activation receptor for cellular immune activation.

[0206] Therefore, in some embodiments, the genetically modified cells expressing CAR are further modified to constitutively express the co-stimulatory receptor. In further embodiments, the genetically modified cells are further modified to express the ligand of the co-stimulatory receptor, thereby promoting cell self-stimulation. Examples of CAR-expressing T cells that also express both the co-stimulatory receptor and its homologous ligand (in order to induce self-stimulation) are known in the art, and particularly include Stephen MT. et al Those published in NatMed, 2007; 13: 1440-9.

[0207] The potency of genetically modified cells containing CARs can be enhanced by further modifying cells to secrete cytokines (preferably pro-inflammatory or pro-proliferative cytokines). This cytokine secretion provides autocrine support to the CAR-expressing cells and alters the local environment surrounding the CAR-expressing cells, thereby recruiting and activating other cells of the immune system. Therefore, in some embodiments of the fourth or fifth aspect of the invention, the genetically modified cells are further modified to secrete cytokines. This secretion can be constitutive or induced upon recognition of a CAR with its homologous antigens.

[0208] While any one or more cytokines may be selected based on the desired immune response, preferred cytokines and / or chemokines include IL-2, IL-7, IL-12, IL-15, IL-17, IL-18 and IL-21, CCL19, CCL21 or combinations thereof.

[0209] The immune cell can be any suitable immune cell or its progenitor cell, or it can be a homogeneous or heterogeneous cell population. In some embodiments, the cell is a leukocyte, peripheral blood mononuclear cell (PBMC), lymphocyte, T cell, CD4+ T cell, CD8+ T cell, natural killer cell, natural killer T cell, or γδ T cell.

[0210] The immune cell may be a T cell, wherein the T cell optionally does not express TcRαβ, PD1, CD3 or CD96 (e.g., by knocking down or knocking out one of these genes at the genetic or functional level).

[0211] Immune cells may not express accessory molecules, which can be checkpoint, exhaustion or apoptosis-related signaling receptors and ligands, such as PD-1, LAG-3, TIGIT, CTLA-4, FAS-L and FAS-R (e.g., by knocking out or knocking down one of these genes at the genetic or functional level).

[0212] Treatment and application methods As further discussed in this document, the invention is applicable to the treatment of a variety of conditions, although it is preferred for the treatment of cancer. The present invention also considers various applications of the CAR of the present invention, including administering genetically modified immune cells expressing nucleic acids encoding the chimeric antigen receptor of the present invention, so that these cells present the CAR on their cell surface. Preferably, the genetically modified immune cells are used to kill target cells.

[0213] In a preferred embodiment, the target cells are cancer cells, and the cell surface molecules of the cancer cells are cancer-associated antigens. The antigen can be a tumor-specific antigen or a tumor-associated antigen. The antigen can be an antigen associated with a specific type of cancer. For example, overexpression of an antigen may be associated with a specific cancer or a specific cancer class. For example, when 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 (e.g., solid tumors) but not with blood (i.e., “liquid”) tumors, and vice versa. The antigen may be associated with a specific lineage of cancer but not with other lineages. For example, the antigen may be associated with sarcoma but not with lymphoma or carcinoma. As used herein, the cancer-related term “associated with” should be understood to mean that the expression of the antigen (whether increased or decreased) is considered a marker of cancer. It should be understood that an antigen may be expressed at low levels, but this does not mean that the antigen is “associated” with a given cancer.

[0214] In a preferred embodiment, the present invention provides a method for killing target cells expressing the nfP2X7 receptor, the method comprising exposing cells expressing the nfP2X7 receptor to the genetically modified cells of the present invention, thereby killing the target cells.

[0215] According to a preferred embodiment of the invention, the antigen recognition domain of the CAR of the present invention is used to directly recognize the nfP2X7 receptor. As used herein, the term "direct recognition" includes the direct binding of the antigen recognition domain of the CAR to the nfP2X7 receptor or its epitope. 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 an antigen-presenting molecule (e.g., the major histocompatibility complex (MHC)).

[0216] It should be understood that, in some cases, the CAR of the present invention can indirectly recognize the nfP2X7 receptor or another antigen on target cells. In such embodiments, the CAR can bind to target cells via an intermediate. Therefore, the present invention also provides a method for killing target cells, the method comprising exposing the target cells to the genetically modified cells of the present invention, thereby killing the target cells. In some embodiments of the present invention, the method for killing target cells further includes the step of exposing the target cells to an intermediate.

[0217] Intermediates can be molecules that directly bind to or interact with the CAR (e.g., via an antigen-binding domain) and also directly bind to or interact with antigens on target cells, such as probes, peptides, or fusion proteins. Non-limiting examples of such intermediates include peptides, antibodies or fragments thereof, antibody Fabs, scFvs, soluble engineered TCRs, or aptamers. Antigens on target cells may contain an nfP2X7 receptor, including an epitope of the nfP2X7 receptor that is different from an epitope recognized by the antigen-recognition domain of the CAR. Antigens on target cells can include antigens that are not the nfP2X7 receptor (e.g., any other tumor-associated or tumor-specific antigens present on the surface of cancer cells, 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 (LI). CAM), CD200 (OX-2), CD221 (IGF1), CD227 (MUC1), CD243 (MRD1), CD246 (ALK), CD271 (LNGFR), CD19, CD20, GD2 and EGFRvIII.

