Chimeric antigen receptors and uses thereof

Abstract

To provide a chimeric antigen receptor (CAR) targeting a tumour-associated antigen which is selectively expressed by cancerous cells but not endogenously expressed on non-cancerous cells.SOLUTION: A chimeric antigen receptor includes domains including an antigen-recognition domain which recognises an epitope, wherein the epitope binds to the dysfunctional P2X7 receptor, a transmembrane domain, and a signalling domain including an intracellular signalling site of an activation receptor and / or an intracellular signalling site of a costimulatory receptor.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Priority claims

[0001] This application claims priority to Australian Provisional Patent Application No. 2015903719 filed on 11 September 2015, the contents of which are incorporated herein by reference.

[0002]

[0002] The present invention relates to chimeric antigen receptors, T cells expressing chimeric antigen receptors, and methods of using chimeric antigen receptors in the prevention and / or treatment of cancer. [Background technology]

[0003]

[0003] The immune system possesses highly evolved and specific mechanisms for protecting against a variety of pathologies. Among these are the detection and elimination of unwanted pathogens, such as bacterial infections, virus-infected cells, and, importantly, mutated cells that can give rise to malignant neoplasms (cancers). The immune system's ability to prevent cancer formation and growth depends on the ability of cells of the immune system to distinguish between "healthy" and "disease-state" (e.g., neoplastic or pre-neoplastic) cells. This is achieved through the recognition of cellular markers (antigens) that mark the transition of cells from a healthy to a disease-state.

[0004]

[0004] Numerous attempts have been made to develop immunotherapeutic approaches to treat cancer by manipulating or directing the immune system to target cells that express cancer cell antigens. Immunotherapeutic approaches have primarily focused on harnessing the humoral immune system using isolated or engineered antibodies, or more recently, on the cellular arm of the immune system.

[0005]

[0005] Early attempts to utilize cellular immunotherapy for the treatment of cancer involved the use of T lymphocytes isolated from tumors and expanded ex vivo. While this approach initially showed some promise in early investigations, there are numerous technical problems associated with it. The ability to isolate and expand T cell populations to clinically relevant numbers is technically challenging, and poor control over the nature of expansion can result in final T cell populations that are significantly heterogeneous and contain only a small number of cancer antigen-specific T cells. As a result, the efficacy of this method is unpredictable and variable.

[0006]

[0006] To address some of the shortcomings associated with the use of ex vivo expanded tumor-isolated T cells, chimeric antigen receptors (CARs or artificial T cell receptors) began to be developed in the late 1980s. Chimeric antigen receptors are created by linking an extracellular domain specific for a desired antigen to a signaling domain, resulting in an antigen-specific receptor capable of inducing T cell function.

[0007]

[0007] By transfecting isolated T cells with CARs, a population of T cells specific to a given antigen can be obtained, resulting in the generation of a large population of antigen-specific T cells that can be used for immunotherapy.

[0008]

[0008] Early clinical trials of CAR-transfected T cells specific for tumor-associated antigens have been promising. However, the efficacy of CAR-transfected T cells has led to significant hypercytokinemia and ultimately death in some patients. These side effects are thought to be primarily induced by on-target, off-tumor activity of CAR-transfected T cells induced as a result of endogenous expression of the CAR's cognate antigen in healthy, non-cancerous cell populations.

[0009]

[0009] Therefore, it is clear that there is a need to develop CARs that target tumor-associated antigens that are selectively expressed by cancerous cells but are not endogenously expressed in non-cancerous cells.

[0010] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. No suggestion or representation is made that any or all of these matters existed prior to the priority date of each claim of this application and therefore formed part of the prior art or were common general knowledge in the art relevant to the present invention. Summary of the Invention

[0011]

[0011] The present invention is based in part on the recognition that due to the significant "on target" but "off tumor" activity of CAR-expressing immune cells, there is a need to develop CARs and genetically modified cells expressing them that target markers specifically associated with various neoplastic (cancerous) or preneoplastic (precancerous) cells. The inventors have recognized that dysfunctional P2X7 receptors are suitable markers to target with CARs.

[0012]

[0012] Therefore, in a first aspect, the present invention provides a chimeric antigen receptor comprising an antigen recognition domain and a signal transduction domain, wherein the antigen recognition domain recognizes a dysfunctional P2X7 receptor.

[0013] In some embodiments, the antigen recognition domain recognizes an epitope associated with the adenosine triphosphate (ATP) binding site of a dysfunctional P2X7 receptor. In some embodiments, the dysfunctional P2X7 receptor has a reduced ability to bind ATP at the ATP binding site compared to the ATP binding ability of a wild-type (functional) P2X7 receptor. In some embodiments, the dysfunctional P2X7 receptor cannot bind ATP at the ATP binding site.

[0014] In some embodiments, the dysfunctional P2X7 receptor has a conformational change that causes the receptor to become dysfunctional. In some embodiments, the conformational change is a change from a trans to a cis amino acid. In some embodiments, the amino acid that has changed from a trans to a cis amino acid is a proline at amino acid position 210 of the dysfunctional P2X7 receptor.

[0015] In some embodiments, the antigen recognition domain recognizes an epitope comprising a proline at amino acid position 210 of a dysfunctional P2X7 receptor. In some embodiments, the antigen recognition domain recognizes an epitope comprising one or more amino acid residues ranging from a glycine at amino acid position 200 to a cysteine ​​at amino acid position 216 (inclusive) of a dysfunctional P2X7 receptor.

[0016] The antigen recognition domain of CAR can be any suitable molecule that can interact with and specifically recognize dysfunctional P2X7 receptor.However, in some embodiments, the antigen recognition domain comprises an amino acid sequence homologous to the amino acid sequence of an antibody or a fragment thereof that binds to dysfunctional P2X7 receptor.In some embodiments, the antigen recognition domain comprises an amino acid sequence homologous to the amino acid sequence of an antigen-binding fragment (Fab) of an antibody that binds to dysfunctional P2X7 receptor.In some embodiments, the antibody is a humanized antibody.

[0017] In some embodiments, the antigen recognition domain comprises an amino acid sequence homologous to the amino acid sequence of a single-chain variable fragment (scFv) or multivalent scFv that binds to a dysfunctional P2X7 receptor. In some embodiments, the multivalent scFv is a bivalent scFv or a trivalent scFv.

[0018]

[0018] In some embodiments, the antigen recognition domain comprises amino acid sequence homology to a single antibody domain (sdAb) that binds to a dysfunctional P2X7 receptor.

[0019] In some embodiments, the antigen recognition domain comprises a binding peptide comprising amino acid sequence homology to one or more CDR regions of an antibody that binds to a dysfunctional P2X7 receptor. In some embodiments, the binding peptide comprises a V region of an antibody that binds to a dysfunctional P2X7 receptor. H Chain and / or V L In some embodiments, the antigen recognition domain comprises one or more amino acid sequences that are at least 50%, 60%, 70%, 80%, 90%, or 94% identical to any one of the regions spanning positions 30 to 35, 50 to 67, or 98 to 108 of the sequence set forth in SEQ ID NO: 10, 32, 33, or 34. In some embodiments, the antigen recognition domain comprises one or more of the sequences spanning positions 30 to 35, 50 to 67, or 98 to 108 of the sequence set forth in SEQ ID NO: 10, 32, 33, or 34. In some embodiments, the antigen recognition domain comprises one or more of the sequences set forth in SEQ ID NO: 10, 32, 33, or 34.

[0019]

[0020] In some embodiments, the signaling domain comprises a portion derived from an activating receptor. In some embodiments, the activating receptor is a member of the CD3 co-receptor complex or an Fc receptor. In some embodiments, the portion derived from the CD3 co-receptor complex is CD3-ζ. In some embodiments, the portion derived from an Fc receptor is FcεRI or FcγRI.

[0020]

[0021] In some embodiments, the signaling domain comprises a portion derived from a costimulatory receptor. In some embodiments, the signaling domain comprises a portion derived from an activating receptor and a portion derived from a costimulatory receptor. In some embodiments, the costimulatory receptor is selected from the group consisting of CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS.

[0021]

[0022] In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor according to the first aspect of the invention.

[0023] In a third aspect, the present invention provides a nucleic acid construct comprising a nucleic acid molecule according to the second aspect of the present invention. In some embodiments, the expression of the nucleic acid molecule is under the control of a transcription control sequence. In some embodiments, the transcription control sequence may be a constitutive promoter or an inducible promoter.

[0022]

[0024] In some embodiments of the third aspect of the invention, the nucleic acid construct further comprises an internal ribosome entry site (IRES) that allows for transcription initiation within the mRNA when expressed from the nucleic acid construct.

[0023]

[0025] In some embodiments of the third aspect of the invention, the nucleic acid construct is a vector, such as a viral vector, that can be used to transform T cells to induce expression of the CAR.

[0024]

[0026] In a fourth aspect, the present invention provides a genetically modified cell comprising a CAR according to the first aspect of the invention. In some embodiments, the cell comprises two or more Includes different CARs.

[0025]

[0027] In a fifth aspect, the present invention provides a genetically modified cell comprising a nucleic acid molecule according to the second aspect of the invention, or a nucleic acid construct according to the third aspect of the invention, or a genomic integrated form of the construct. In some embodiments, the nucleic acid molecule or nucleic acid construct encodes two or more different CARs.

[0026]

[0028] In some embodiments of the fourth and fifth aspects of the invention, the two or more different CARs have different signaling domains.

[0029] In some embodiments of the fourth and fifth aspects of the present invention, the cell comprises a first CAR having a signaling domain comprising a portion derived from an activating receptor and a second CAR having a signaling domain comprising a portion derived from a costimulatory receptor. In some embodiments, the activating receptor is a member of the CD3 coreceptor complex or an Fc receptor. In some embodiments, the costimulatory receptor is selected from the group consisting of CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS.

[0027]

[0030] In some embodiments of the fourth and fifth aspects of the invention, the cells are further modified to constitutively express a costimulatory receptor, hi some embodiments, the cells are further modified to express a ligand for the costimulatory receptor, thereby promoting self-stimulation of the cell.

[0028]

[0031] In some embodiments of the fourth and fifth aspects of the invention, the cells are further modified to secrete a cytokine, hi some embodiments, the cytokine is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-17, and IL-21, or a combination thereof.

[0029]

[0032] In some embodiments of the fourth and fifth aspects of the invention, the cell is a leukocyte, hi some embodiments, the cell is a peripheral blood mononuclear cell (PBMC), a lymphocyte, a T cell (including a CD4+ T cell or a CD8+ T cell), a natural killer cell, or a natural killer T cell.

[0030]

[0033] In a sixth aspect, the present invention provides a method of killing cells expressing a dysfunctional P2X7 receptor, the method comprising exposing the cells expressing the dysfunctional P2X7 receptor to genetically modified cells having a chimeric antigen receptor, wherein the chimeric antigen receptor is directed against the dysfunctional P2X7 receptor.

[0031]

[0034] In some embodiments of the sixth aspect of the present invention, CAR directly recognizes dysfunctional P2X7 receptor, or recognizes dysfunctional P2X7 receptor through intermediate.In some embodiments, intermediate is a probe that binds to dysfunctional P2X7 receptor, and CAR recognizes the probe.In some embodiments, the probe is an antibody or aptamer.In some embodiments, the probe comprises a tag, and CAR recognizes the tag.

[0032]

[0035] In a seventh aspect, the present invention provides a method of killing cells expressing dysfunctional P2X7, comprising exposing cells expressing a dysfunctional P2X7 receptor to a genetically modified cell according to the fourth or fifth aspect of the invention.

[0033]

[0036] In some embodiments of the sixth and seventh aspects of the invention, cells expressing dysfunctional P2X7 receptors are exposed to genetically modified cells together with exogenous cytokines. In some embodiments, the genetically modified cell is a genetically modified cell that is autologous to the cell expressing the dysfunctional P2X7 receptor.

[0034] In some embodiments of the sixth and seventh aspects of the present invention, the cell that expresses dysfunctional P2X7 receptor is cancer cell.In some embodiments, cancer is selected from the group consisting of brain cancer, esophageal cancer, oral cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cervical cancer, epithelial cell cancer, skin cancer, leukemia, lymphoma, myeloma, breast cancer, ovarian cancer, endometrial cancer and testicular cancer.In some embodiments, cancer is selected from the group consisting of lung cancer, esophageal cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer, cervical cancer, vaginal cancer, epithelial cell cancer, skin cancer, blood-related cancer, breast cancer, endometrial cancer, uterine cancer and testicular cancer.

[0035]

[0037] In some embodiments of the sixth and seventh aspects of the invention, the cancer is metastatic, hi some embodiments, the cancer is stage III cancer or stage IV cancer.

[0036]

[0038] In an eighth aspect, the present invention provides a method of expanding in vitro a genetically modified cell according to the fourth or fifth aspect of the invention, comprising exposing the cell to an antigen of the CAR. In some embodiments, the method comprises the further step of exposing the cell to a cytokine.

[0037]

[0039] In a ninth aspect, the present invention provides a method of expanding in vitro a genetically modified cell according to the fourth or fifth aspect of the invention, comprising exposing the cell to an antigen of the CAR and simultaneously exposing the cell to a cytokine.

[0038]

[0040] In some embodiments of the eighth and ninth aspects of the invention, the cytokine is a member of the IL-2 subfamily, the interferon subfamily, the IL-10 subfamily, the IL-1 subfamily, the IL-17 subfamily, or the TGF-β subfamily.

[0039]

[0041] In some embodiments of the eighth and ninth aspects of the invention, the cytokine is selected from the group consisting of IFN-γ, IL-2, IL-5, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, TNF-α, TGF-β1, TGF-β2, TGF-β3, and GM-CSF, or a combination thereof.

[0040]

[0042] In a tenth aspect, the present invention provides a method of expanding genetically modified cells according to the fourth or fifth aspects of the invention in vitro, comprising exposing the cells to immobilized anti-CD3 and anti-CD28 antibodies. In some embodiments of the tenth aspect of the invention, the antibodies are immobilized on a bead substrate (e.g., "human activator" Dynabeads™). In some embodiments of the tenth aspect of the invention, the antibodies are immobilized on the surface of a tissue culture vessel, such as the surface of a culture flask, plate, or bioreactor.

[0041]

[0043] In an eleventh aspect, the present invention provides a pharmaceutical composition comprising a genetically modified cell according to the fourth or fifth aspect of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a suitable adjuvant, which may comprise a cytokine. In some embodiments, the pharmaceutical composition may also comprise a mediator described herein.