[0218] CARs may be able to directly recognize intermediates, or intermediates may have a tag that can be recognized by the CAR. In either case, the intermediate provides specificity for target cells, while the genetically modified cell with the CAR provides efficacy and directs the immune response against the target cell. Examples of CARs that recognize cells via intermediates are known in the art, for example, European patent application EP2651442. Such intermediates are also referred to in the art as “bridging molecules.” Other examples suitable for guiding the CAR of the present invention, which binds to the nfP2X7 receptor, or CAR-guided bridging molecules (also known as BRiDGE molecules) to target cells are described in WO 2022 / 187906, which is incorporated herein by reference. Such bridging molecules typically comprise a polypeptide or a derivative thereof containing an E200 sequence that can be recognized by the antigen-recognition domain of the CAR of the present invention.

[0219] The term "aptamer" as used throughout the specification refers to any oligonucleotide, polynucleotide, peptide, or polypeptide that specifically binds to or preferentially forms a complex with a target (particularly mesothelin).

[0220] In some embodiments of the invention, the target cells are in a subject. In some embodiments, the subject is a human. In some embodiments, the method further includes exposing the target cells to genetically modified cells along with exogenous cytokines.

[0221] In some embodiments of the present invention, the genetically modified cells are autologous genetically modified cells derived from the target cells of the subject.

[0222] In another aspect, the present invention provides a method for treating or preventing cancer in a subject, the method comprising providing the subject with genetically modified cells of the present invention, thereby treating or preventing cancer. Optionally, the method further comprises the step of exposing target cells to an intermediate to enable the genetically modified cells to bind to the target cells.

[0223] As used herein, the term “treat, treating, or treatment” should be understood to include, within its scope, one or more of the following outcomes: (i) to some extent inhibiting the growth of a primary tumor in a subject, including slowing and complete growth arrest, and including reducing the growth of a primary tumor after resection; (ii) to some extent inhibiting the growth and formation of one or more secondary tumors in a subject; (iii) reducing the number of tumor cells in a subject; (iv) reducing the size of a tumor in a subject; (v) inhibiting (i.e. reducing, slowing, or completely stopping) tumor cell infiltration into peripheral organs; (vi) inhibiting (i.e. reducing, slowing, or completely stopping) metastasis; (vii) improving the life expectancy of a subject compared to an untreated state; (viii) improving the quality of life of a subject compared to an untreated state; (ix) alleviating, reducing, or improving at least one symptom of cancer in a subject; (x) causing cancer regression or remission in a subject; (xi) alleviating the condition caused by cancer in a subject; and (xii) terminating cancer-related symptoms in a subject.

[0224] As used herein, the term “prevent (or preventing)” should be understood within its scope to include inhibiting the formation of primary tumors in subjects, inhibiting the formation of one or more secondary tumors in subjects, or reducing or eliminating the recurrence of cancer in subjects in remission.

[0225] As used herein, the term "inhibition" means a reduction or decrease in the growth of cancer, cancerous cells, or tumors compared to the growth of a control (e.g., untreated cells or a subject). In some embodiments, growth may be reduced or decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% relative to an untreated control.

[0226] Inhibition of cancer, tumor, or cancer cell growth can be assessed using a range of methods known in the art. For example, for cancer cells in vitro, cell growth can be determined by appropriate proliferation assays or by assessing the extent to which tritium-containing thymidine is incorporated into cellular DNA over a given time period. For tumors or cancer cells in vivo, tumor or cell growth can be determined, for example, by appropriate imaging methods known in the art.

[0227] As used herein, the term "subject" refers to any animal capable of developing cancer. Specific subjects of interest are humans, as well as scientifically relevant species such as mice, rats, ferrets, guinea pigs, hamsters, non-human primates, dogs, pigs, and sheep, or economically relevant animals such as horses, dogs, cats, and cattle. In a preferred embodiment of the invention, the subject is a human.

[0228] The reference to "provided to a subject" refers to the administration of genetically modified cells to a subject. Alternatively, the genetically modified cells can be generated within the subject. For example, the genetically modified cells can be generated in vivo, resulting in the subject having an endogenous population of genetically modified cells. Suitable methods for such in vivo generation are known in the art and include gene therapy for the subject.

[0229] Providing genetically modified cells that express a CAR targeting cells expressing or displaying the nfP2X7 receptor may be sufficient to provide an effective immunotherapy against precancerous or cancerous cells. Adjuvants, when used with the genetically modified cells, may induce an immune response that can further enhance the immunotherapy. Cytokines (preferably pro-inflammatory cytokines) are particularly suitable adjuvants to be provided to the subject along with genetically modified cells containing the CAR.

[0230] Therefore, in some embodiments of the invention, genetically modified cells are administered to a subject together with cytokines. It should be understood that, as used throughout the specification, the term "together with" includes genetically modified cells administered simultaneously with or in combination with cytokines. Thus, when administered in combination with cytokines, this can be considered to include combination therapy, whereby the subject's immunotherapy comprises both treatment with cytokines and treatment with genetically modified cells having a CAR targeting cells expressing or displaying the nfP2X7 receptor or its epitopes. In some forms, the cytokines and genetically modified cells are administered on different days (>24 hours). In other forms, the cytokines and genetically modified cells are administered on the same day (within 24 hours). In a further form, the cytokines and genetically modified cells are administered within 18 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 2 minutes, or 1 minute of each other.

[0231] Suitable cytokines for administration with genetically modified cells include IL-2, IL-4, IL-6, IL-7, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, IFNα, IFNβ, IFNγ, GM-CSF, TGFβ, and TNFα. Preferred cytokines include IL-7 and IL-15. Furthermore, cytokines can be administered in recombinant form, natural form, or via delivery systems (e.g., fusion with proteins); as nucleic acid sequences expressed in genetically modified cells; or conjugated with polymers (e.g., polyethylene glycol (PEG)).

[0232] The cells to be genetically modified can be obtained from any suitable source. In some embodiments of the invention, the cells to be genetically modified are autologous cells, which are cells that are homologous to cells expressing or displaying the nfP2X7 receptor or its epitopes. Advantageously, autologous cells are not recognized as “non-autologous” by the subject’s immune system and are therefore tolerated by the subject. However, in some forms of cancer, suitable autologous cells may not be readily available. Therefore, in some embodiments of the invention, the cells to be genetically modified are allogeneic or xenologous cells.

[0233] It will be clearly understood that although this specification relates particularly to application in humans, the invention can also be used for veterinary purposes. Therefore, the invention is applicable in all respects to livestock (e.g., cattle, sheep, horses, and poultry); companion animals (e.g., cats and dogs); and zoo animals. Thus, the general term "subject" or "subject to / being treated" should be understood to include all animals (e.g., humans, apes, dogs, cats, horses, and cattle).

[0234] The term "application" refers to the administration of a therapeutically effective dose of the above-described composition containing the corresponding cells to an individual. "Therapeutically effective dose" refers to the dose that produces its therapeutic effect. The exact dose will depend on the purpose of treatment and will be determined by those skilled in the art using known techniques. As is known in the art and described above, adjustments may be necessary for systemic and local delivery, age, weight, general health condition, sex, diet, timing of administration, drug interactions, and severity of the condition, and will be determined by those skilled in the art through routine experiments.

[0235] Subjects requiring treatment include those already diagnosed with benign, precancerous, or non-metastatic tumors, as well as those seeking to prevent cancer development or recurrence. Subjects may have metastatic cells, including those present in ascites and / or lymph nodes.

[0236] 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 and preferably stop) the infiltration of cancer cells into peripheral organs; inhibit (i.e., slow down and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more of the symptoms associated with the disease to some extent.

[0237] Treatment efficacy can be measured by assessing survival duration, time to disease progression, response rate (RR), duration of response, and / or quality of life.

[0238] This method is particularly useful for prolonging the time it takes for the disease to progress.

[0239] This method is particularly useful for prolonging human survival (including overall survival and progression-free survival).

[0240] This 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.

[0241] This method is particularly useful for providing a partial response to a therapy, in which the body responds to the therapy by reducing the size of one or more tumors or lesions, or by reducing the degree of cancer.

[0242] The goal or outcome of treatment can be any one or more of the following: - 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 and preferably stop) tumor metastasis to some extent; - It can inhibit tumor growth to a certain extent; - To some extent, it can alleviate one or more of the symptoms associated with the condition.

[0243] In one implementation plan, subjects requiring treatment include those with benign, precancerous, non-metastatic tumors.

[0244] In one implementation, the cancer is precancerous or pretumoral.

[0245] In one implementation, the cancer is either secondary or metastatic. Secondary cancer can be located in any organ or tissue, and is particularly common in organs or tissues with relatively high hemodynamic pressure, such as the lungs, liver, kidneys, pancreas, intestines, and brain. Secondary cancer can be detected in ascites and / or lymph nodes.

[0246] In one implementation, cancer may be virtually undetectable.

[0247] "Precancerous" or "precancerous" generally refers to a condition or growth that usually precedes or develops into cancer. Precancerous growth can be characterized by cells with abnormal cell cycle regulation, proliferation, or differentiation, which can be identified by cell cycle markers.

[0248] Cancer can be a solid tumor or a "liquid" tumor. In other words, cancer can grow in tissues (carcinoma, sarcoma, adenoma, etc.), or it can be cancer that exists in bodily fluids (such as blood or bone marrow) (e.g., lymphoma and leukemia). The term "cancer" should be understood to include benign, precancerous, pre-tumoral, non-metastatic, or metastatic tumors. In some implementations, cancer refers to metastatic cancer, such as stage III or IV cancer.

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

[0250] In one implementation, the cancer is either secondary or metastatic. Secondary cancer can be located in any organ or tissue, and is particularly common in organs or tissues with relatively high hemodynamic pressure, such as the lungs, liver, kidneys, pancreas, intestines, and brain. Secondary cancer can be detected in ascites and / or lymph nodes.

[0251] In some implementations, the cancer that needs to be treated may be a cancer characterized by low levels of dysfunctional expression of the P2X7 receptor.

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

[0253] Precancerous, neoplastic, and metastatic cancers are specific examples to which the methods of the present invention can be applied. Broad examples include breast tumors, colorectal tumors, adenocarcinoma, mesothelioma, bladder tumors, prostate tumors, germ cell tumors, liver / cholecystitis, carcinoma, malignant tumors, neuroendocrine tumors, pituitary tumors, small round cell tumors, squamous cell carcinoma, melanoma, atypical fibrous xanthoma, seminoma, non-seminomatous tumor, stromal-stromal tumor, Sertoli cell tumors, skin tumors, kidney tumors, testicular tumors, brain tumors, ovarian tumors, gastric tumors, pancreatic tumors, oral tumors, bladder tumors, bone tumors, cervical tumors, esophageal tumors, laryngeal tumors, liver tumors, lung tumors, vaginal tumors, and Wilms' tumors. Preferred examples include pancreatic tumors, ovarian tumors, gastric tumors, lung tumors, liver tumors, colorectal tumors, cervical tumors, endometrial tumors, kidney tumors, breast tumors, and testicular tumors.