[0042]

[0044] For a fuller understanding of the aspects and advantages of the present invention, reference should be made to the following detailed description taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0043] [Figure 1]

[0045] FIG. 1 is a schematic diagram showing the configuration of an anti-non-functional (nf) P2X7 receptor chimeric antigen receptor (CAR) according to an embodiment of the present invention. [Figure 2]

[0046] FIG. 2 is a schematic diagram showing the BLIV plasmid used to express the anti-nf P2X7 receptor CAR of FIG. 1. [Figure 3]

[0047] Electrophoresis gel showing restriction fragments derived from BamHI-restricted DNA isolated from E. coli clones transformed with BLIV plasmids. [Figure 4]

[0048] Electrophoresis gel showing restriction fragments from EcoRI, BamHI, and PstI restricted DNA isolated from selected E. coli clones transfected with BLIV plasmids. [Figure 5]

[0049] Figure 1 shows microscopic images of 293T cells transfected with the plasmids required for the construction of the lentiviral vector containing the BLIV-CAR-short hinge construct and 293T cells transduced with the supernatant containing the lentiviral vector. [Figure 6]

[0050] Figure 1 shows microscopic images of 293T cells transfected with the plasmids required for the construction of the lentiviral vector containing the BLIV-CAR-long hinge construct and 293T cells transduced with the supernatant containing the lentiviral vector. [Figure 7]

[0051] FACS analysis of the cell purity of T cells purified with the RosetteSep Human CD8+ T Cell Enrichment Kit. [Figure 8]

[0052] FACS analysis of a killing assay involving co-culture of CD8+ T cells and BT549 cells. [Figure 9]

[0053] Graphs depicting the percentage of dye-labeled target cells that disappeared after 48 hours of co-culture of CD8+ T cells transduced with lentiviral vectors containing BLIV-CAR-short hinge plasmid and BLIV-CAR-long hinge plasmid compared to untransduced CD8+ T cells and CD8+ T cells transduced with an empty BLIV plasmid. [Figure 10]

[0054] Alignment of PEP2-2-1-1, PEP2-472-2, and PEP2-2-12 binding peptides with antibodies directed against the nf-P2X7 receptor. [Figure 11]

[0055] FIG. 1 is a schematic diagram showing the configuration of an anti-nf P2X7 receptor CAR according to a further embodiment of the present invention. [Figure 12]

[0056] FIG. 12 is a schematic diagram showing the pCDH plasmid used to express the anti-nf P2X7 receptor CAR of FIG. 11. [Figure 13]

[0057] FIG. 1 is an electrophoresis gel showing restriction fragments derived from EcoRI and NotI restricted DNA isolated from selected Sure 2 clones transformed with pCDH plasmid. [Figure 14]

[0058] FACS analysis of transfection efficiency of HEK293T cells. [Figure 15]

[0059] Representative histograms of FACS analysis of lentiviral transduction efficiency. [Figure 16]

[0060] FACS analysis of the percentage of transduced CD8 cells expressing GFP. [Figure 17]

[0061] FIG. 1 is a diagram of fusion protein scaffolds for the generation of non-functional and functional P2X7 receptors. [Figure 18]

[0062] FIG. 1 is an electrophoresis gel showing restriction fragments derived from BamHI and PmeI restricted DNA isolated from selected E. cloni® 10G clones transformed with EXD2_K193A or EXD2_WT containing the pDONR-107 vector. [Figure 19]

[0063] FIG. 1 is an electrophoresis gel showing restriction fragments derived from Bam HI-restricted DNA isolated from selected E. cloni® 10G clones transformed with EXD2_K193A or EXD2_WT containing the pLV-416 vector. [Figure 20]

[0064] FACS analysis of transduction of lentiviral packaging of pLV-416-EXD2_K193A and pLV-416-EXD2_WT into HEK293 cells. [Figure 21]

[0065] FACS analysis of lentiviral transduction into HEK293 containing either the pLV-416-EXD2_K193A or pLV-416-EXD2_WT construct. [Figure 22]

[0066] Graph depicting killing of nfP2X7-expressing HEK target cells and 231 breast cancer cells by T cells expressing PEP2-2-1-1, PEP2-472-2 CARs. DETAILED DESCRIPTION OF THE INVENTION

[0044]

[0067] Nucleotide and polypeptide sequences referred to herein are represented by sequence identifier numbers (SEQ ID NOs). A summary of the sequence identifiers is provided in Table 1. A sequence listing is further provided at the end of the specification.

[0045] [Table 1-1]

[0046] [Table 1-2]

[0047]

[0068] The present inventors have recognized that due to the significant "on target" but "off tumor" activity of immune cells expressing chimeric antigen receptors (CARs), there is a need to develop CARs and genetically modified cells expressing them that target markers specifically associated with neoplastic (cancerous) or preneoplastic (precancerous) cells. The present inventors have demonstrated that dysfunctional P2X7 receptors may be involved in the development of CAR-expressing immune cells in various cancers. It has been recognized that these are suitable markers for targeting.

[0048]

[0069] Thus, in a first aspect, the present invention provides a chimeric antigen receptor (CAR) comprising an antigen recognition domain and a signaling domain, wherein the antigen recognition domain recognizes a dysfunctional P2X7 receptor.

[0049]

[0070] Chimeric antigen receptor is an artificially constructed protein that can induce antigen-specific cellular responses when expressed on the surface of a cell.CAR comprises at least two domains: the first domain is an antigen recognition domain that specifically recognizes an antigen, or more specifically, the epitope portion of the antigen, and the second domain is a signal transduction domain that can induce or participate in the induction of intracellular signal transduction pathways.

[0050]

[0071] The combination of these two domains determines the antigen specificity of CAR and the ability of CAR to induce desired cellular responses, and the latter also depends on the host cell of CAR.For example, when a CAR is expressed in T helper cells and has a signaling domain containing a CD3 activation domain, it can induce CD4+ T helper cells to secrete various cytokines when activated by encountering its cognate antigen.In another example, when the same CAR is expressed in CD8+ cytotoxic T cells, it can induce the release of cytokines when activated by cells expressing the cognate antigen, which ultimately leads to the induction of apoptosis of antigen-expressing cells.

[0051]

[0072] In addition to antigen recognition domain and signal transduction domain, CAR may further comprise additional components or parts.For example, CAR may comprise a transmembrane domain, which may comprise or be associated with a portion of the signal transduction domain of CAR.The transmembrane domain is typically one or more hydrophobic helices, which span the lipid bilayer of the cell, and embed the CAR in the cell membrane.The transmembrane domain of CAR may be one of the determining factors of the expression pattern of CAR when combined with cells.For example, the use of a CD3 co-receptor-related transmembrane domain can enable the expression of CAR in naive T cells, while the use of a CD4 co-receptor-derived transmembrane domain can induce the expression of CAR in T helper cells but not in cytotoxic T cells.

[0052]

[0073] An additional component or portion of a CAR can be a linker domain. The linker domain (also known as a spacer or hinge domain) spans the extracellular side of the transmembrane domain to the antigen recognition domain, thereby linking the antigen recognition domain to the transmembrane domain. In some cases, a linker domain is not required for a functional CAR (i.e., the antigen recognition domain can be directly connected to the transmembrane domain), but in some situations, the use of a linker domain can increase the efficacy of the CAR. The linker domain can have various functionalities, including allowing flexibility of the CAR to allow the orientation of the antigen recognition domain of the CAR necessary for antigen binding. As a result, the linker domain can be any amino acid sequence that performs this function. One non-limiting example of a linker domain is a domain having amino acid sequence homology to the hinge region of an IgG antibody, e.g., the IgG1 hinge region. Another example includes an amino acid sequence having sequence homology to the CH2CH3 region of an antibody, or to portions of the CD3 co-receptor complex, the CD4 co-receptor, or the CD8 co-receptor.

[0053]

[0074] The P2X7 receptor (P2X purinergic receptor, ligand-gated ion channel 7) is an ATP-gated ion channel expressed in several species, including humans. This receptor is encoded by a gene whose official code is P2RX7. This gene is also referred to as P2X purinergic receptor 7, ATP receptor, P2Z receptor, P2X7 receptor, and purinergic receptor P2X7 variant A. For purposes of this disclosure, this gene and the encoded receptor will be referred to herein as P2X7 and P2X7, respectively.

[0054]

[0075] The mRNA sequence, coding (cDNA) sequence, and amino acid sequence of the human P2X7 gene are set forth in SEQ ID NOs: 1 to 3, respectively. The mRNA sequence and amino acid sequence of the human P2X7 gene are also represented by GenBank accession numbers NM_002562.5 and NP_002553.3, respectively. The P2X7 gene is conserved in chimpanzees, rhesus monkeys, dogs, cows, mice, rats, pigs, chickens, zebrafish, and frogs. Further details of the P2X7 gene in humans and other species can be found at the National Centre for Biotechnology Information. It can be accessed from the GenBank database at National Center for Biological Information (NCBI) (www.ncbi.nlm.nih.gov). For example, the gene identifier number of human P2X7 is 5027, that of chimpanzee is 452318, that of monkey is 699455, that of dog is 448778, that of cow is 286814, that of mouse is 18439, that of zebrafish is 387298, and that of frog is 398286. In addition, at least 73 organisms have orthologs of the human P2X7 gene.

[0055]

[0076] Further details about the P2X7 gene in humans and other species can also be found in NCBI's UniGene portal (for example, for human P2X7, refer to UniGene Hs.729169-http: / / www.ncbi.nlm.nih.gov / UniGene / clust.cgi?UGID=4540770&TAXID=9606&SEARCH).Alternatively, the nucleotide sequence and amino acid sequence details of P2X7 gene can be accessed from UniProt database (www.uniprot.org), and the UniProt identifier of human P2X7 gene is Q99572.The contents of GenBank and UniProt records are incorporated herein by reference.

[0056]

[0077] P2X7 receptor is formed by three protein subunits (monomers), and in human natural receptor, at least one of these monomers has the amino acid sequence described in SEQ ID NO:3.It should be understood that " P2X7 receptor " referred to herein also includes the naturally occurring variant of this receptor, including splice variant, the naturally occurring truncated form and allelic variant of this receptor.P2X7 receptor can also include the subunit with modified amino acid sequence, for example, comprise the truncated, amino acid deleted or modified amino acid described in SEQ ID NO:3.

[0057]

[0078] A "variant" of a P2X7 gene or encoded protein may, for example, exhibit a nucleic acid or amino acid sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to a native P2X7 receptor, respectively.

[0058]

[0079] The P2X7 receptor is activated by ATP binding to the ATP binding site of the receptor. This causes the rapid opening of the channel (within milliseconds), which selectively allows small cations to move across the membrane. After a short time (within seconds), a large pore is formed in the cell membrane, which allows molecules up to 900 Da in size to permeate the cell membrane. This pore formation ultimately leads to cell depolarization and, in many cases, cytotoxicity and cell death. This role is played by the P2X7 receptor. This has led to the idea that the signal transduction pathway is involved in apoptosis in a variety of cell types.

[0059]

[0080] Similar to other molecules involved in apoptosis, such as Bcl2 and Bax, the reduction or loss of P2X7 receptor function can lead to cells that are relatively resistant to induced apoptosis.In many cases, this resistance to apoptosis is important in the transition from normal "healthy" cells to mutated precancerous or cancerous cells.As a result, the ability of P2X7 receptor to target cells with reduced or lost function provides it a promising target for cancer therapy.

[0060]

[0081] Therefore, in the first aspect of the present invention, CAR recognizes dysfunctional P2X7 receptor.With respect to P2X7 receptor, the term " dysfunctional " used throughout this specification comprises the reduction of receptor function compared with its corresponding function in normal non-tumor cell.In some embodiments, the function of P2X7 receptor can be reduced by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 99%.In some embodiments, the term " dysfunctional " can include non-functional P2X7 receptor.This means that P2X7 receptor cannot be induced to allow cation or other molecules to pass through cell membrane.

[0061]

[0082] Any change in wild-type or natural form of receptor that causes P2X7 dysfunctional receptor is encompassed herein.For example, dysfunctional receptor may be the result of mutation or modification in one or more amino acids of receptor that are related to the binding of ATP to receptor.In fact, P2X7 receptor is dysfunctional when the ability to bind ATP at ATP-binding site is reduced or it cannot bind to it.In this case, the antigen recognition domain of chimeric antigen receptor is expected to recognize the epitope associated with the ATP-binding site of dysfunctional P2X7 receptor.As a result, in some embodiments of the first aspect of the present invention, the antigen recognition domain of chimeric antigen receptor recognizes the epitope associated with the ATP-binding site of dysfunctional P2X7 receptor.In some embodiments, dysfunctional P2X7 receptor has reduced ability to bind ATP compared with the ATP-binding ability of wild-type (functional) P2X7 receptor.In some embodiments, dysfunctional P2X7 receptor cannot bind to ATP.

[0062]

[0083] The modification of one or more amino acids of P2X7 receptor may include a change in conformation of one or more amino acids of this receptor.Therefore, in some embodiments of the first aspect of the present invention, the chimeric antigen receptor binds to a dysfunctional P2X7 receptor that has a change in conformation that makes the receptor dysfunctional.Specifically, this change in conformation may be a change from trans to cis configuration of one or more amino acids of P2X7 receptor.In some embodiments, the proline at position 210 of P2X7 receptor changes from trans to cis configuration.In this case, the antigen recognition domain of CAR may recognize an epitope comprising the proline at amino acid position 210 of P2X7 receptor.In some embodiments of the first aspect of the present invention, the antigen recognition domain recognizes an epitope comprising one or more amino acids ranging from the glycine at amino acid position 200 to the cysteine ​​at amino acid position 216 (inclusive) of the dysfunctional P2X7 receptor. In some embodiments of the first aspect of the present invention, the antigen recognition domain recognizes an epitope comprising one or more of the proline at position 210 of the dysfunctional P2X7 receptor and the amino acid residues ranging from the glycine at amino acid position 200 to the cysteine ​​at amino acid position 216 (inclusive) of the dysfunctional P2X7 receptor.

[0063]

[0084] Without wishing to be bound by theory, it is believed that position 210 of the P2X7 receptor As a result of the change in the configuration of the proline at position 210 of SEQ ID NO: 3, the three-dimensional structure of the receptor may be altered. This alteration in the three-dimensional structure allows the antigen-recognition domain of CAR to bind to amino acids or epitopes that were previously inaccessible in the native three-dimensional structure of P2X7 receptor. Thus, in some embodiments, CAR recognizes one or more epitopes of P2X7 receptor that are exposed to the antigen-recognition domain as a result of the change in the configuration of the proline at position 210 of SEQ ID NO: 3 from trans to cis. These epitopes may include one or more of the amino acids at positions 200 to 210 or 297 to 306 (inclusive) of P2X7 receptor. Thus, in some embodiments of the first aspect of the present invention, the antigen-recognition domain recognizes an epitope that includes one or more of the amino acids at positions 200 to 210 and / or 297 to 306 of P2X7 receptor.

[0064]

[0085] The term "recognize" used throughout this specification refers to the ability of antigen-recognizing domain to bind with dysfunctional P2X7 receptor, its part, or its epitope.In some embodiments, antigen-recognizing domain may directly bind with dysfunctional P2X7 receptor or its epitope.In other embodiments, antigen-recognizing domain may bind with the processed form of dysfunctional P2X7 receptor.When used in this context, the term "processed form" refers to the form of P2X7 receptor that is cut or digested as a result of intracellular processing.As a result, the recognition of the "processed form" of dysfunctional P2X7 receptor may result from being presented in combination with major histocompatibility complex (MHC).

[0065]

[0086] The antigen recognition domain can be any suitable domain that can recognize dysfunctional P2X7 receptor or its epitope.The term "antigen recognition domain" used throughout this specification refers to the part of CAR that provides CAR specificity for dysfunctional P2X7 receptor.The antigen recognition domain can be the entire extracellular region of CAR, or just a part thereof.Suitable antigen recognition domains include, but are not limited to, polypeptides that have sequence homology to the antigen-binding site of an antibody or its fragment that binds to dysfunctional P2X7 receptor.Therefore, in some embodiments of the first aspect of the present invention, the antigen recognition domain comprises an amino acid sequence that has homology to an antibody or its fragment that binds to dysfunctional P2X7 receptor.In some embodiments, a part of the antigen recognition domain comprises an amino acid sequence that has homology to an antibody or its fragment that binds to dysfunctional P2X7 receptor.The original homologous antibody sequence can be any suitable sequence of an antibody that has affinity for P2X7 receptor. For example, the sequence may share sequence homology with antibodies originating from one or more of the following species: human, non-human primate, mouse, rat, rabbit, sheep, goat, ferret, dog, chicken, cat, guinea pig, hamster, horse, cow, or pig. The antigen recognition domain may share sequence homology with the sequence of a monoclonal antibody produced from a hybridoma cell line. If the species from which the homologous antibody sequence originates is not human, the antibody is preferably a humanized antibody. The homologous antibody sequence may also be derived from non-mammalian species, such as cartilaginous fish (e.g., shark IgNAR antibodies, see WO 2012 / 073048). Alternatively, the antigen-binding domain may comprise a modified protein scaffold that provides functionality similar to that of a shark antibody, such as an i-body with a binding moiety based on a shark IgNAR antibody (see WO 2005 / 118629). In addition, the antigen recognition domain may be, be derived from, or share sequence homology with any other suitable binding molecule or peptide that can selectively interact with the dysfunctional P2X7 receptor with sufficient affinity to activate the CAR signaling domain.Methods for identifying antigen-binding proteins are known in the art, such as panning of phage display libraries, protein affinity chromatography, co-immunoprecipitation, and the yeast two-hybrid system, among others (Srinivasa Ra. See O, V. et al., Int J Proteomics, 2014; article number 147648).