[0254] Specific examples of cancers include, but are not limited to, adenocarcinoma, adenoma, adenofibroma, adenolymphoma, odontoma, AIDS-related cancers, acoustic neuroma, acute lymphoblastic leukemia, acute myeloid leukemia, adenocystic carcinoma, adrenocortical carcinoma, idiopathic myelomectomy, alopecia, alveolar soft tissue sarcoma, ameloblastoma, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, sclerosing hemangioma, hemangiomatosis, amine precursor uptake and decarboxylation cell tumors, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, and ataxia-telangiectasia. Basal cell carcinoma (skin), bladder cancer, bone cancer, colorectal cancer, brainstem glioma, brain and CNS tumors, breast cancer (preferably triple-negative breast cancer), branchial protozoa, CNS tumors, carcinoid tumors, cervical cancer, childhood brain tumors, childhood cancers, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, cutaneous T-cell lymphoma, malignant tumors (e.g., Walker's carcinoma, basal cell carcinoma, squamous basal cell carcinoma, Brown-Pierce carcinoma, ductal carcinoma, Ehrlich tumor, Krebs tumor). 2. Merkel cell carcinoma, mucinous carcinoma, non-small cell lung cancer, oat cell carcinoma, papillary carcinoma, scleroderma, bronchiolar carcinoma, bronchial carcinoma, squamous cell carcinoma and transitional cell carcinoma), carcinosarcoma, cervical abnormalities, phyllodes tumor of the breast, cementum tumor, chordoma, mammillary tumor, chondrosarcoma, chondroblastoma, craniopharyngioma, chondroma, bile duct carcinoma, cholesteatoma, cylindrica, cystadenocarcinoma, cystadenoma, dermatofibrosarcoma protuberans, fibroblastic small round cell tumor, ductal carcinoma. Cancer, dysgerminoma, endocrine system cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing sarcoma, extrahepatic bile duct cancer, eye cancer, melanoma, retinoblastoma, fallopian tube cancer, Fanconi anemia, fibroma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumors, urogenital cancer, germ cell tumors, gestational trophoblastic disease, glioma, gynecological cancer, giant cell tumor, ganglioma, glioma, glomus tumor, granuloma Ovarian amphipathic germ cell tumor, hematologic malignancies, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, vesicular birth defects, hypercalcemia, hypopharyngeal cancer, hamartoma, hemangioendothelioma, hemangioma, hemangiopericytoma, angiosarcoma, angiosarcoma, histiocytic disorders, malignant histiocytosis, histiocytoma, hepatocellular carcinoma, hidradenoma, immunoproliferative microcystin, OPoma, eye Intracellular melanoma, pancreatic islet cell carcinoma, Kaposi's sarcoma, renal cell carcinoma, Langerhans cell histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Liposarcoma, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leiomyosarcoma, leukemia (e.g., B-cell leukemia, mixed-cell leukemia, naked-cell leukemia, T-cell leukemia, T-cell chronic leukemia, HTLV-II-related leukemia),Lymphangiosarcoma (leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, mast cell leukemia and granulocytic leukemia), leukemic sarcoma, Ledich's cell tumor, liposarcoma, leiomyoma, leiomyosarcoma, lymphangioma, lymphangiocytoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma, male breast cancer, malignant renal rhabdomyosarcoma, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, oral cancer, multiple endocrine tumors, mycosis fungoides, myelodysplastic syndrome, myeloma, myeloproliferative disorders, malignant carcinoid syndromes Cancer, heart disease, medulloblastoma, meningioma, melanoma, stromal tumor, mesonephroma, mesothelioma, myoblastoma, fibroma, sarcoma, myxoma, myxosarcoma, nasal carcinoma, nasopharyngeal carcinoma, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen's rupture syndrome, non-melanoma skin cancer, non-small cell lung cancer (NSCLC), schwannoma, neuroblastoma, neuroepithelial tumor, neurofibromatosis, neurofibroma, neuroma, tumors (e.g., bone tumors, breast tumors, digestive system tumors, colorectal tumors, liver tumors), eye cancer, esophageal cancer, oral cancer Nasopharyngeal carcinoma, osteosarcoma, colostomy, ovarian cancer, pancreatic cancer, nasal cancer, parathyroid carcinoma, parotid gland cancer, penile cancer, peripheral neuroectodermal tumors, pituitary cancer, polycythemia vera, prostate cancer, osteoma, osteosarcoma, ovarian cancer, papilloma, paraganglioma, non-pheochromocytic paraganglioma, pineal tumor, plasmacytoma, proto-oncogenes, rare cancers and related disorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, reticuloendothelial proliferation, rhabdomyosarcoma, salivary gland carcinoma, sarcoma, schwannoma, Cezari syndrome, skin cancer. Small cell lung cancer (SCLC), small intestinal cancer, soft tissue sarcoma, spinal cord tumors, squamous cell carcinoma (skin), gastric cancer, synovial sarcoma, sarcoma (e.g., Ewing's experimental sarcoma, Kaposi's sarcoma, and mast cell sarcoma), Sertoli cell tumor, synovial tumor, testicular cancer, thymic carcinoma, thyroid cancer, transitional cell carcinoma (bladder), transitional cell carcinoma (renal pelvis / ureter), trophoblastic carcinoma, teratoma, follicular cell tumor, thymoma, trophoblastic tumor, urethral cancer, urinary system cancer, urothelial carcinoma, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor. Preferred specific examples include pleural or peritoneal mesothelioma, gastric cancer, endometrial cancer, colorectal cancer, non-small cell lung adenocarcinoma, bile duct cancer, ovarian cancer, esophageal cancer, pancreatic ductal adenocarcinoma, triple-negative breast cancer, and any other mesothelin-positive cancer.

[0255] In some embodiments of the present invention, the method is used for the prevention or treatment of one or more cancers selected from the following: brain cancer, esophageal cancer, oral cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cervical cancer, epithelial cell carcinoma, 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 cancer, epithelial cell carcinoma, skin cancer, blood-related cancers, breast cancer, endometrial cancer, uterine cancer, and testicular cancer.