[0066]

[0087] In some embodiments, the antigen recognition domain of CAR comprises the amino acid sequence homology with the amino acid sequence of the antigen binding fragment (Fab) part of the antibody that binds to dysfunctional P2X7 receptor.As understood in the art, the Fab part of antibody is composed of one constant region and one variable region of each of antibody heavy chain and light chain.Fab is the antigen determinant region of antibody, and can be generated by enzymatically cutting Fc region from antibody.

[0067]

[0088] In some embodiments of the first aspect of the present invention, the antigen-recognition domain comprises an amino acid sequence homologous to the amino acid sequence of single-chain variable fragment (scFv) that binds to dysfunctional P2X7 receptor.As understood in the art, scFv is a fusion protein that comprises two parts that can share homology with or be identical to the variable heavy chain (VH) and variable light chain (VL) of antibody, and these two parts are connected together by a linker peptide.For example, scFv can comprise the VH and VL amino acid sequences derived from an antibody that recognizes dysfunctional P2X7 receptor.In this context, it is expected that the term "derived from" does not refer to the origin of the polypeptide itself, but rather refers to the origin of the amino acid sequence that constitutes a part of the antigen-binding region.As a result, the term "derived from" includes synthetic, artificial, or other polypeptides that share sequence identity with the antibody that binds to dysfunctional P2X7 receptor.

[0068]

[0089] In some embodiments of the first aspect of the present invention, the antigen recognition domain comprises amino acid sequence homology to the amino acid sequence of a multivalent scFv that binds to dysfunctional P2X7 receptor. In some embodiments, the multivalent scFv is a bivalent scFv or a trivalent scFv.

[0069]

[0090] In some embodiments of the first aspect of the present invention, the antigen recognition domain has the amino acid sequence of a single chain antibody domain (sdAb) that binds to a dysfunctional P2X7 receptor.

[0091] In some embodiments, the antigen recognition domain comprises the amino acid sequence set forth in SEQ ID NO: 10, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34, or a functional variant thereof.

[0070]

[0092] In some embodiments, the antigen recognition domain comprises a binding peptide comprising an amino acid sequence homologous to one or more CDR regions of an antibody that binds to a dysfunctional P2X7 receptor. In some embodiments, the binding peptide comprises a V region of an antibody that binds to a dysfunctional P2X7 receptor. H Chain and / or V LThe antigen-recognition domain comprises one or more regions having sequence homology to the CDR1, 2, and 3 domains of the corresponding chain. In some embodiments, the antigen-recognition domain comprises one or more sequences that are at least 50%, 60%, 70%, 80%, 90%, or 94% identical to any one of the CDR regions spanning positions 30 to 35, 50 to 67, or 98 to 108 of the sequence set forth in SEQ ID NO: 10, 32, 33, or 34. In some embodiments, the antigen-recognition domain comprises one or more of the sequences spanning positions 30 to 35, 50 to 67, or 98 to 108 of the sequence set forth in SEQ ID NO: 10, 32, 33, or 34. Any public sequence that allows for proper formation and positioning of the CDR regions can be present between the CDR regions of the antigen-binding peptide set forth in SEQ ID NO: 10, 32, 33, or 34. In some embodiments, the antigen recognition domain comprises a sequence that is 50%, 60%, 70%, 80%, or 90%, 95%, or 99% identical to one of the sequences set forth in SEQ ID NO: 10, 32, 33, or 34.

[0071]

[0093] The antibody directed to dysfunctional P2X7 receptor, from which suitable amino acid sequence can be derived, and the method for producing such antibody have been described in the art (see, for example, WO2001 / 020155, WO2003 / 020762, WO2008 / 043145, WO2008 / 043146, WO2009 / 033233, WO2011 / 020155 and WO2011 / 075789).The method for producing polyclonal and monoclonal antibodies against specific epitopes (such as those mentioned above) is expected to be known to those skilled in the art.In summary, the desired epitope (such as the segment of dysfunctional P2X7 receptor that contains the proline at position 210) is injected into suitable host animal in the presence of suitable immunogenic carrier protein and adjuvant. Serum can then be collected from immunized animal, and antibody can be isolated based on its antibody class or its antigen specificity.After evaluating the compatibility and specificity of purified antibody, antibody can be further processed to isolate antigen binding fragment, or be sequenced to identify related VH and VL domain.The epitope suitable for producing antibody directed to dysfunctional P2X7 receptor is known in the art (see for example WO2008 / 043146, WO2010 / 000041 and WO2009 / 033233).

[0072]

[0094] The signal transduction domain of a CAR can be any suitable domain that can induce or participate in the induction of an intracellular signal transduction cascade when the CAR is activated as a result of antigen recognition by the antigen recognition domain of the CAR. The signal transduction domain of a CAR will be specifically selected according to the intracellular outcome desired after CAR activation. Although there are many possible signal transduction domains, when used in immunotherapy and cancer therapy, signal transduction domains can be classified into two general categories based on the receptors from which they are derived: activating receptors and costimulatory receptors (see further details below). Thus, in some embodiments of the first aspect of the present invention, the signal transduction domain comprises a portion derived from an activating receptor. In some embodiments, the signal transduction domain comprises a portion derived from a costimulatory receptor.

[0073]

[0095] As used throughout this specification, the term "portion," when used in reference to an activating or costimulatory receptor, relates to any segment of the receptor that includes sequences that are responsible for or involved in the initiation / induction of an intracellular signaling cascade following interaction of the receptor with its cognate antigen or ligand. An example of the initiation / induction of a T cell receptor (TCR) intracellular signaling cascade via CD3 is outlined below.

[0074]

[0096] Without wishing to be bound by theory, the extracellular portion of the TCR is primarily composed of heterodimers of either clonoplasmic TCRα and TCRβ chains (TCRα / β receptors) or TCRγ and TCRδ chains (TCRγδ receptors). These TCR heterodimers generally lack intrinsic signaling capability, and therefore, they noncovalently associate with multiple signaling subunits of CD3 (primarily CD3-zeta, CD3-gamma, CD3-delta, and CD3-epsilon). The CD3 gamma, delta, and epsilon chains each have an intracellular (cytoplasmic) portion that contains a single immunoreceptor tyrosine-based activation motif (ITAM), while the CD3-zeta chain contains three tandem ITAMs. Upon binding of the TCR to its cognate antigen in the presence of MHC and essential co-receptors such as CD4 or CD8, signaling is initiated, resulting in the phosphorylation of two tyrosine residues within the intracellular ITAM of the CD3 chain by a tyrosine kinase (i.e., Lck). Consequently, a second tyrosine kinase (ZAP-70, itself activated by Lck phosphorylation) is recruited to biphosphorylate the ITAM. As a result, multiple A number of downstream target proteins are activated, which ultimately leads to changes in intracellular configuration, calcium mobilization, and actin cytoskeletal rearrangements, which, in combination, ultimately result in the activation of transcription factors and the induction of T cell immune responses.

[0075]

[0097] The term "activating receptor" as used throughout this specification relates to a receptor or co-receptor that forms a component of or is involved in the formation of a T cell receptor (TCR) complex, or a receptor that is involved in the specific activation of an immune cell as a result of recognition of an antigenic or other immunogenic stimulus.

[0076]

[0098] Non-limiting examples of such activating receptors include components of the T cell receptor-CD3 complex (CD3-zeta, CD3-gamma, CD3-delta, and CD3-epsilon), CD4 coreceptors, CD8 coreceptors, Fc receptors, or natural killer (NK) cell-associated activating receptors such as LY-49 (KLRA1), natural cytotoxicity receptors (NCRs, preferably NKp46, NKp44, NKp30, or NKG2, or CD94 / NKG2 heterodimers). Consequently, in some embodiments of the first aspect of the invention, the signaling domain comprises a portion derived from any one or more of a member of the CD3 coreceptor complex (preferably, the CD3-zeta chain or a portion thereof), a CD4 coreceptor, a CD8 coreceptor, an Fc receptor (FcR) (preferably, FcεRI or FcγRI), or an NK-associated receptor such as LY-49.

[0077]

[0099] Specific intracellular signaling portions of each CD3 chain are known in the art. For example, the intracellular cytoplasmic region of the CD3 zeta chain spans amino acids 52 to 164 of the sequence set forth in SEQ ID NO:4, with three ITAM regions spanning amino acids 61 to 89, 100 to 128, and 131 to 159 of SEQ ID NO:4. Furthermore, the intracellular portion of the CD3 epsilon chain spans amino acids 153 to 207 of the sequence set forth in SEQ ID NO:5, with a single ITAM region spanning amino acids 178 to 205 of SEQ ID NO:5. The intracellular portion of the CD3 gamma chain spans amino acids 138 to 182 of the sequence set forth in SEQ ID NO:6, with a single ITAM region spanning amino acids 149 to 177 of SEQ ID NO:6. The intracellular portion of CD3 delta spans amino acids 127 to 171 of the sequence set forth in SEQ ID NO:7, with a single ITAM region spanning amino acids 138 to 166 of SEQ ID NO:7.

[0078]

[0100] In some embodiments of the first aspect of the invention, the signaling domain comprises a portion derived from either CD3 (CD3-zeta chain or a portion thereof) or an Fc receptor (preferably FcεRI or FcγRI). In some embodiments, the portion of the CD3-zeta co-receptor complex comprises the amino acid sequence set forth in SEQ ID NO: 22 or a functional variant thereof.

[0079]

[0101] The intracellular portions of Fc receptors are known in the art. For example, the intracellular portion of FcεR1 spans amino acids 1 to 59, 118 to 130, and 201 to 244 of the sequence set forth in SEQ ID NO: 8. Furthermore, the intracellular portion of FcγRI spans amino acids 314 to 374 of the sequence set forth in SEQ ID NO: 9.

[0080]

[0102] Various combinations of portions of the activating receptor can be used to combine the transmembrane (TM) and intracellular (IC) portions of the CAR, e.g., CD3ζ TM and CD3ζ IC (Landmeier S. et al., Cancer Res. 2007;67:8335-43; Guest RD. et al., J Immunother. 2005,28:203-11; Hombach AA. et al., J Immunol. 2007;178:4650-7), CD4 TM and CD3ζ IC (James SE. et al., J Immunol. 2008;180:7028-38), CD8 TM and CD3ζ IC (Patel SD. et al., Gene Ther. 1999;6:412-9), and FcεRIγ TM and Fcε RIγ IC (Haynes NM. et al., J Immunol. 2001; 166: 182-7, Annenkov AE. et al., J Immunol. 1998; 161: 6604-13) can be formed.

[0081]

[0103] The term "costimulatory receptor" as used throughout this specification refers to a receptor or coreceptor that assists in the activation of immune cells upon antigen-specific induction of an activating receptor. As will be understood, costimulatory receptors do not require the presence of an antigen, nor are they antigen-specific, but are typically one of two signals, the other being an activation signal required for the induction of an immune cell response. In the context of an immune response, costimulatory receptors are typically activated by the presence of their ligands expressed on the surface of antigen-presenting cells (APCs), such as dendritic cells or macrophages. Specifically with respect to T cells, costimulation is necessary to result in cell activation, proliferation, differentiation, and survival (all of which are commonly referred to as T cell activation), but the presentation of antigen to T cells in the absence of costimulation can result in anergy, clonal deletion, and / or the development of antigen-specific tolerance. Importantly, costimulatory molecules can direct T cell responses to simultaneously encountered antigens. In general, antigens encountered in the context of "positive" costimulatory molecules are expected to result in T cell activation and a cellular immune response aimed at eliminating cells expressing that antigen, whereas antigens encountered in the context of "negative" co-receptors are expected to result in the induction of a state of tolerance to the simultaneously encountered antigen.

[0082]

[0104] Non-limiting examples of T cell costimulatory receptors include CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS. Specifically, CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS all represent "positive" costimulatory molecules that enhance the activation of T cell responses. Thus, in some embodiments of the first aspect of the invention, the signaling domain comprises a portion derived from any one or more of CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS.

[0083]

[0105] In some embodiments of the first aspect of the present invention, the signaling domain comprises a portion derived from a CD28, OX40, or 4-1BB costimulatory receptor. In some embodiments, the signaling domain comprises a portion of a CD28 costimulatory receptor. In some embodiments, the signaling domain comprises a portion of an OX40 costimulatory receptor. In some embodiments, the portion of the OX40 costimulatory receptor comprises the amino acid sequence set forth in SEQ ID NO: 20 or a functional variant thereof.

[0084]

[0106] Various combinations of costimulatory receptor moieties can be used to form the transmembrane (TM) and intracellular (IC) portions of the CAR, such as CD8 TM and DAP10 IC or CD8 TM and 4-1BB IC (Marin V. et al., Exp Hematol. 2007; 35: 1388-97), CD28 TM and CD28 IC (Wilkie S. et al., J Immunol. 2008; 180: 4901-9, Maher J. et al., Nat Biotechnol. 2002; 20: 70-5), and CD8 TM and CD28 IC (Marin V. et al., Exp Hematol. 2007; 35: 1388-97).

[0085]

[0107] Sequence information for the above-mentioned activating and costimulatory receptors is readily accessible in various databases. For example, examples of human amino acid, gene, and mRNA sequences for these receptors are provided in Table 2.

[0086] [Table 2]

[0087]

[0108] Although Table 2 is provided with respect to human activating and costimulatory receptors, one skilled in the art will understand that homologous and orthologous forms of each receptor exist in the majority of mammalian and vertebrate species. Thus, the above-referenced sequences are provided merely as non-limiting examples of receptor sequences that may be included in a CAR of the first aspect of the invention, as well as homologous and orthologous sequences from any desired species that may be used to generate a CAR suitable for a given species.

[0088]

[0109] In some embodiments of the first aspect of the present invention, the signaling domain comprises a portion derived from an activating receptor and a portion derived from a costimulatory receptor. Although it is not intended to be construed as a general rule, in this context, antigen recognition by the antigen recognition domain of the CAR is expected to simultaneously induce both an intracellular activation signal and an intracellular costimulatory signal. As a result, this is expected to stimulate antigen presentation by APCs expressing costimulatory ligands. Alternatively, the CAR may have a signal transduction domain that induces a portion derived from either an activating receptor or a costimulatory receptor. In this alternative form, the CAR only induces either an activating intracellular signal transduction cascade or a costimulatory intracellular signal transduction cascade.

[0089]

[0110] In some embodiments of the first aspect of the present invention, the CAR is expected to have a signaling domain comprising one portion derived from a single activating receptor and multiple portions derived from multiple costimulatory receptors. In some embodiments, the CAR is expected to have a signaling domain comprising multiple portions derived from multiple activating receptors and one portion derived from a single costimulatory receptor. In some embodiments, the CAR is expected to have a signaling domain comprising multiple portions derived from multiple activating receptors and multiple portions derived from multiple costimulatory receptors. In some embodiments, the CAR is expected to have a signaling domain comprising one portion derived from a single activating receptor and multiple portions derived from two costimulatory receptors. In some embodiments, the CAR is expected to have a signaling domain comprising one portion derived from a single activating receptor and multiple portions derived from three costimulatory receptors. In some embodiments, the CAR is expected to have a signaling domain comprising multiple portions derived from two activating receptors and one portion derived from a costimulatory receptor. In some embodiments, the CAR is expected to have a signaling domain comprising multiple portions derived from two activating receptors and multiple portions derived from two costimulatory receptors. As will be appreciated, there are a number of additional variations in the activating and costimulatory receptors from which signaling domains can be derived, and the above examples are not intended to limit the possible combinations encompassed herein.

[0090]

[0111] In some embodiments of the first aspect of the invention, the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54, or a functional variant of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54. In some embodiments, the functional variant comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54.

[0091]

[0112] As set forth above, the present invention includes functional variants of any one of SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54. In the context of the present invention, a "functional variant" may include any amino acid sequence so long as it maintains the function of any one of SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54.

[0092]

[0113] Thus, a functional variant can be, for example, an insertion, deletion, or substitution of one or more amino acids relative to one of SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54; SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54, so long as the functional variant maintains the function of one of SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 , a mutant form or allelic variant of one of SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54; an ortholog of one of SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54; a homeologue of one of SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54; an analog of one of SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54, and the like.

[0093]

[0114] For example, with respect to SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54, the function of the chimeric antigen receptor comprising SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54 is to recognize dysfunctional P2X7 receptor without significant recognition of functional P2X7 receptor, and induce intracellular signals that lead to the activation of T cells expressing CAR.As will be understood by those skilled in the art, changes to the part of the amino acid sequence of the chimeric antigen receptor described in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54 can be made without significant alteration of the recognition of dysfunctional P2X7 receptor and / or the activation of T cells expressing CAR.Such changes may include, but are not limited to, changes in the hinge region of the chimeric antigen receptor, changes in the transmembrane domain, and changes in the part of the activating receptor and / or costimulatory receptor that constitutes the intracellular domain of the chimeric antigen receptor.

[0094]

[0115] As shown above, functional variants may include individual amino acid substitutions, deletions, or insertions compared to one of SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54. For example, those skilled in the art will recognize that any amino acid can be replaced with a chemically (functionally) similar amino acid and retain the function of the polypeptide. Such conservative amino acid substitutions are well known in the art. The groups in Table 3 below each contain amino acids that are conservative substitutions for each other.

[0095] [Table 3]

[0096]

[0116] Furthermore, if desired, unnatural amino acids or chemical amino acid analogs can be introduced as substitutions or additions into the polypeptides encompassed herein, including, but not limited to, D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6-aminohexanoic acid, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, Cα-methylamino acids, and Nα-methylamino acids, as well as common amino acid analogs.

[0097]

[0117] As noted above, a functional variant of any one of SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54 may comprise an amino acid sequence that is at least 80% identical to any one of SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54. In other embodiments, a functional variant may comprise at least 85% amino acid sequence identity, at least 90% amino acid sequence identity, at least 91% amino acid sequence identity, at least 92% amino acid sequence identity, at least 93% amino acid sequence identity, at least 94% amino acid sequence identity, at least 95% amino acid sequence identity, at least 96% amino acid sequence identity, at least 97% amino acid sequence identity, at least 98% amino acid sequence identity, at least 99% amino acid sequence identity, or at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% amino acid sequence identity to any one of SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54.

[0098]

[0118] When comparing amino acid sequences, the sequences should be compared over a comparison window determined by the length of the polypeptide. For example, a comparison window of at least 20 amino acid residues, at least 50 amino acid residues, at least 75 amino acid residues, at least 100 amino acid residues, at least 200 amino acid residues, at least 300 amino acid residues, at least 400 amino acid residues, at least 500 amino acid residues, at least 600 amino acid residues, or the entire length of any one of SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54 can be used. The comparison window can contain about 20% or less of additions or deletions (i.e., gaps) compared to the reference sequence (without additions or deletions) for optimal alignment of the two sequences. Alignment of sequences optimally aligned to a comparison window can be performed by computer implementations of algorithms such as the BLAST family of programs disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389-3402. Global alignment programs can also be used to align similar sequences of approximately equal size. Examples of global alignment programs include the EMBOSS package (Rice P et al., 2000, Trends Genet., 16:276-277), and the GGSEARCH program (fasta.bioch.virginia.edu / fa (Available at sta_www2 / fasta_www.cgi?rm=compare&pgm=gnw). Both of these programs are based on the Needleman-Wunsch algorithm, which is used to find the optimal alignment of two sequences (including gaps) along their entire length. A detailed discussion of sequence analysis can also be found in Unit 19.3 of Ausubel et al. ("Current Protocols in Molecular Biology," John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998).

[0099]

[0119] In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor according to the first aspect of the invention. In some embodiments, the nucleic acid molecule is a non-naturally occurring nucleic acid molecule.

[0100]

[0120] In some embodiments of the second aspect of the invention, the nucleic acid molecule comprises a nucleotide sequence that encodes the amino acid sequence set forth in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54, or encodes a functional variant of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54. In some embodiments, the functional variant comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54.

[0101]

[0121] Nucleic acid molecules can contain any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified or modified RNA or DNA. For example, nucleic acid molecules include single-stranded and / or double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA that can be single-stranded, more typically double-stranded, or a mixture of single-stranded and double-stranded regions. In addition, nucleic acid molecules can contain triple-stranded regions containing RNA or DNA, or both RNA and DNA. Nucleic acid molecules can also contain one or more modified bases, or DNA or RNA backbones modified for stability or other reasons. Various modifications can be made to DNA and RNA, and therefore the term "nucleic acid molecule" encompasses chemically, enzymatically, or metabolically modified forms.

[0102]

[0122] In some embodiments of the second aspect of the invention, the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.

[0103]

[0123] Those skilled in the art will understand that any nucleotide sequence encoding a chimeric antigen receptor having the amino acid sequence set forth in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, or a functional variant of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, is contemplated by the present invention. For example, variants of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37 that contain one or more nucleic acids that differ from SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37 but still encode the same amino acid sequence, are contemplated. Due to the degeneracy of the genetic code, a large number of nucleic acids can encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at all positions in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37 where alanine is specified by a codon, the codon may be altered to any of the corresponding codons described without altering the encoded polypeptide. Thus, Thus, every nucleotide sequence herein encoding a chimeric antigen receptor having the amino acid sequence set forth in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, or a functional variant of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, describes every possible silent variation in the nucleotide sequence. One of ordinary skill in the art will understand that each codon in the nucleic acid (except AUG, which is normally the only codon for methionine, and TGG, which is normally the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Accordingly, every silent variation in a nucleotide sequence encoding a polypeptide is implicit in each described sequence.

[0104]

[0124] In a third aspect, the present invention provides a nucleic acid construct comprising a nucleic acid molecule according to the second aspect of the invention. The nucleic acid construct may further comprise one or more host origins of replication, a selectable marker gene active in one or more hosts, and / or one or more transcription control sequences.

[0105]

[0125] As used herein, the term "selectable marker gene" includes any gene that confers a phenotype on cells in which it is expressed, such that it facilitates the identification and / or selection of cells that have been transfected or transformed with the construct.

[0106]

[0126] A "selectable marker gene" includes any nucleotide sequence that, when expressed by cells transformed with a construct, confers a phenotype on the cells that facilitates identification and / or selection of those transformed cells. Various nucleotide sequences encoding suitable selectable markers are known in the art (e.g., Mortesen, R.M. and Kingston, R.E. Curr Protoc Mol Biol, 2009; Unit 9.5). Exemplary nucleotide sequences encoding selectable markers include the adenosine deaminase (ADA) gene; the cytosine deaminase (CDA) gene; the dihydrofolate reductase (DHFR) gene; the histidinol dehydrogenase (hisD) gene; the puromycin-N-acetyltransferase (PAC) gene; the thymidine kinase (TK) gene; the xanthine-guanine phosphoribosyltransferase (XGPRT) gene, or antibiotic resistance genes, such as the ampicillin resistance gene, the puromycin resistance gene, the bleomycin resistance gene, the hygromycin resistance gene, the kanamycin resistance gene, and the ampicillin resistance gene, fluorescent reporter genes, such as green, red, yellow, or blue fluorescent protein-encoding genes; and luminescence-based reporter genes, such as the luciferase gene, that allow optical selection of cells using techniques such as fluorescence-activated cell sorting (FACS), among others.

[0107]

[0127] Furthermore, it should be noted that the selectable marker gene may be in a different open reading frame in the construct or may be expressed as a fusion protein with another polypeptide (e.g., CAR).

[0108]

[0128] As mentioned above, the nucleic acid construct may also contain one or more transcription control sequences. The term "transcription control sequence" should be understood to include any nucleic acid sequence that performs transcription of an operably connected nucleic acid. Transcription control sequences may include, for example, a leader, a polyadenylation sequence, a promoter, an enhancer or upstream activation sequence, and a transcription terminator. Typically, a transcription control sequence includes at least a promoter. The term "promoter" as used herein refers to any nucleic acid that confers, activates, or enhances expression of a nucleic acid in a cell.

[0109]

[0129] In some embodiments, at least one transcription control sequence is For purposes of this specification, a transcriptional control sequence is said to be "operably linked" to a given nucleic acid molecule if the transcriptional control sequence is capable of promoting, inhibiting, or otherwise regulating transcription of the nucleic acid molecule. Thus, in some embodiments, the nucleic acid molecule is under the control of a transcriptional control sequence, such as a constitutive promoter or an inducible promoter.

[0110]

[0130] A "nucleic acid construct" can be in any suitable form, such as a plasmid, phage, transposon, cosmid, chromosome, or vector, that is capable of replication when associated with appropriate control elements and allows the genetic sequences contained within the construct to be moved between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. In some embodiments, the nucleic acid construct is a vector. In some embodiments, the vector is a viral vector.

[0111]

[0131] A promoter can constitutively or differentially control the expression of an operably linked nucleic acid molecule in a cell, tissue, or organ in which expression occurs. Thus, a promoter can include, for example, a constitutive promoter or an inducible promoter. A "constitutive promoter" is a promoter that is active under most environmental and physiological conditions. An "inducible promoter" is a promoter that is active under specific environmental and physiological conditions. The present invention contemplates the use of any promoter that is active in a cell of interest. Therefore, it is expected that a wide range of promoters can be easily identified by those skilled in the art.

[0112]

[0132] Constitutive mammalian promoters can include, but are not limited to, simian virus 40 (SV40), cytomegalovirus (CMV), P-actin, ubiquitin C (UBC), elongation factor-1 alpha (EF1A), phosphoglycerate kinase (PGK), and CMV early enhancer / chicken beta actin (CAGG).

[0113]

[0133] Inducible promoters can include, but are not limited to, chemically inducible promoters and physically inducible promoters.Chemically inducible promoters include promoters whose activity is controlled by chemicals, such as alcohol, antibiotics, steroids, metal ions, or other compounds.Examples of chemically inducible promoters include, among others, tetracycline-regulated promoters (see, for example, U.S. Patent No. 5,851,796 and U.S. Patent No. 5,464,758); steroid-responsive promoters, such as glucocorticoid receptor promoters (see, for example, U.S. Patent No. 5,512,483), ecdysone receptor promoters (see, for example, U.S. Patent No. 6,379,945); and metal-responsive promoters, such as metallothionein promoters (see, for example, U.S. Patent No. 4,940,661, U.S. Patent No. 4,579,821, and U.S. Patent No. 4,601,978).

[0114]

[0134] As mentioned above, the control sequence can also include a terminator. The term "terminator" refers to the DNA sequence at the end of a transcription unit that signals the end of transcription. A terminator is generally a 3' untranslated DNA sequence that contains a polyadenylation signal, which promotes the addition of a polyadenylation sequence to the 3' end of the primary transcript. Similar to a promoter sequence, a terminator can be any terminator sequence that is functional in the cells, tissues, or organs intended for use. Suitable terminators are expected to be known to those skilled in the art.

[0115]

[0135] As will be appreciated, the nucleic acid construct of the third aspect of the invention may further comprise the additional sequence: For example, it may further comprise sequences that allow for enhanced expression, cytoplasmic or membrane transport, and location signals. Non-limiting examples include an internal ribosome entry site (IRES).

[0116]

[0136] The present invention extends to all genetic constructs essentially as described herein, which may further comprise nucleotide sequences intended for the maintenance and / or replication of the genetic construct in eukaryotes and / or for the integration of the genetic construct or portions thereof into the genome of eukaryotic cells.

[0117]

[0137] Methods for studying the introduction (transfection / transduction) of exogenous genetic material, such as the nucleic acid construct of the third aspect of the present invention, into eukaryotic cells are known in the art.As can be understood, the most suitable method for introducing nucleic acid constructs into desired host cells depends on many factors, such as the size of nucleic acid constructs, the type of host cells, the desired transfection / transduction efficiency, and the desired or necessary final viability of transfected / transduced cells.Non-limiting examples of such methods include chemical transfection using chemicals such as cationic polymers, calcium phosphate, or structures such as liposomes and dendrimers; non-chemical methods such as electroporation, sonoporation, heat shock, or optical transfection; particle-based methods such as "gene gun" delivery, magnetofection, or impalefection, or viral transduction.

[0118]

[0138] The nucleic acid construct will be selected depending on the desired transfection / transduction method. In some embodiments of the third aspect of the present 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 using viral transduction to induce CAR expression in PBMCs (Parker, LL. et al., Hum Gene Ther. 2000; 11:2377-87), and more generally, methods using retroviral systems for transduction of mammalian cells (Cepko, C. and Pear, W. Curr Protoc Mol Biol. 2001, unit 9.9), are known in the art. In other embodiments, the nucleic acid construct is a plasmid, cosmid, artificial chromosome, etc., and can be transfected into cells by any suitable method known in the art.

[0119]

[0139] In a fourth aspect, the present invention provides a genetically modified cell comprising a chimeric antigen receptor according to the first aspect of the invention.

[0140] In some embodiments of the fourth aspect of the invention, the genetically modified cells comprise two or more different CARs.

[0120]

[0141] In a fifth aspect, the present invention provides a genetically modified cell comprising a nucleic acid molecule according to the second aspect of the invention, or a nucleic acid construct according to the third aspect of the invention, or a genomically integrated form of the nucleic acid construct.

[0121]

[0142] In some embodiments of the fifth aspect of the invention, the genetically modified cell comprises a nucleic acid molecule or nucleic acid construct encoding two or more different CARs. In some embodiments of the fifth aspect of the invention, the genetically modified cell comprises two or more nucleic acid molecules or two or more nucleic acid constructs, each encoding a different CAR.

[0122]

[0143] As referred to herein, "genetically modified cells" include those encompassed by the present invention. The term "nucleic acid molecule" includes any cell that contains a non-naturally occurring and / or introduced nucleic acid molecule or nucleic acid construct. The introduced nucleic acid molecule or nucleic acid construct can be maintained in the cell as a separate DNA molecule or can be integrated into the genomic DNA of the cell.

[0123]

[0144] The genomic DNA of a cell should be understood in a broad sense to include any endogenous DNA that makes up the genetic complement of the cell. Thus, the genomic DNA of a cell should be understood to include chromosomes, mitochondrial DNA, etc. Thus, the term "genomic integration" contemplates chromosomal integration, mitochondrial DNA integration, etc. The "genomically integrated form" of a construct may be all or part of the construct. However, in some embodiments, the genomic integrated form of a construct comprises at least the nucleic acid molecule of the second aspect of the present invention.