[0256] The presence, improvement, treatment, or minimization of cancer progression can be determined by any clinical or biochemical method as described herein or known in the art. A positive response to treatment or minimization of cancer progression can be determined by any method known in the art and may include determining: - A reduction in the number of cancer cells; - Reduction in tumor size; - Inhibit (i.e., slow down to some extent and preferably stop) the infiltration of cancer cells into peripheral organs; - Inhibit (i.e., slow down and preferably stop) tumor metastasis to some extent; - Reduce or completely prevent tumor metastasis after removal of the primary tumor; - It can inhibit tumor growth to a certain extent; - To some extent alleviate one or more of the symptoms associated with cancer; and / or -Increase the survival rate of test subjects.

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

[0258] Subjects who have received cancer treatment may be in partial or complete remission. In other words, subjects who have received cancer treatment as described above may have measurable parameters of tumor growth reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater, as detectable by physical examination, radiological studies, or by biomarker levels from blood or urine tests. Alternatively, in the case of complete remission, all detectable disease manifestations completely disappear, leaving the subject with no detectable signs of cancer. Subjects may have substantially undetectable signs of cancer. “Substantially undetectable” cancer generally refers to a situation where the therapy has reduced the size, volume, or other physical measure of the cancer to such an extent that, as a result of the therapy, the cancer cannot be clearly detected using relevant standard detection techniques (e.g., in vivo imaging).

[0259] 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 and preferably stop) the infiltration of cancer cells into peripheral organs; inhibit (i.e., slow down and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more of the symptoms associated with the disease to some extent.

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

[0261] In one implementation, this method is particularly useful for delaying cancer progression. In another implementation, this method is particularly useful for prolonging subject survival (including overall survival and progression-free survival). It should be understood that overall survival is the length of time a patient diagnosed with cancer remains alive from the date of cancer diagnosis or the start of treatment. It should be understood that progression-free survival refers to the length of time a patient has the disease but whose condition has not worsened during and after cancer treatment.

[0262] Survival analysis can be performed using techniques well-known in the art, including the Kaplan-Meier method. The Kaplan-Meier method estimates a survival function based on lifespan data. In medical research, it can be used to measure the proportion of patients who survive a certain period after treatment. A survival function plotted using the Kaplan-Meier method appears as a series of horizontal steps with decreasing amplitude; when the sample size is sufficiently large, this plot will approximate the true survival function of the population. It is assumed that the value of the survival function is constant between consecutive different sampling observations (“clicks”).

[0263] A key advantage of the Kaplan-Meier curve is that it allows for the consideration of data loss due to “censoring” (e.g., if a patient drops out of the study) before the final results are observed. On the graph, small vertical scale marks indicate data loss, specifically cases where patient data has been censored. When no truncation or censoring occurs, the Kaplan-Meier curve corresponds to the empirical distribution.

[0264] In one implementation, 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. In one implementation, the method is particularly useful for providing a partial response to therapy, where, in response to the therapy, the size of one or more tumors or lesions decreases, or the degree of cancer decreases.

[0265] Composition and application The delivery or administration of genetically modified cells according to the present invention can be either individual cell delivery or administration, or cell delivery or administration formulated into a suitable pharmaceutical composition. Therefore, the present invention provides a pharmaceutical composition comprising the genetically modified cells of the present invention and a pharmaceutically acceptable carrier.

[0266] Methods for providing CAR-containing cells for immunotherapy are known in the art (see, 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). Furthermore, protocols and methods for preparing, amplifying, and evaluating mammalian cells expressing CARs are known in the art (see, for example, Cheadle, EJ). et al (Antibody Engineering: Methods and Protocols, Second Edition, Methods in Molecular Biology, vol. 907: 645-66), and summarized in the following examples.

[0267] Considering the specific physical and chemical properties of the cells to be administered, the pharmaceutical composition may also contain one or more pharmaceutically acceptable additives, including pharmaceutically acceptable salts, amino acids, peptides, polymers, solvents, buffers, excipients, and fillers. In some embodiments, the pharmaceutical composition comprises a suspension of the genetically modified cells of the present invention in a suitable medium (e.g., an isotonic saline solution). In some embodiments, the pharmaceutical composition may contain suitable adjuvants, such as one or more cytokines as described above. In some embodiments, the pharmaceutical composition may also contain intermediates as described above.

[0268] The drug composition can also be administered via parenteral routes, including intravenous, intraventricular, intraperitoneal, intramuscular, intrapleural, or intracranial injection, or local injection to the site of a tumor or cancerous mass.

[0269] It should be understood that the invention disclosed and defined in this specification includes all alternative combinations of two or more individual features mentioned in or clearly apparent from the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0270] Example Example 1 : Identification of surrogate sdAb sequences for inclusion in CARs Single-domain CARs for binding nfP2X7 are known, including CARs containing an sdAb named "BIL03". The inventors sought to identify alternative CAR-based sdAbs for targeting cancer cells. Many different anti-nfP2X7 sdAb sequences were tested in the context of CAR.

[0271] Three suitable sdAb sequences (4A7, 3aB9, and 3aD9, as defined in Table 3 herein) were identified as providing efficacy in the context of CAR, including in the context of CAR T cell / BRiDGE systems (as disclosed in WO 2022 / 187906), preferably wherein the CAR has a hinge region of CD8A, a transmembrane domain of CD28, and costimulatory / signal transduction domains of CD28, 41BB (CD137), and CD3ζ.

[0272] Interestingly, the inventors discovered that when sdAb was paired with different light chain sequences, none of these sdAb sequences provided a suitable CAR construct.

[0273] Example 2: BIL03 heavy chain light chain pairing identification Although the single-domain antigen-binding domain contained in anti-nfP2X7 receptor CARs is known, it is unclear whether its binding affinity can be improved by adding a pairing variable light chain. As shown in Example 1, in some cases, in the case of CARs, pairing of the light chain with the heavy chain does not result in improved CAR function. Furthermore, suitable light chains that can pair with known heavy chains are not readily apparent from the prior art.