[0124]

[0145] As used herein, the term "different CARs" or "different chimeric antigen receptors" refers to any two or more CARs that have either non-identical antigen recognition domains and / or non-identical signaling domains. In one example, "different CARs" includes two CARs that have the same antigen recognition domain (e.g., both CARs can recognize dysfunctional P2X7 receptors), but have different signaling domains, such as one CAR has a signaling domain that includes a portion of an activating receptor, and the other CAR has a signaling domain that includes a portion of a costimulatory receptor. As will be understood, at least one of the two or more CARs in this embodiment has an antigen recognition domain that recognizes dysfunctional P2X7 receptors, and the other CARs can take any suitable form and be directed to any suitable antigen.

[0125]

[0146] Thus, in some embodiments of the fourth and fifth aspects of the invention, the two or more different CARs have different signaling domains and may have the same or different antigen recognition domains. Specifically, a genetically modified cell according to the fourth or fifth aspect of the invention may comprise a first chimeric antigen receptor having a signaling domain comprising a portion derived from an activating receptor, and a second chimeric antigen receptor having a signaling domain comprising a portion derived from a costimulatory receptor.

[0126]

[0147] In some embodiments of the fourth or fifth aspect of the invention, the activating receptor (from which a portion of the signaling domain is derived) is a CD3 co-receptor complex or an Fc receptor.

[0127]

[0148] In some embodiments of the fourth or fifth aspect of the invention, the costimulatory receptor (from which a portion of the signaling moiety is derived) is selected from the group consisting of CD27, CD28, CD-30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS.

[0128]

[0149] In some embodiments of the fourth or fifth aspect of the invention, the costimulatory receptor (from which a portion of the signaling moiety is derived) is selected from the group consisting of CD28, OX40, or 4-1BB.

[0129]

[0150] In some embodiments of the fourth and fifth aspects of the invention, the genetically modified cells are further modified to constitutively express a costimulatory receptor.

[0151] As mentioned above, a cellular immune response is typically induced only when an activating signal (typically in response to an antigen) and a costimulatory signal are simultaneously experienced. Thus, the above-described embodiments, including two or more CARs, in which two or more CARs combine to provide both an intracellular activating signal and an intracellular costimulatory signal, can be used. By having genetically modified cells with a portion of the CAR signaling domain, it is ensured that a sufficient immune response can be induced in response to the recognition of alloantigen by CAR. Alternatively, the genetically modified cells can only contain one CAR with an antigen recognition domain that recognizes dysfunctional P2X7 receptor, and constitutively express a costimulatory receptor, thereby increasing the possibility that costimulation will occur simultaneously when the CAR is activated. Alternatively, the genetically modified cells can be further modified to constitutively express both the costimulatory receptor and its ligand. In this case, the cells will continuously receive costimulation, and only the activation of the CAR with a signaling domain that includes a portion derived from the activating receptor is required for the immune activation of the cells.

[0130]

[0152] Thus, in some embodiments of the fourth or fifth aspect of the present invention, the genetically modified cells are further modified to constitutively express a costimulatory receptor. In a further embodiment, the genetically modified cells are further modified to express a ligand of the costimulatory receptor, thereby promoting self-stimulation of the cells. Examples of CAR-expressing T cells that express both costimulatory receptors and their cognate ligands (so as to induce self-stimulation) are known in the art, and are described, inter alia, in Stephen MT. et al., Nat. Med, 2007;13:1440-9.

[0131]

[0153] The ability of genetically modified cells containing CAR can be enhanced by further modifying the cells to secrete cytokines, preferably pro-inflammatory or pro-proliferative cytokines. This cytokine secretion both provides autocrine support for the cells expressing CAR and changes the local environment surrounding the CAR-expressing cells so that other cells of the immune system are recruited and activated. As a result, in some embodiments of the fourth or fifth aspect of the present invention, the genetically modified cells are further modified to secrete cytokines. This secretion can be constitutive or can be inducible when the CAR recognizes its cognate ligand antigen.

[0132]

[0154] Although any one or more cytokines may be selected depending on the desired immune response, preferred cytokines include IL-2, IL-7, IL-12, IL-15, IL-17, and IL-21, or combinations thereof.

[0133]

[0155] The genetically modified cells of the fourth or fifth aspects of the invention can be any suitable immune cell, or can be a homogeneous or heterogeneous cell population. In some embodiments, the cells are leukocytes, peripheral blood mononuclear cells (PBMCs), lymphocytes, T cells, CD4+ T cells, CD8+ T cells, natural killer cells, or natural killer T cells.

[0134]

[0156] In a sixth aspect, the present invention provides a method for killing cells expressing a dysfunctional P2X7 receptor, comprising exposing the cells expressing the dysfunctional P2X7 receptor to genetically modified cells having a chimeric antigen receptor, wherein the chimeric antigen receptor is directed against the dysfunctional P2X7 receptor.

[0135]

[0157] Thus, in some embodiments of the sixth aspect of the invention, the CAR directly recognizes the dysfunctional P2X7 receptor, hi other embodiments, the CAR indirectly recognizes the dysfunctional P2X7 receptor.

[0136]

[0158] As used herein, the term "directly recognize" refers to the antigen recognition domain of the CAR directly binding to the dysfunctional P2X7 receptor or its epitope when it is present in its natural form. In another non-limiting example, antigen recognition The recognition domain may bind directly to a processed form of the dysfunctional P2X7 receptor that can be presented by antigen-presenting molecules such as the major histocompatibility complex (MHC).

[0137]

[0159] As an alternative to direct recognition of cells with dysfunctional P2X7 receptors by the CAR, the CAR may be directed to cells with dysfunctional P2X7 receptors by indirect means.

[0138]

[0160] As a result, in some embodiments of the sixth aspect of the present invention, chimeric antigen receptor recognizes dysfunctional P2X7 receptor through an intermediary. The intermediary can be a molecule such as a probe that directly binds to or interacts with dysfunctional P2X7 receptor. Non-limiting examples of such probes include antibody, antibody Fab, scFv, soluble engineered TCR, or aptamer. CAR can directly recognize the probe, or the probe can have a tag that is recognized by CAR. In either case, the probe provides specificity to target cells (i.e., cells with dysfunctional P2X7 receptor), while the genetically modified cells with CAR provide efficacy and direct immune response to target cells. Alternatively, the intermediary can be a cell-intrinsic marker that is associated with dysfunctional P2X7 receptor or whose expression correlates with dysfunctional P2X7 receptor. The abnormal regulation of the marker can be the result or the cause of dysfunctional P2X7 receptor.

[0139]

[0161] In some embodiments of the sixth aspect of the invention, the method of killing cells having dysfunctional P2X7 receptors further comprises the step of exposing the cells having dysfunctional P2X7 receptors to an intermediary.

[0140]

[0162] In some embodiments of the sixth aspect of the present invention, the intermediary is a probe that binds to the dysfunctional P2X7 receptor, and the chimeric antigen receptor recognizes the probe. Preferably, the probe is an antibody or an aptamer.

[0141]

[0163] The term "aptamer" as used throughout this specification refers to any oligonucleic acid, polynucleic acid, peptide, or polypeptide that specifically binds to or preferentially complexes with a target (specifically, a dysfunctional P2X7 receptor).

[0142]

[0164] In some embodiments of the sixth aspect of the present invention, the probe comprises a tag and the chimeric antigen receptor recognizes the tag. Examples of CARs that recognize cells using an intermediary are known in the art, for example, European Patent Application No. 2651442.

[0143]

[0165] In some embodiments of the sixth aspect of the present invention, the cell with dysfunctional P2X7 receptor is in the body of an object.In some embodiments, the object is human.In some embodiments, the method further comprises exposing the cell that expresses dysfunctional P2X7 receptor to gene modification together with exogenous cytokine.

[0144]

[0166] In some embodiments of the sixth aspect of the present invention, the genetically modified cells are genetically modified cells that are autologous to the cells expressing the dysfunctional P2X7 receptor derived from the subject.

[0145]

[0167] In some embodiments of the sixth aspect of the present invention, the cell that expresses dysfunctional P2X7 receptor is in the body of a subject.In some embodiments of the sixth aspect of the present invention, the cell that expresses dysfunctional P2X7 receptor is a cancer cell.

[0146]

[0168] In some embodiments of the sixth aspect, the present invention provides a method for treating or preventing cancer in a subject. provides a method for preventing the disease, the method comprising providing to a subject genetically modified cells having a chimeric antigen receptor, wherein the chimeric antigen receptor is directed to target cells having a dysfunctional P2X7 receptor.

[0147]

[0169] The terms "treat," "treating," or "treatment," as used herein, should be understood to include within their scope one or more of the following results: (i) inhibiting to some extent the growth of a primary tumor in a subject (including delaying and completely halting growth, including reducing the growth of a primary tumor after resection), (ii) inhibiting to some extent the growth and formation of one or more secondary tumors in a subject, (iii) reducing the number of tumor cells in a subject, (iv) reducing the size of a tumor in a subject, (v) inhibiting (i.e., reducing, delaying, or completely halting) tumor cell invasion into peripheral organs, (vi) inhibiting (i.e., reducing, delaying, or completely halting) metastasis, (vii) improving the subject's life expectancy compared to an untreated condition, (viii) improving the subject's quality of life compared to an untreated condition, (ix) alleviating, attenuating, or relieving at least one symptom of cancer in a subject, (x) causing regression or remission of cancer in a subject, (xi) alleviating a condition in a subject caused by cancer, and (xii) halting symptoms associated with cancer in a subject.

[0148]

[0170] The terms "prevent" or "preventing," as used herein, should be understood to include within its scope inhibiting the formation of a primary tumor in a subject, inhibiting the formation of one or more secondary tumors in a subject, or reducing or eliminating the recurrence of cancer in a subject in remission.

[0149]

[0171] The term "inhibiting," as used herein, is intended to mean a decrease or reduction in the growth of a cancer, cancerous cell, or tumor as compared to growth in a control, such as an untreated cell or subject. In some embodiments, growth may be decreased or reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% as compared to an untreated control.

[0150]

[0172] The inhibition of the growth of cancer, tumor or cancerous cell can be evaluated by a wide range of methods known in the art.For example, for cancerous cells in vitro, the growth of cell can be determined by suitable proliferation assay or by the method of evaluating the degree of tritiated thymidine incorporation into cellular DNA over a given period.For tumor cells or cancerous cells existing in vivo, the growth of tumor or cell can be determined by suitable imaging methods known in the art, for example.

[0151]

[0173] The term " subject " as used herein can refer to any animal that can suffer from cancer.Particularly, the subject of interest is human beings, and scientifically relevant species such as mice, rats, ferrets, guinea pigs, hamsters, non-human primates, dogs, pigs and sheep, or economically relevant animals such as horses, dogs, cats and cattle.In the preferred embodiment of the sixth aspect of the present invention, the subject is human beings.

[0152]

[0174] References to "providing to a subject" relate to administering genetically modified cells to a subject. Alternatively, genetically modified cells can be generated within a subject. For example, genetically modified cells can be generated in vivo such that a subject has an endogenous population of genetically modified cells. Suitable means for such in vivo generation are known in the art and include gene therapy of a subject.

[0153]

[0175] As used throughout this specification, a target having a dysfunctional P2X7 receptor is referred to as " The reference to "directed" CAR intends to selectively target immune response to a certain cell based on the cell that has dysfunctional P2X7 receptor.Importantly, this targeting is not limited to the direct recognition of dysfunctional P2X7 receptor by CAR.That is, CAR itself does not need to directly recognize or bind to dysfunctional P2X7 receptor, but simply needs to be able to selectively recognize and be activated by the cell that expresses dysfunctional P2X7 receptor.

[0154]

[0176] Thus, in some embodiments of the sixth aspect of the invention, the CAR directly recognizes the dysfunctional P2X7 receptor, hi other embodiments, the CAR indirectly recognizes the dysfunctional P2X7 receptor.

[0155]

[0177] As used herein, the term " directly recognize " includes that when dysfunctional P2X7 receptor or its epitope exists in its natural form, the antigen recognition domain of CAR directly binds thereto.In another non-limiting example, antigen recognition domain may directly bind to the processed form of dysfunctional P2X7 receptor, which can be presented by antigen-presenting molecules such as major histocompatibility complex (MHC).

[0156]

[0178] As an alternative to direct recognition of cells with dysfunctional P2X7 receptors by the CAR, the CAR may be directed to target cells with dysfunctional P2X7 receptors by indirect means.

[0157]

[0179] As a result, in some embodiments of the sixth aspect of the present invention, chimeric antigen receptor recognizes dysfunctional P2X7 receptor through an intermediary. The intermediary can be a molecule such as a probe that directly binds to or interacts with dysfunctional P2X7 receptor. Non-limiting examples of such probes include antibody, antibody Fab, scFv, soluble engineered TCR, or aptamer. CAR can directly recognize the probe, or the probe can have a tag that is recognized by CAR. In either case, the probe provides specificity to target cells (i.e., cells with dysfunctional P2X7 receptor), while the genetically modified cells with CAR provide efficacy and direct immune response to target cells. Alternatively, the intermediary can be a cell-intrinsic marker that is associated with dysfunctional P2X7 receptor or whose expression correlates with dysfunctional P2X7 receptor. The abnormal regulation of the marker can be the result or the cause of dysfunctional P2X7 receptor.

[0158]

[0180] In some embodiments of the sixth aspect of the invention, the method of treating or preventing cancer in a subject further comprises providing an intermediary to the subject.

[0181] In some embodiments of the sixth aspect of the present invention, the intermediary is a probe that binds to the dysfunctional P2X7 receptor, and the chimeric antigen receptor recognizes the probe. Preferably, the probe is an antibody or an aptamer.

[0159]

[0182] The term "aptamer" as used throughout this specification refers to any oligonucleic acid, polynucleic acid, peptide, or polypeptide that specifically binds to or preferentially complexes with a target (specifically, a dysfunctional P2X7 receptor).

[0160]

[0183] In some embodiments of the sixth aspect of the present invention, the probe comprises a tag and the chimeric antigen receptor recognizes the tag. Examples of CARs that recognize cells using an intermediary are known in the art, for example, European Patent Application No. 2651442.

[0161]

[0184] In a seventh aspect, the present invention provides a method of treating or preventing cancer in a subject, comprising administering to the subject genetically modified cells according to the fourth or fifth aspect of the invention. A method is provided, comprising the steps of:

[0162]

[0185] Although providing genetically modified cells that express CAR and have dysfunctional P2X7 receptor target cells can be sufficient to provide effective immunotherapy for precancerous or cancerous cells, by providing adjuvant together with genetically modified cells, the induction of immune response can be further enhanced, and immunotherapy can be enhanced.Cytokine, preferably pro-inflammatory cytokine, is the adjuvant that is particularly suitable for providing to subject with genetically modified cells that have CAR.

[0163]

[0186] Therefore, in some embodiments of the sixth and seventh aspects of the present invention, genetically modified cells are administered to a subject together with cytokines.As used throughout this specification, the term "together" should be understood to include that genetically modified cells are administered simultaneously with cytokines or in combination with cytokines.As a result, when administered together with cytokines, this can be considered to include combination therapy, and in this case, the immunotherapy of the subject includes both treatment with cytokines and treatment with genetically modified cells having CAR directed to target cells that express dysfunctional P2X7 receptors.In some forms, cytokines are administered on a different day (more than 24 hours) from the administration of genetically modified cells.In other forms, cytokines are administered on the same day (within 24 hours) as genetically modified cells.In further forms, 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.

[0164]

[0187] 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-2 and IFNα. Furthermore, cytokines can be administered in recombinant form, in natural form, or via delivery systems such as fusions with proteins that are expressed in genetically modified cells or delivered as nucleic acid sequences conjugated with polymers such as polyethylene glycol (PEG).

[0165]

[0188] The cell to be genetically modified can be obtained from any suitable source.In some embodiments of the sixth or seventh aspect of the present invention, the cell to be genetically modified is autologous cell, which is the cell that is autologous to the cell that expresses dysfunctional P2X7 receptor.Advantageously, autologous cell is not recognized as "non-self" by the immune system of the subject, and therefore is expected to be tolerated by the subject.However, in some forms of cancer, suitable autologous cell may not be easily available.Therefore, in some embodiments of the present invention, the cell to be genetically modified is allogeneic cell or xenogeneic cell.

[0166]

[0189] P2X7 dysfunction is a common molecular alteration in various cancers.As a result, the method of the sixth or seventh aspect of the present invention can be used for the prevention and treatment of various cancers.

[0167]

[0190] In some embodiments of the sixth or seventh aspect of the invention, the method is used to prevent or treat a cancer selected from one or more of 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 cancer, skin cancer, leukemia, lymphoma, myeloma, breast cancer, ovarian cancer, endometrial cancer, testicular cancer. Preferably, the cancer is lung cancer, Selected from one or more of esophageal cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer, cervical cancer, vaginal cancer, epithelial cell cancer, skin cancer, blood-related cancer, breast cancer, endometrial cancer, uterine cancer, and testicular cancer.

[0168]

[0191] In some embodiments of the sixth or seventh aspect of the invention, the cancer is a metastatic cancer, for example a stage III or stage IV cancer.

[0192] When producing genetically modified cells according to the fourth or fifth aspect of the present invention, it may be desirable to expand the cell population in vitro to increase the total number of cells available for therapy. This can be done using a step of exposing the cells to an antigen of the CAR. Thus, in an eighth aspect, the present invention provides a method of expanding genetically modified cells according to the fourth or fifth aspect of the present invention in vitro, comprising a step of exposing the cells to an antigen of the CAR. In some embodiments, the method comprises the further step of exposing the cells to a cytokine.

[0169]

[0193] In a ninth aspect, the present invention provides a method of expanding in vitro a genetically modified cell according to the fourth or fifth aspect of the invention, comprising exposing the cell to an antigen of the CAR and simultaneously exposing the cell to a cytokine.

[0170]

[0194] Preferred cytokines for use in the eighth or ninth aspects of the invention may include members of the IL-2 subfamily, the interferon subfamily, the IL-10 subfamily, the IL-1 subfamily, the IL-17 subfamily, or the TGF-β subfamily. In some embodiments of the eighth or ninth aspects of the invention, the cytokine is selected from the group consisting of IFN-γ, IL-2, IL-5, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, TNF-α, TGF-β1, TGF-β2, TGF-β3, and GM-CSF, or a combination thereof.

[0171]

[0195] In a tenth aspect, the present invention provides a method of expanding genetically modified cells according to the fourth or fifth aspects of the invention in vitro, comprising exposing the cells to immobilized anti-CD3 and anti-CD28 antibodies. In some embodiments of the tenth aspect of the invention, the antibodies are immobilized on a bead substrate (e.g., "human activator" Dynabeads™). In some embodiments of the tenth aspect of the invention, the antibodies are immobilized on an alternative surface, such as the surface of a tissue culture vessel, culture flask, plate, or bioreactor.

[0172]

[0196] As understood by those skilled in the art, depending on the signal transduction domain of CAR, the recognition of its cognate antigen by CAR causes intracellular signal transduction, which can ultimately cause cell proliferation.Therefore, a small number of cells, or even individual cells, can proliferate (or in the case of single cells, undergo clonally expanding) to form therapeutically significant numbers.This process can be further enhanced by providing cytokines.

[0173]

[0197] The delivery or administration of genetically modified cells according to the fourth or fifth aspect of the invention may be the delivery or administration of the cells alone, or the delivery or administration of the cells formulated in a suitable pharmaceutical composition. Thus, in an eleventh aspect, the present invention provides a pharmaceutical composition comprising genetically modified cells according to the fourth or fifth aspect of the invention and a pharmaceutically acceptable carrier.

[0174]

[0198] Methods for providing cells containing a CAR for immunotherapy are known in the art (e.g., Kershaw, M.H. et al., Clin Cancer Res. 2013). 06;12(20):6106-15; Parker LL. et al., Hum Gene Ther 2000;11:2337-87.) Additionally, protocols and methods for the preparation, growth, and evaluation of mammalian CAR-expressing cells are known in the art (e.g., Cheadle, EJ. et al., Antibody Engineering: Methods and Protocols, Second Edition, Methods in Molecular Biology, vol. 907:645-66) and are outlined in the Examples below.

[0175]

[0199] The pharmaceutical composition may also contain one or more pharmaceutically acceptable additives, including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients, and bulking agents, taking into account the specific physical and chemical characteristics of the cells to be administered. In some embodiments, the pharmaceutical composition comprises a suspension of genetically modified cells according to the fourth or fifth aspect of the present invention in a suitable medium, such as isotonic saline. In some embodiments, the pharmaceutical composition may contain a suitable adjuvant, such as one or more cytokines described above. In some embodiments, the pharmaceutical composition may also contain the mediator described above.

[0176]

[0200] Administration of the pharmaceutical composition may also be via parenteral means, including intravenous, intraventricular, intraperitoneal, intramuscular, or intracranial injection, or local injection at the site of a tumor or cancer mass.

[0177]

[0201] Throughout this specification, unless the context requires otherwise, it is expected that the use of "comprise" or variations such as "comprises" or "comprising" will imply the inclusion of a stated element or integer or group of elements or integers, but not the exclusion of any other element or integer or group of elements or integers.

[0178]

[0202] Finally, reference is made to standard textbooks in molecular biology, including methods for carrying out the basic techniques encompassed by the present invention, e.g., Green MR and Sambrook J, Molecular Cloning: A Laboratory Please refer to the Manual (4th ed.), Cold Spring Harbor Laboratory Press, 2012.

[0179]

[0203] Although the present invention has been described in some detail for purposes of clarity and understanding, it is expected that it will be apparent to those skilled in the art that various modifications and alterations to the embodiments and methods described herein can be made without departing from the scope of the inventive concepts disclosed herein.

[0180]

[0204] The present invention is further illustrated in the following examples, which are for the purpose of illustrating particular embodiments only and are not intended to be limiting to the foregoing description. [Example]

[0181] Example 1 Protocol for the design and expression of PEP2-2-3-binding peptide chimeric antigen receptor (CAR)

[0205] Exemplary protocols detailing the process of designing and expressing an anti-nonfunctional (nf) P2X7 receptor CAR according to embodiments of the present invention are detailed below.

[0182] Design of PEP2-2-3 (anti-nf P2x7) chimeric antigen receptor

[0206] An anti-nfP2x7 chimeric antigen receptor (CAR) was designed according to the schematic diagram illustrated in Figure 1.

[0183]

[0207] An antigen recognition domain 1 of a CAR was generated that contains the amino acid sequence of a PEP2-2-3 binding peptide (amino acid sequence set forth in SEQ ID NO: 10, nucleotide sequence set forth in SEQ ID NO: 11). The PEP2-2-3 sequence was shown to have specific affinity for dysfunctional P2X7 receptors expressed on cancer cells, such as prostate LNCap cells, without significant affinity for monocytes or lymphocytes.

[0184]

[0208] CD8a signaling peptide 2 (having the amino acid sequence set forth in SEQ ID NO: 12 and the nucleotide sequence set forth in SEQ ID NO: 13) was linked to the N-terminus of PEP2-2-3 antigen recognition domain 1. CD8a signaling peptide 2 contains a Kozak consensus sequence at positions 1 to 13 of SEQ ID NO: 13. CD8a signaling peptide 2, including the Kozak sequence, acts to facilitate ribosomal recognition of the transcribed RNA and provides a translation initiation site, thereby facilitating translation of the CAR transcribed RNA sequence into protein.

[0185]

[0209] The antigen recognition domain 1 of CAR is linked to the transmembrane domain 3 via one of two hinge regions, designated as long hinge 4 and short hinge 5. Providing long hinge 4 may allow the mobility of the antigen recognition domain, which may be required for the antigen recognition domain to interact with its cognate ligand (dysfunctional P2X7). The amino acid sequence and nucleotide sequence of long hinge 4 are set forth in SEQ ID NO: 14 and SEQ ID NO: 15, respectively. The amino acid sequence and nucleotide sequence of short hinge 5 are set forth in SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0186]

[0210] Transmembrane domain 3 and a portion of intracellular domain 6 of the CAR are provided by a portion 7 of the CD28 costimulatory receptor (amino acid sequence set forth in SEQ ID NO: 18 and nucleotide sequence set forth in SEQ ID NO: 19). The intracellular domain further comprises a portion 8 of the costimulatory receptor OX40 (amino acid sequence set forth in SEQ ID NO: 20 and nucleotide sequence set forth in SEQ ID NO: 21), and a portion 9 of the activating receptor CD3 zeta (amino acid sequence set forth in SEQ ID NO: 22 and nucleotide sequence set forth in SEQ ID NO: 23).

[0187]

[0211] P2A sequence 10 (amino acid sequence set forth in SEQ ID NO: 24 and nucleotide sequence set forth in SEQ ID NO: 25) was added to the C-terminus of CAR to allow post-translational excision of any peptide sequence added to the C-terminus of CAR. The amino acid sequences of the constructed anti-nfP2X7CAR-long hinge and anti-nfP2X7CAR-short hinge are set forth in SEQ ID NOs: 26 and 27, respectively.

[0188] Lentiviral vector design and assembly

[0212] The designed CAR was incorporated into the BLIV lentiviral plasmid (System Biosciences, California, USA), which contains the fluorescent and bioluminescent reporter proteins green fluorescent protein (GFP) and firefly luciferase (FLuc), as illustrated in Figure 2. The BLIV plasmid further contains a T2A coding sequence between the coding sequences for the GFP and FLuc reporter proteins, allowing post-translational separation of the FLuc and GFP proteins.

[0189]

[0213] Sequences with homology to the sequences upstream and downstream of the NheI restriction site of the BLIV vector were added to the 5' and 3' ends of the designed CAR to form SEQ ID NO:2. The final nucleotide sequences were obtained as set forth in SEQ ID NO:8 (CAR-long hinge) and SEQ ID NO:29 (CAR-short hinge). Inclusion of the 5' and 3' sequences allowed for the incorporation of the anti-nf P2X7CAR into a BLIV vector using Gibson cloning.

[0190]

[0214] The nucleotide sequences of anti-nf P2X7CAR-long hinge and anti-nf P2X7CAR-short hinge were constructed using gene block technology (gBlock™ Gene Fragments - Integrated DNA Technologies, Iowa, USA) and assembled using a Gibson Assembly Cloning Kit (New England Biolabs inc. Ipswich MA, USA - Catalog No. E5510S) according to the manufacturer's instructions.

[0191]

[0215] The BLIV plasmid was restricted at the NheI cloning site and the anti-nf P2X7CAR coding sequence was incorporated using Gibson assembly. Cloning and evaluation of BLIV-CAR vectors

[0216] The resulting BLIV-CAR vector was transformed into New England Biolabs 5-alpha competent E. coli cells (provided in the Gibson Assembly Cloning Kit) according to the manufacturer's instructions. - A tube of NEB 5-alpha competent E. coli cells was thawed on ice for 10 minutes. - 1-5 μl containing 1 pg-100 ng of BLIV-CAR plasmid DNA was added to the cell mixture and mixed by rocking the tube side to side 4-5 times. - The mixture of E. coli and plasmid was left on ice for 30 minutes without mixing. - The mixture of cells and plasmid was heat shocked at 42°C for 30 seconds and then left on ice for 5 minutes without mixing. - 950 μl of SOC was added to the mixture, which was then heated to 37°C for 60 minutes and shaken vigorously. - Selection plates were prepared and heated to 37°C. - Ten-fold serial dilutions of cells were prepared in SOC solution. - 50-100 μl of each dilution was plated onto selective plates and incubated overnight at 37°C.

[0192]

[0217] After incubating the transformed E. coli cells, 10 bacterial colonies transformed with the BLIV-CAR-short hinge plasmid and 10 bacterial colonies transformed with the BLIV-CAR-long hinge plasmid were isolated, and the plasmid DNA was purified and digested with BamHI. The digested DNA was analyzed by gel electrophoresis for the appropriate size restriction fragments. As shown in Figure 3, colonies 2 through 9 of the bacterial clones transformed with the BLIV-CAR-long hinge plasmid contained the appropriate size restriction fragments (7.8 kb and 2.8 kb), whereas only colony 4 of the bacterial clones transformed with the BLIV-CAR-short hinge plasmid yielded the appropriate size restriction fragments (7.4 kb and 2.8 kb).

[0193]

[0218] Bacterial clones 2 to 4 (L2 to L4) containing the BLIV-CAR-long hinge plasmid and bacterial clone 4 (S4) containing the BLIV-CAR-short hinge plasmid were selected for further confirmation of plasmid identity using the restriction enzymes EcoRI, BamHI, and PstI. All colonies displayed restriction fragments of the expected lengths, as described in Table 4 and Figure 4.

[0194] [Table 4]

[0195] Lentiviral vector construction and validation

[0219] Lentivirus was packaged using a three-plasmid protocol using 293T cells according to the following method. Day 1: 293T cells were seeded in 35 ml of DMEM medium with 10% serum in a T-225 flask so that the cells would be 90-95% confluent the next day. Day 2: 30 μg of either the resulting BLIV-CAR plasmid (or unmodified BLIV plasmid), 30 μg of gag-pol plasmid Delta8.2, and 15 μg of VSV-G plasmid (pMD2.G) were added to OptiMEM medium to a final volume of 750 μl and mixed. 300 μl of PEI solution was added and incubated at room temperature for at least 20 minutes. The mixture was then added to confluent 293T cells and incubated at 37°C. Day 3: 24 hours after adding the plasmid mixture, the supernatant was decanted from the 293T cells and stored at 4° C. The decanted mixture was replaced with 35 ml of fresh medium and then further incubated at 37° C. Day 4: 48 hours after addition of the plasmid mixture, the medium was removed and combined with the supernatant harvested at 24 hours. The combined supernatant was spun at 1500g for 15 minutes to remove any remaining cellular debris. The supernatant was filtered through a 0.45um filter and then spun at 17,000rpm in a WX ultracentrifuge for 1 hour. After centrifugation, the supernatant was manually decanted, leaving 50-200ul in the tube. The centrifuge tube was placed into a 50ml screw-cap tube to prevent contamination and evaporation, and the virus was resuspended overnight at 4°C. Day 5: The virus was resuspended from the bottom of the centrifuge tube and transferred to a new 1.5 ml tube. The resuspended virus was spun in a microcentrifuge tube at 5000 rpm for 5 minutes to remove any remaining debris.

[0196]

[0220] Transfection of BLIV-CAR-short hinge and BLIV-CAR-long hinge vectors into 293T cells was assessed after 24 hours of incubation in the presence of GFP fluorophore (see Figures 5A and 6A). Supernatants collected on day 5 (as described above) containing short-hinge and long-hinge BLIV-CAR lentiviral vectors were incubated with fresh 293T cells and visualized for GFP fluorescence to test for transduction capacity (see Figures 5B and 6B).