[0274] The inventors sought to identify suitable light chain pairings for a single-domain heavy chain protein BIL03 (as defined in Table 1 herein), which is disclosed in the prior art in the context of sdAb-based CAR.

[0275] More than 10 different light chain pairings were tested, including the variable light chain defined as GB1 in Table 1 of this paper.

[0276] The inventors discovered that specific light chains paired with WT B1 (defined in Table 1) provide stable expression and reduce aggregation. Surprisingly, this light-heavy chain pairing also significantly enhances binding affinity to the target antigen, as shown in the table below.

[0277] Table 2: Binding affinity of BIL03 heavy chains compared to their affinity when pairing with various light chain variable domains.

[0278] The binding affinity of two different antigens derived from the dysfunctional P2X7 receptor was assessed: E200 peptide (GHNYTTRNILPGLNITC) (SEQ ID NO: 143) and Ext peptide 17 (GHNYTTRNILPGLNITSTFHKTSGSGK) (SEQ ID NO: 102).

[0279] Example 3: scFv CAR design and production CAR construction: Two CARs were designed. The first one (CAR10A) was designed to contain the hinge region of the single-domain antibody BIL03 (2-2-1) (sequence provided in Table 1) and CD8A, the transmembrane domain of CD28, and the co-stimulatory / signal transduction domains of CD28, 41BB (CD137) and CD3ζ.

[0280] The second (CAR 12AV1) was designed to contain the scFv light-heavy chain pair identified in Example 2: WTB1-BIL03 (sequences provided in Table 1) and the hinge region of CD8A, the transmembrane domain of CD28, and the co-stimulatory / signal transduction domains of CD28, 41BB, and CD3ζ.

[0281] Both CARs contain C-terminal tEGFR to facilitate CAR detection.

[0282] The nucleic acid encoding CAR was designed, synthesized, and cloned into a transfer vector using computer technology, and then packaged into self-inactivated (SIN) lentiviral particles (3rd generation LV system) in HEK293T cells. The supernatant was concentrated by ultracentrifugation according to standard protocol.

[0283] Production of CAR T cells CAR T cells were generated by transducing CD4 / CD8-positive selected T cells (1:1 ratio) with lentivirus. These T cells were then magnetically activated cell sorting (MACS) and stimulated with TransAct (all according to the manufacturer's instructions), followed by culture in TexMACS medium supplemented with IL7 / IL15 (10 ng / mL). The donor source was erythrocyte sedimentation rate (ESR) brown-yellow layer.

[0284] Activated untransduced T cells (aUTs) do not express any receptors that can bind to EGFR.

[0285] In luciferase-based killing assays, reporter cell lines are incubated with effector cells, with or without BRiDGE molecules (e.g., as disclosed in WO 2022 / 187906). Relevant controls are used to calculate viability and / or specific lysis (e.g., only 100%, 75%, 50%, 25%, 10%, and 0% of target cells) to calculate a standard curve used to ultimately determine target cell lysis.

[0286] In flow-based killing assays, reporter cell lines are incubated together with effector cells at a specified concentration of BRiDGE molecules (e.g., as disclosed in WO 2022 / 187906). Relevant controls are used to calculate viability and / or specific lysis.

[0287] The results showed that cells transduced with CAR12AV1 were able to kill cells more directly than cells transduced with CAR10A, although both CARs enabled cells to kill indirectly when combined with BRiDGE molecules.

[0288] Figure 1 This study presents a direct comparison of the cytotoxic capacity of T cells expressing CAR10A compared to those expressing CAR12AV1. The target cells used were the AML cell line MOLM-13. The ET ratio was 5:1, and the CAR to target ratio was 2.1:1. Cells were deprived of cytokines for 3 days prior to exposure to CAR T cells.

[0289] The results also showed that CAR12AV1-T cells exhibited significantly higher direct cell killing (as indicated by the reduction in MOL-13 cell counts) compared to CAR10A-T cells. Co-incubation with 200 ng / mL of a Fab-based anti-CD33 BRiDGE molecule (as disclosed in WO 2022 / 187906) enabled T cells expressing either CAR to kill MOLM-13 cells with greater efficacy.

[0290] Example 4: Cell killing by T cells expressing CAR molecules of the application CAR T cells expressing CAR10A and CAR12A were generated according to the method described in Example 3 above. Furthermore, T cells expressing single-domain CAR 3a-B9-CAR and 4A7-CAR were generated.

[0291] Assess the ability of CAR T cells to directly kill JeKo-1 and MOLM-13 cells, or to kill JeKo-1 and MOLM-13 cells in the presence of anti-CD19 or anti-CD33BRiDGE molecules (as disclosed in WO 2022 / 187906).

[0292] Figure 2The study demonstrated that T cells expressing any of CAR10A, CAR12A, 3a-B9-CAR, and 4A7-CAR induced cell killing in the absence of BRiDGE molecules, indicating that these CAR T cells can directly kill cells. Co-incubation with different concentrations of anti-CD19 BRiDGE molecules enabled T cells to kill JeKo-1 cells with greater potency. The results also showed that the cell-killing efficacy increased in a dose-dependent manner in response to increased BRiDGE molecule concentration.

[0293] Figure 3 This study demonstrated that T cells expressing 3a-B9-CAR and 4A7-CAR were also able to directly and indirectly kill MOLM-13 cells when co-incubated with anti-CD33 BRiDGE molecules. Similar results were achieved using CAR-positive cell to cancer cell ratios of 5:1 and 2.5:1 (data not shown).