[0197] Screening for CAR T cell function

[0221] 10 8 CD8 T cells were isolated from 50 ml of human blood using the RosetteSep™ Human CD8+ T Cell Isolation Kit (Stemcell technologies, Vancouver, Canada) according to the manufacturer's instructions. Purity analysis showed that 76.6% of the purified cells were CD8+, as depicted in Figure 7.

[0198]

[0222] CD8+ T cells were cultured at 10 per well. 5 CD8+ T cells were incubated with T cell proliferation (CD3 / CD28) beads at a 1:1 ratio. CD8+ cells were then incubated overnight with lentiviral preparations containing either unmodified BLIV plasmid, BLIV-CAR-short hinge plasmid, or BLIV-CAR-long hinge plasmid at a multiplicity of infection (MOI) of 5 or higher. After incubation, CD8+ T cells were washed and then co-cultured with target cells.

[0199]

[0223] Target cells expressing non-functional P2X7 receptors were obtained from the mammalian cancer cell line BT549 (ATCC HTB-122). These cells were dye-labeled using the fluorescent membrane-intercalating dye eFluor™ 670 (affymetrix eBioscience) according to the manufacturer's instructions. - BT549 cells were prepared as a single cell suspension and washed twice in PBS to remove any residual serum. - Cells were resuspended in room temperature PBS. - A 10 μM solution of the cell proliferation dye eFluor® 670 was prepared in room temperature PBS. An equal volume of 10 μM dye solution was added to the prepared BT549 cells to obtain a final concentration of 5 μM dye solution. - BT549 cells in the dye solution were incubated in the dark at 37°C for 10 minutes, after which labeling was stopped by adding 4 volumes of cold culture medium containing 10% serum, followed by incubation on ice in the dark for 5 minutes. - Finally, the cells were washed three times in culture medium and then resuspended in culture medium at the desired concentration.

[0200]

[0224] After dye labeling, the target cells were co-cultured with prepared CD8+ T cells at ratios of 10:1, 5:1, 1:1, and 0:1 (T cell:target).

[0225] After 24 hours of co-culture, cells were harvested and analyzed using fluorescence-activated cell sorting (FACS). The number of target cells containing membrane-intercalating dye was quantified to assess whether the co-cultured T cells resulted in target cell killing or cessation of cell proliferation. The gating and analysis strategy used to quantify the effectiveness of CD8+ T cells in killing target cells is illustrated in Figure 8 and quantified in Figure 9. Figure 8A illustrates the gating and histogram analysis of labeled CD8+ T cells. Figure 8B illustrates the gating and histogram analysis of labeled BT549 target cells. Figure 8C illustrates the gating and histogram analysis of control CD8+ T cells and BT549 targets after 24 hours of co-culture. Figure 8D illustrates the gating and histogram analysis of BLIV-CAR-long hinge-transduced CD8+ T cells and BT549 target cells after 24 hours of co-culture. Figure 8E illustrates gating and histogram analysis after 24 hours of co-culture of BLIC-CAR-short hinge-transduced CD8+ T cells and BT549 target cells.

[0201]

[0226] As can be seen in Figure 9, when target cells were co-cultured with CD8 T cells transduced with lentivirus containing either BLIV-CAR-long hinge or BLIV-CAR-short hinge, target cells were shaped more efficiently than non-transduced cells or controls. There was an increase in the number of BT549 target cells eliminated (killed) compared to when co-cultured with transduced (unmodified BLIV vector) CD8 T cells.

[0202]

[0227] Considering the results presented in Figure 9, it is clear that CD8+ T cells transduced with anti-nfP2X7 CAR receptors (with short or long hinge) exhibit increased levels of cytotoxic activity against non-functional P2X7-expressing target cells, indicating the ability of CAR-T cells to kill cancer cell targets.

[0203] Example 2 Design of alternative anti-nfP2X7 chimeric antigen receptors

[0228] Further exemplary protocols detailing the process of designing anti-nonfunctional (nf) P2X7 receptor CARs and expressing them in T cells according to embodiments of the present invention are detailed below.

[0204]

[0229] Three anti-nonfunctional P2X7 binding peptides were used to design anti-nfP2X7 CARs. Specifically, the CARs were designed to contain antigen recognition domains with sequence homology to peptides PEP2-2-1-1, PEP2-472-2, or PEP2-2-12 (having the amino acid sequences set forth in SEQ ID NOs: 32, 33, and 34, respectively). These binding peptides have been shown to bind to nonfunctional P2X7 receptors (Barden, JA, Sluyter, R., Gu, BJ & Wiley, JS 2003. Specific detection of non-functional P2X7 receptors). human P2X(7) receptors in HEK293 cells and B-lymphocytes.FEBS Lett 538,159-162).

[0205]

[0230] The alignment of the above-mentioned binding peptide with the heavy chain variable region of an antibody that recognizes a non-functional P2X7 receptor is shown in Figure 10. The alignment of the sequences of the complementarity determining regions (CDR1 to 3) is shown in boxes.

[0206]

[0231] A specific example of the construction of a CAR with the PEP2-2-1-1 sequence is detailed below: The same CAR structure and sequence was used for CARs with the PEP2-472-2 sequence or the PEP2-2-12 sequence as the binding peptide, instead of PEP2-2-1-1 as an alternative binding peptide.

[0207]

[0232] A DNA sequence encoding the PEP2-2-1-1 binding peptide was synthesized in frame with other DNA sequences to generate a CAR with the structure described below.

[0233] Referring to Figure 11, an antigen recognition domain was prepared by linking leader sequence 11 of Homo sapiens CD8a molecule (CD8A) transcript variant 1 (having the amino acid sequence set forth in SEQ ID NO:30 and the nucleotide sequence set forth in SEQ ID NO:31) to the N-terminus of PEP2-2-1-1 binding peptide 12 (having the amino acid sequence set forth in SEQ ID NO:32 and the nucleotide sequence set forth in SEQ ID NO:35).

[0208]

[0234] The antigen recognition domain was then linked to the transmembrane domain via a modified IgG4 hinge-CH2-CH4 13 having the sequence of the long hinge described in Example 1 above (i.e., the amino acid sequence set forth in SEQ ID NO: 14 and the nucleotide sequence set forth in SEQ ID NO: 15).

[0209]

[0235] The extracellular domain containing the CD8 leader sequence 11 and PEP2-2-1 binding peptide 12 was linked to a transmembrane domain 14 provided by a portion 15 of human CD28 (having the amino acid sequence set forth in SEQ ID NO: 18 and the nucleotide sequence set forth in SEQ ID NO: 19) that also contains a portion 16 of the CD28 cytoplasmic domain.

[0210]

[0236] The intracellular portion 17 of the CAR was provided by linking a portion 14 of the human CD28 molecule described above and the cytoplasmic domain 18 of Homo sapiens tumor necrosis factor receptor superfamily member 4 (TNFRSF4 / OX40—having the amino acid sequence set forth in SEQ ID NO:20 and the nucleotide sequence set forth in SEQ ID NO:21) to the cytoplasmic domain 19 of the Homo sapiens CD247 molecule (T cell surface glycoprotein CD3 zeta chain, having the amino acid sequence set forth in SEQ ID NO:22 and the nucleotide sequence set forth in SEQ ID NO:23).

[0211] Lentiviral vector design and assembly

[0237] The nucleotide sequences of the designed PEP2-2-1-1, PEP2-472-2, and PEP2-2-12 CARs were assembled using gene block technology (gBlock™ Gene Fragments - Integrated DNA Technologies, Iowa, USA) and ligated using the Gibson Assembly Cloning Kit (New The CARs were assembled using a cloning vector (England Biolabs Inc. Ipswich MA, USA - Catalog No. E5510S) according to the manufacturer's instructions. The sequences of the nucleotide constructs of PEP2-2-1-1, PEP2-472-2, or PEP2-2-12 CARs for incorporation into the cloning vector (including restriction sites) are set forth in SEQ ID NOs: 35, 36, and 37, respectively.

[0212]

[0238] The CAR nucleotide construct was incorporated into the pCDH-CMV-MCS-T2A (pCDH) vector (System Biosciences, California, USA, catalog number CD524A-1), which contains the fluorescent reporter protein green fluorescent protein (GFP), as illustrated in Figure 11. The pCDH vector further contains a T2A coding sequence between the cloning site and the GFP, allowing post-translational separation of the cloned CAR and GFP proteins.

[0213]

[0239] To integrate the nucleotide constructs of PEP2-2-12 and PEP2-472-2 CAR into the pCDH vector, the pCDH vector was digested with EcoRI and NotI and gel-purified (QIAquick Gel Extraction Kit, QIAGEN). The PEP2-2-12 and PEP2-472-2 CAR nucleotide gBlock constructs were also digested with EcoRI and NotI digestion enzymes. The digested gBlock fragments were then purified using a QIAquick PCR Purification Kit according to the manufacturer's instructions. The digested vector was ligated to the digested CAR construct at a 3:1 molar insert-to-vector ratio. The ligation mix was transformed into chemically competent SURE2 cells (Agilent).

[0214]

[0240] The PEP2-2-1-1 CAR construct contains an internal EcoR1 restriction site and was therefore incorporated into the pCDH vector in a different manner than the PEP2-2-12 and PEP2-472-2 CAR nucleotide constructs. The pCDH vector was digested with EcoR1, and the resulting 5'-overhang was filled in with T4 DNA polymerase in the presence of 100uM dNTPs (12°C for 15 minutes). The reaction was terminated (75°C for 20 minutes in the presence of 10mM EDTA), and the digested vector was column purified (QIAquick PCR Purification Kit, QIAGEN). The purified vector was then further digested with NotI and gel purified (QIAquick Gel Extraction Kit, QIAGEN). The PEP2-2-1-1 CAR construct fragment was first digested with SmaI, followed by digestion with NotI (both at 25°C). The gBlock fragment was digested with restriction enzymes and then purified using QIAquick The vector was purified using a PCR purification kit according to the manufacturer's instructions. was ligated with the CAR construct at an insert to vector molar ratio of 3:1.

[0215] Cloning and evaluation of pCDH-CAR vector

[0241] The ligation mix of each of the three CAR constructs described above was transformed into chemically competent SURE2 cells (Agilent) according to the manufacturer's instructions. - SURE2 cells were thawed on ice. After thawing, the cells were gently mixed and 100 μl aliquots of cells were placed into pre-chilled 14 ml round-bottom tubes. - 2 μl of β-mercaptoethanol was added to each cell aliquot. - The tubes were mixed and incubated on ice for 10 minutes, swirling gently every 2 minutes. - 0.1 to 50 ng of each pCDH-CAR vector was added to the cell aliquots. The aliquots were mixed gently and then incubated on ice for 30 minutes. - The tubes were heat pulsed at 42°C in a water bath for 30 seconds and then incubated on ice for 2 minutes. - 0.9 ml of pre-warmed (42°C) NZY+ medium was added to each tube, followed by incubation at 37°C for 1 hour with vigorous mixing at 225-250 rpm. - Up to 200 μl of the transformation mixture was plated onto LB agar plates containing antibiotics and then incubated overnight at 37°C. -Colonies were picked and further cultured overnight. - Plasmid DNA was isolated from the cultured clones using the Quicklyse Miniprep Kit (QIAGEN) and digested with EcoRI / Not I digestion to identify clones with the appropriate size of the CAR-pCDH vector.

[0216]

[0242] After incubating the transformed (SURE2) cells, 5–6 colonies of cells transformed with pCDH-CAR were isolated for each of the PEP2-2-1-1, PEP2-472-2, or PEP2-2-12 binding peptides and further incubated overnight. Plasmid DNA was isolated from each cultured colony using the Quicklyse Miniprep Kit (QIAGEN) and digested with EcoRI / NotI restriction enzymes. The digested DNA was analyzed by gel electrophoresis for appropriately sized restriction fragments.

[0217]

[0243] As shown in Figure 13, colony 3 of the PEP2-2-1-1 pCDH-CAR construct, colonies 1 and 3 of the PEP2-472-2 pCDH-CAR construct, and colonies 1, 3, and 5 of the PEP2-2-12 pCDH-CAR construct contained the appropriate size restriction fragment.

[0218]

[0244] Each selected clone was sequenced to confirm CAR integration using appropriate primers selected from Table 5.

[0219] [Table 5]

[0220]

[0245] The sequencing data for each selected colony was aligned with the computer-derived recombinant clones of each of the PEP2-2-1-1, PEP2-472-2, or PEP2-2-12 CAR constructs, and the appropriate construct was verified for at least one of each of the selected colonies. Large-scale endotoxin-removal plasmid isolation of verified clones was performed using the NucleoBond® Xtra Midi EF kit, Macherey-Nagel, according to the manufacturer's instructions.

[0221] Viral vector construction and validation

[0246] Lentiviral packaging was performed in transiently transfected Hek293T cells using Lipofectamine 2000 reagent (Invitrogen) according to standard laboratory protocols (Brown, CY et al., 2010. Robust, reversible gene knockdown using a single lentiviral short hairpin RNA vector. Hum Gene Ther 21, 1005-1017). - 12.5ug of lentiviral vector DNA was mixed with 3.75ug of pMD2.g (VSV-G envelope expression vector), 6.25ug of pRSV-Rev, and 7.5ug of pCMVdelta8.2 per transfection in a T75cm flask with 75ul of Lipofectin according to the manufacturer's protocol and incubated overnight. The next morning, the medium was changed and the virus-containing supernatant was collected after 48 hours. The collected supernatant was centrifuged at 300×g for 5 minutes and then filtered through a 0.45 μm filter. - Viral particles from the filtered supernatant were concentrated by ultracentrifugation (68,000 x g for 90 minutes at 4°C in a Beckman SW32 rotor). The supernatant was removed and the viral pellet was gently resuspended in DMEM on ice. - 100ul virus aliquots were stored at -70°C until required.

[0222]

[0247] To assess viral transfection rates, transfected Hek293T cells were harvested and the percentage of GFP-positive cells (pCDH vector-containing cells) was determined by flow cytometry. Representative results from Hek293T cells transfected with the LV-PEP2-472-2 packaging mix are shown in Figure 14.

[0223]

[0248] Viral titers were calculated by transducing a known number of Hek293T cells with serial dilutions (1:50 and 1:100) of concentrated LV stock. Transduction was performed overnight in the presence of 8 μg / ml of polybrene (hexadimethrine bromide). The following day, the medium containing virus and polybrene was replaced with fresh medium, cells were harvested 24 hours later, and the percentage of GFP-positive cells was determined by flow cytometry. Viral titers were calculated using the formula: transducing units / ml (TU) = (F x C / V) x D, where F = frequency of GFP+ cells (%GFP+ / 100), C = number of cells at the time of virus addition, V = transduction volume in mL, and D = dilution factor. Representative flow data for LV-PEP2-472-2 transduction are shown in Figure 15. The TU for each of the PEP2-2-1-1, PEP2-12-2, and PEP2-472-2 CAR viral vectors is provided in Table 6 below.

[0224] [Table 6]

[0225] Screening for nf-P2X7CAR T cell function Generation of CD8 T cells expressing anti-nf-P2X7CAR

[0249] Human CD8 cells were purified and transduced according to the following method.

[0226]

[0250] Human CD8 T cells were purified from mononuclear cells (MNCs) isolated from Buffy Coats (Australian Red Cross Blood Services) from an anonymous donor. MNCs were isolated using Ficoll-Paque™ density gradient medium. CD8 T cells were purified from MNCs using Dynabeads® Untouched™ Human CD8 T Cell Kit (Invitrogen) according to the manufacturer's instructions. The purity of the isolated cells, assessed by flow cytometry, was 85% or higher.