[0294] Figures 4(A) and 4(B) further demonstrate that T cells expressing 3a-B9-CAR and 4A7-CAR were able to directly and indirectly kill JeKo-1 cells when co-incubated with anti-CD19 BRiDGE molecules. The results also show that this effect is not induced by co-incubation of cell-specific BRiDGE molecules with anti-CD33 BRiDGE. JeKo-1 cells do not express CD33 but do express CD19; therefore, inducing greater cell killing with anti-CD19 BRiDGE molecules shows that this effect is controlled by specific CAR / BRiDGE interactions. Similar results were achieved with CAR-positive cell to cancer cell ratios of 5:1 and 2.5:1 (Figure 4(B)).

[0295] It should be understood that the invention disclosed and defined in this specification includes all alternative combinations of two or more individual features mentioned in or clearly apparent from the text or drawings. All these different combinations constitute various alternative aspects of the invention.

Claims

1. A chimeric antigen receptor (CAR) comprising: i) An antigen recognition domain that recognizes a dysfunctional P2X7 receptor or an epitope derived therefrom; ii) Transmembrane domains; and iii) Intracellular domains, The antigen recognition domain comprises a complementarity-determining region (CDR) from VH and a CDR from VL, wherein VH comprises a sequence as shown in SEQ ID NO: 4 and VL comprises a sequence as shown in SEQ ID NO:

12.

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

12.

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

4. The CAR according to any one of claims 1 to 3, wherein the antigen recognition domain comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 4 or a functional variant thereof, the functional variant comprising a sequence that is 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 the amino acid sequence; the light chain variable domain comprises as shown in SEQ ID NO:

4. The amino acid sequence shown in 12 or a functional variant thereof, or composed thereof, wherein the functional variant comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence. Furthermore, the antigen recognition domain retains the ability to bind to the nfP2X7 receptor.

5. The CAR according to any one of claims 1 to 3, wherein the antigen recognition domain comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 4, and the light chain variable domain comprising or consisting of the amino acid sequence shown in SEQ ID NO:

12. Compared with the amino acid sequence shown in SEQ ID NO: 4 or 12, the heavy chain variable domain and / or the light chain variable domain respectively contain 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 residues that have been substituted, deleted or added, and wherein the antigen recognition domain retains the ability to bind to the nfP2X7 receptor.

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

7. The CAR according to any one of claims 1 to 6, wherein the antigen recognition domain comprises, is substantially composed of, or is composed of the amino acid sequences of SEQ ID NO:4 and 12 (in the order of N-terminus to C-terminus or C-terminus to N-terminus).

8. The CAR according to any one of claims 1 to 7, wherein the antigen recognition domain comprises, is substantially composed of, or is composed of SEQ ID NO: 12 and SEQ ID NO: 4 (i.e., VL to VH) in the order from N-terminus to C-terminus.

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

10. The CAR according to any one of claims 1 to 9, wherein the antigen recognition domain is a single-stranded variable fragment (scFv).

11. A chimeric antigen receptor (CAR) comprising: i) An antigen recognition domain that recognizes a dysfunctional P2X7 receptor or an epitope derived therefrom; ii) Transmembrane domains; and iii) Intracellular domains, The antigen recognition domain includes the complementarity-determining region (CDR) from the VH, wherein the VH includes a sequence as shown in SEQ ID NO: 135, 136 or 137.

12. The CAR of claim 11, wherein the antigen recognition domain comprises: (i) VH, wherein VH comprises complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR 1 comprises a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 124; wherein CDR 2 comprises a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 124; and wherein CDR 3 ... The sequences shown in NO:128, 129 or 130 are 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 sequences; (ii) VH, wherein the VH comprises at least about 95% or 96% or 97% or 98% or 99% identical to the sequence shown in SEQ ID NO: 135, 136 or 137; (iii) VH, wherein VH comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO: 124, CDR2 comprises the sequence shown in SEQ ID NO: 125, 126, or 127, and CDR3 comprises the sequence shown in SEQ ID NO: 128, 129, or 130; or (iv) VH, wherein the VH comprises the sequence shown in SEQ ID NO: 135, 136 or 137.

13. The CAR according to claim 11 or 12, wherein the antigen recognition domain further comprises at least one of the following: (i) VH, which comprises frame regions (FR) 1, FR2, FR3, and FR4, wherein FR1 comprises 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% of the sequence shown in SEQ ID NO: 131; FR2 comprises 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% of the sequence shown in SEQ ID NO: 132; FR3 comprises 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% of the sequence shown in SEQ ID NO: 133; and FR4 .... The sequence shown in 134 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. (ii) VH, wherein VH comprises FR1, FR2, FR3 and FR4, wherein FR1 comprises the sequence shown in SEQ ID NO: 131, FR2 comprises the sequence shown in SEQ ID NO: 132, FR3 comprises the sequence shown in SEQ ID NO: 133 and FR4 comprises the sequence shown in SEQ ID NO:

134.

14. The CAR according to any one of claims 11 to 13, wherein the antigen recognition domain comprises, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 135, 136, or 137.

15. The CAR according to any one of claims 11 to 13, wherein the antigen-recognizing domain comprises a heavy chain variable domain, the heavy chain variable domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 135 or a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or constitutes thereof; wherein the heavy chain does not contain any sequence changes in the CDR compared to the sequence of SEQ ID NO: 135, and / or wherein the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

16. The CAR according to any one of claims 11 to 13, wherein the antigen-recognizing domain comprises a heavy chain variable domain, the heavy chain variable domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 135, wherein, compared to the amino acid sequence shown in SEQ ID NO: 135, the heavy chain variable domain of the antigen-binding domain comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residues substituted, deleted, or added; preferably, wherein the amino acid substitution, deletion, or addition is not in the CDR, and / or wherein the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

17. The CAR according to any one of claims 11 to 13, wherein the antigen-recognizing domain comprises a heavy chain variable domain, the heavy chain variable domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 136 or a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or constitutes thereof; wherein the heavy chain does not contain any sequence changes in the CDR 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. The CAR according to any one of claims 11 to 13, wherein the antigen-recognizing domain comprises a heavy chain variable domain, the heavy chain variable domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 136, wherein, compared to the amino acid sequence shown in SEQ ID NO: 136, the heavy chain variable domain of the antigen-binding domain comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residues substituted, deleted, or added; preferably, wherein the amino acid substitution, deletion, or addition is not in the CDR, and / or wherein the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

19. The CAR according to any one of claims 11 to 13, wherein the antigen recognition domain comprises a heavy chain variable domain, the heavy chain variable domain comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 137 or a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or constitutes thereof; wherein, Compared to the sequence of SEQ ID NO: 137, the heavy chain does not contain any sequence changes in the CDR, and / or wherein, The antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

20. The CAR according to any one of claims 11 to 13, wherein the antigen-recognizing domain comprises a heavy chain variable domain, the heavy chain variable domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 137, wherein, compared to the amino acid sequence shown in SEQ ID NO: 137, the heavy chain variable domain of the antigen-binding domain comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residues substituted, deleted, or added; preferably, wherein the amino acid substitution, deletion, or addition is not in the CDR, and / or wherein the antigen-binding domain retains the ability to bind to the nfP2X7 receptor.

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

22. The CAR of claim 21, wherein the hinge region is derived from the CD8a, CD28, or IgG4 hinge region.

23. The CAR of claim 22, wherein the hinge region comprises or is composed of the amino acid sequence shown in SEQ ID NO:

88.

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

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

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

27. The CAR of claim 26, wherein the transmembrane domain comprises or is composed of the amino acid sequence of SEQ ID NO:

89.

28. The CAR according to any one of claims 1 to 27, wherein the signal transduction domain of the CAR comprises a portion derived from an activating receptor.

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

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

31. The CAR of claim 30, wherein the signal transduction domain comprises the amino acid sequence shown in SEQ ID NO:

92.

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

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

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

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

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

37. The CAR of claim 36, wherein the signal transduction domain comprises an amino acid sequence containing the sequence shown in SEQ ID NO: 90, 91 and / or 92 and combinations thereof.

38. The CAR according to any one of claims 1 to 37, wherein the CAR comprises (from the N-terminus to the C-terminus) the antigen recognition domain, CD8a hinge, CD28 transmembrane domain, CD28 signal transduction domain, 4-1BB signal transduction domain and CD3ζ (zeta) signal transduction domain as defined in any one of claims 1 to 20.

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

12.

40. The CAR of claim 38, wherein the CAR comprises the amino acid sequence shown in SEQ ID NO: 138 or a functional variant thereof, the functional variant comprising a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 135; wherein the functional variant comprises an antigen recognition domain as defined herein, preferably comprising a VH, the VH comprising the amino acid sequence shown in SEQ ID NO:

135.

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

136.

42. The CAR of claim 38, wherein the CAR comprises the amino acid sequence shown in SEQ ID NO: 140 or a functional variant thereof, the functional variant comprising a sequence that is 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 the amino acid sequence shown in SEQ ID NO: 137; wherein the functional variant comprises an antigen recognition domain as defined herein, preferably comprising a VH, the VH comprising the amino acid sequence shown in SEQ ID NO:

137.

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

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

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

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

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

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

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

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

51. A method for producing genetically modified cells, the method comprising transducing the cells, preferably immune cells, with the nucleic acid construct of claim 44, such that the transduced cells express the CAR, thereby producing genetically modified cells.

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

53. A method for killing cells expressing the nfP2X7 receptor, the method comprising exposing the cells expressing the nfP2X7 receptor to genetically modified cells according to any one of claims 45 to 50, thereby killing the cells expressing the nfP2X7 receptor.

54. The method of claim 53, wherein the cell expressing the nfP2X7 receptor is a cancer cell.

55. The method according to claim 54, wherein the cancer is selected from the group consisting of brain cancer, esophageal cancer, oral cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cervical cancer, epithelial cell carcinoma, skin cancer, leukemia, lymphoma, myeloma, breast cancer, ovarian cancer, endometrial cancer, thymic cancer, and testicular cancer.

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

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

58. A method, the method being: To treat, prevent, or minimize cancer progression in subjects. To minimize, reduce, or prevent tumor growth in the subject. Minimize, reduce, or prevent subject transfer, or Increase the survival of test subjects. The method comprises administering to the subject any of the CARs of claims 1 to 42, the nucleic acid of claim 43, the nucleic acid construct of claim 44, the genetically modified cells of any of claims 45 to 50, or the pharmaceutical composition of claim 57, thereby: To treat, prevent, or minimize cancer progression in subjects. To minimize, reduce, or prevent tumor growth in the subject. Minimize, reduce, or prevent subject transfer, or Increase the survival rate of test subjects.

59. Use in the preparation of a medicament of any one of claims 1 to 42, the nucleic acid of claim 43, the nucleic acid construct of claim 44, the genetically modified cell of any one of claims 45 to 50, or the pharmaceutical composition of claim 57, wherein the medicament is used for: To treat, prevent, or minimize cancer progression in subjects. To minimize, reduce, or prevent tumor growth in the subject. Minimize, reduce, or prevent subject transfer, or Increase the survival rate of test subjects.

60. The CAR of any one of claims 1 to 42, the nucleic acid of claim 43, the nucleic acid construct of claim 44, the genetically modified cell of any one of claims 45 to 50, or the pharmaceutical composition of claim 57, wherein it is used for: To treat, prevent, or minimize cancer progression in subjects. To minimize, reduce, or prevent tumor growth in the subject. Minimize, reduce, or prevent subject transfer, or Increase the survival rate of test subjects.

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