[0227]

[0251] 2×10 6 Purified cells were preincubated with CD3 / CD28 beads (bead-to-cell ratio 3:1) and IL2 (500 U / ml) for 30 minutes, followed by the addition of 1 to 2 multiplicity of infection (MOI) units of virus-containing LV-PEP2-2-1-1, LV-PEP2-472-2, or empty LV vector (GFP control virus) along with 8 μg / ml polybrene. Cells were incubated with virus for 16 hours, after which the virus-containing medium was removed. The remaining cells and beads were incubated in fresh medium containing IL2 for 40 hours before analyzing GFP fluorescence levels.

[0228]

[0252] As illustrated in Figure 16, GFP+ CD8 cells, indicating successful transduction, were detected within 8 days. % to 43%. Generation of target cells expressing nf-P2X7 receptors or wild-type (WT) P2X7 receptors

[0253] To evaluate the efficacy of CD8 cells expressing anti-nf-P2X7-CAR, Hek293T cells were prepared that overexpressed either a non-functional P2X7 receptor (with the K193A mutation) or the extracellular domain of the wild-type P2X7 receptor on the cell surface.

[0229]

[0254] The gBlock gene fragments of EXD2_K193A (nf-P2X7) and EXD2_WT (functional P2X7) (SEQ ID NOs: 47 and 48, respectively) were ordered from Integrated DNA technologies (IDT). The EXD2 domain was designed to be expressed in frame with a DNA sequence encoding a fusion protein consisting of IgK-leader-HA-MYC-PDGFR-transmembrane domain from pDisplay (Invitrogen, Figure 17). These fusion proteins were designed for surface expression. The gene fragments of EXD2_K193A and EXD2_WT were cloned between the HA and MYC epitope tags to form a fusion gene block. Gateway attB1 and attB2 sequences were included at the 5' and 3' ends of the fusion gene block for cloning into the LV-416-IRES-puro vector (Clontech).

[0230]

[0255] Cloning was performed using Gateway® (ThermoFisher) and all steps were performed according to the manufacturer's protocol. First, an entry clone was generated by BP recombination between the attB flanking DNA fragments (EXD2_K193A, SEQ ID NO: 47, and EXD2_WT, SEQ ID NO: 48) and the attP-containing pDONR-107 vector. The BP recombination reaction was used to transform chemically competent E. cloni® 10G cells (Lucigen®) according to the manufacturer's protocol. - The transformed cells were plated on LB agar plates containing 50ug / ml kanamycin (Sigma) and incubated overnight at 37°C. - Two clones were selected from each plate and microcultures (2 mL) were prepared in LB medium with kanamycin (SIGMA) (50 μg / ml). After overnight incubation at 37°C, agitation was performed. - The next day, plasmid DNA was extracted from the microcultures using a QIAGEN QuickLyse miniprep kit. Diagnostic Bam H1-HF (NEB) and PmeI (NEB) digests were performed to identify recombinant clones. After Bam H1 and Bam H1 / PmeI digestion, both EXD2_K193A and EXD2_WT clones were confirmed to be correctly digested by gel electrophoresis (Figure 18).

[0231]

[0256] One clone from each construct (EXD2_K193A and EXD2_WT) was selected for LR recombination reaction (described below) to insert the EXD2_K193A and EXD2_WT constructs into the destination pLV-416 vector.

[0232]

[0257] After clone selection, a subsequent LR recombination reaction was performed to transfer each EXD2 insert from the pDONR-107 entry clone into the target pLV-416 vector to generate an expression vector. The final LR recombination reaction was used to transform chemically competent E. cloni® 10G cells (Lucigen®) according to the manufacturer's protocol. - The transformed cells were plated on LB agar plates containing 100ug / ml ampicillin (SIGMA) and incubated overnight at 37°C. - Six clones were selected from each plate to prepare microcultures (2 mL) in LB broth with ampicillin (50 μg / ml), which were incubated overnight at 37°C with agitation. Incubated. - The next day, plasmid DNA was isolated and Bam H1 digested to identify recombinant clones. Recombinant clones were identified by the presence of three bands of the appropriate size (3431, 1056, and 5844 bp, see Figure 19). As can be seen in Figure 19, all six clones selected from each plate yielded restriction enzyme fragments of the appropriate size. - Two clones transduced with pLV-416 constructs containing EXD2_K193A or EXD2_WT were sequenced with the primers listed in Table 7 to confirm that the constructs were correct.

[0233] [Table 7]

[0234]

[0258] The following protocol was used to produce viral particles for transduction of HEK293 cells and generation of suitable HEK293 cell lines expressing functional or non-functional P2X7 receptors. - HEK293 cells were seeded the day before transfection (7 × 10 per flask). 6 cells). - HEK293T cells were transfected with lentiviral packaging vectors and either pLV-416-EXD2 or pLV-416-EXD2_WT. A GFP expression plasmid (1 μg) was also included to monitor transfection efficiency. After overnight incubation, the medium containing the transfection reagent was removed and replaced with 10 ml of fresh medium (DMEM with 10% FCS). 10 ml of medium was harvested after 24 hours and stored in 2 ml aliquots at -80°C until required. Another 10 ml of fresh medium (DMEM with 10% FCS) was added to the flask, which was harvested after another 24 hours. - Viral particles were isolated from the harvested medium by centrifuging the medium at 1200 rpm and then filtered through a 0.45 μm filter. The filtered medium together with the viral particles was used to transfect HEK293 cells.

[0235]

[0259] To assess transfection efficiency, cells were harvested after removing the second 10 ml of medium, and the percentage of GFP-positive cells was determined by flow cytometry. Figure 20 illustrates that HEK293 cells were transfected with pLV-416-EXD2_K193A and pLV-416-EXD2_WT with 97% and 85% efficiency.

[0236]

[0260] To generate stable HEK293 cells overexpressing functional and non-functional P2X7 extracellular domains on the cell surface, the following protocol was used. - HEK293 cells were seeded into T25 flasks the day before transduction (1 flask 7 x 10 5 cells). - The next day, the medium was removed from each flask and new medium containing viral particles produced according to the protocol described above was added according to the ratios listed in Table 8. - Polybrene was added to each flask to a final concentration of 8ug / mL.

[0237] [Table 8]

[0238] - 24 hours after transduction, the medium was removed from each flask and fresh medium (DMEM with 10% FCS) supplemented with 1600 μg / mL G418 was added to all flasks except the flask containing the control GFP-expressing lentivirus (LV-411-GFP). - HEK293T cells transduced with the control pLV-411-GFP virus were monitored for GFP expression for 72 hours after transduction (see Figure 21). - All untransduced cells died after 4 days of culture in G418-supplemented medium. Transduced cell lines continued to grow normally in medium containing G418.

[0239]

[0261] The extracellular domain of transfected P2X7 receptor contains HA-epitope tag and MYC-epitope tag.Therefore, these cells can be stained with anti-HA and anti-MYC monoclonal antibody, and confirm the surface expression of extracellular domain by flow cytometry.

[0240] Screening for CAR T cell function

[0262] To assess the functionality of the nf-P2X7-CAR, CD8 cells transduced with either the PEP2-2-1-1 or PEP2-472-2 CAR constructs (prepared as described above) were cultured at 1 × 10 cells expressing the nf-P2X7 receptor in 96-well round-bottom culture plates. 4 The target cells (prepared as described above) were co-cultured with MDA-MB-231 breast cancer cells expressing non-functional P2X7 receptors (231 P2X7 cells) at a 1:1 ratio for 4 hours.

[0241]

[0263] The percentage of cytotoxicity was determined in the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wisconsin, USA) according to the manufacturer's instructions. Briefly: 45 minutes before and for 4 hours, 10 μl of lysis solution (10x) per 100 μl of target cells was added to each well. After a further 45 minutes, the plates were centrifuged at 250 x g for 4 minutes. - A 50 μl aliquot was taken from each well and transferred to a 96-well flat-bottom plate. - 50 μl of CytoTox 96® reagent was added to each well of the plate containing the transferred aliquot and the plate was covered with foil for 30 minutes at room temperature. After 30 minutes, 50 μl of stop solution was added to each well and the absorbance at 490 nm was read from each well.

[0242]

[0264] The absorbance values ​​for each well were corrected according to the manufacturer's instructions, and the percentage of cytotoxicity was calculated using the following formula and normalized to empty vector-transduced T cells to obtain the fold change in cell killing.

[0243]

number

[0244]

[0265] As shown in Figure 22A, both CD8 T cells expressing PEP2-2-1-1 CAR and CD8 T cells expressing PEP2-472-2 CAR killed approximately 15-fold and 11-fold (respectively) more HEK cells expressing non-functional P2X7 receptors than CD8 cells transduced with empty vector. Furthermore, as shown in Figure 22B, PEP2-2-1-1 CAR-expressing CD8 T cells and PEP2-472-2 CAR-expressing CD8 T cells killed approximately 2.5-fold and 2.25-fold (respectively) more 231 P2X7 cells than CD8 cells transduced with empty vector.

[0245]

[0266] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illustrate example embodiments and does not impose limitations on the scope of the claimed invention. No language herein should be construed as indicating any non-claimed element as essential.

[0246]

[0267] The description provided herein relates to several embodiments that may share common properties and characteristics. It should be understood that one or more features of one embodiment may be combinable with one or more features of other embodiments. In addition, a single feature or combination of features of an embodiment may constitute an additional embodiment.

[0247]

[0268] The headings used herein are included solely for the reader's ease of reference and are not intended to limit the subject matter found throughout this disclosure or the claims. The headings are not intended to be used to interpret the scope of the claims or their limitations.

[0248]

[0269] Those skilled in the art will recognize that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes such variations and modifications. The invention also includes all of the steps, features, compositions, and compounds referenced or shown in this specification, individually or collectively, as well as any combination of any two or more of the steps or features.

[0249]

[0270] Additionally, it should be noted that as used herein, the singular forms "a," "an," and "the" include plural aspects unless the context already indicates otherwise.

[0250]

[0271] Future patent applications may be filed based on this application, for example, by claiming priority from this application, by claiming divisional status, and / or by claiming continuation status, and it will be understood that the following claims are not intended to limit the scope to which any such future applications may be patented. Aspects of the invention [Aspect 1] A chimeric antigen receptor comprising an antigen recognition domain and a signal transduction domain, wherein the antigen recognition domain recognizes a dysfunctional P2X7 receptor. [Aspect 2] The chimeric antigen receptor of claim 1, wherein the antigen recognition domain recognizes an epitope associated with the adenosine triphosphate (ATP) binding site of a dysfunctional P2X7 receptor. [Embodiment 3] The chimeric antigen receptor of claim 1 or 2, wherein the dysfunctional P2X7 receptor has a reduced ability to bind to ATP compared to the ATP binding ability of a wild-type (functional) P2X7 receptor. [Aspect 4] The chimeric antigen receptor of any one of claims 1 to 3, wherein the dysfunctional P2X7 receptor has a conformational change that causes the receptor to be dysfunctional. [Aspect 5] The chimeric antigen receptor of claim 4, wherein the change in configuration is a change from a trans configuration to a cis configuration of an amino acid. [Aspect 6] The chimeric antigen receptor of claim 5, wherein the amino acid changed from trans to cis configuration is proline at amino acid position 210 of the dysfunctional P2X7 receptor. [Aspect 7] The chimeric antigen receptor according to any one of claims 1 to 6, wherein the antigen recognition domain recognizes an epitope comprising a proline at amino acid position 210 of the dysfunctional P2X7 receptor. [Embodiment 8] The chimeric antigen receptor according to any one of claims 1 to 7, wherein the antigen recognition domain recognizes an epitope comprising one or more amino acid residues ranging from glycine at amino acid position 200 to cysteine ​​at amino acid position 216 of the dysfunctional P2X7 receptor. [Embodiment 9] The chimeric antigen receptor of any one of claims 1 to 8, wherein the antigen recognition domain comprises an amino acid sequence homologous to the amino acid sequence of an antibody or fragment thereof that binds to a dysfunctional P2X7 receptor. [Embodiment 10] The chimeric antigen receptor of any one of claims 1 to 9, wherein the antigen recognition domain comprises an amino acid sequence homologous to the amino acid sequence of an antigen-binding fragment (Fab) portion of an antibody that binds to a dysfunctional P2X7 receptor. [Aspect 11] The chimeric antigen receptor of claim 9 or 10, wherein the antibody is a humanized antibody. [Embodiment 12] The chimeric antigen receptor of any one of claims 1 to 9, wherein the antigen recognition domain comprises an amino acid sequence homologous to the amino acid sequence of a single-chain variable fragment (scFv) that binds to a dysfunctional P2X7 receptor. [Embodiment 13] The chimeric antigen receptor of any one of claims 1 to 9, wherein the antigen recognition domain comprises an amino acid sequence homologous to the amino acid sequence of a multivalent single-chain variable fragment (scFv) that binds to a dysfunctional P2X7 receptor. [Aspect 14] The chimeric antigen receptor of claim 13, wherein the multivalent single-chain variable fragment (scFv) is a bivalent scFv or a trivalent scFv. [Embodiment 15] The chimeric antigen receptor of any one of claims 1 to 9, wherein the antigen recognition domain comprises an amino acid sequence homologous to the amino acid sequence of a single antibody domain (sdAb) that binds to a dysfunctional P2X7 receptor. [Embodiment 16] The chimeric antigen receptor of any one of claims 1 to 15, wherein the signaling domain comprises a portion derived from an activating receptor. [Aspect 17] The chimeric antigen receptor of claim 16, wherein the activating receptor is a member of the CD3 coreceptor complex. [Aspect 18] The chimeric antigen receptor of claim 17, wherein the portion derived from the CD3 co-receptor complex is CD3-zeta. [Aspect 19] The chimeric antigen receptor of claim 16, wherein the activating receptor is an Fc receptor. [Aspect 20] The chimeric antigen receptor of claim 19, wherein the portion derived from an Fc receptor is FcεRI or FcγRI. [Embodiment 21] The chimeric antigen receptor of any one of claims 1 to 15, wherein the signaling domain comprises a portion derived from a costimulatory receptor. [Embodiment 22] The chimeric antigen receptor of any one of claims 1 to 21, wherein the signaling domain comprises a portion derived from an activating receptor and a portion derived from a costimulatory receptor. [Aspect 23] The chimeric antigen receptor of claim 21 or 22, wherein the costimulatory receptor is selected from the group consisting of CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS. [Aspect 24] A nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor of any one of claims 1 to 23. [Aspect 25] A nucleic acid construct comprising the nucleic acid molecule of claim 24. [Aspect 26] The nucleic acid construct of claim 25, wherein expression of the nucleic acid molecule is under the control of a transcriptional regulatory sequence. [Aspect 27] The nucleic acid construct of claim 26, wherein the transcriptional regulatory sequence is a constitutive promoter. [Embodiment 28] The nucleic acid construct of claim 26, wherein the transcriptional control sequence is an inducible promoter. [Aspect 29] A nucleic acid construct described in any one of claims 25 to 28, further comprising an internal ribosome entry site (IRES). [Aspect 30] A nucleic acid construct described in any one of claims 25 to 29, wherein the nucleic acid construct is a vector. [Aspect 31] The nucleic acid construct of claim 30, wherein the vector is a viral vector. [Aspect 32] A genetically modified cell comprising the chimeric antigen receptor described in any one of claims 1 to 23. [Aspect 33] The genetically modified cell of claim 32, comprising two or more different chimeric antigen receptors. [Aspect 34] A genetically modified cell comprising the nucleic acid molecule of claim 24, or the nucleic acid construct of any one of claims 25 to 31, or a genomically integrated form of the nucleic acid construct. [Embodiment 35] The genetically modified cell of claim 34, wherein the nucleic acid molecule or nucleic acid construct encodes two or more different chimeric antigen receptors. [Embodiment 36] The genetically modified cell of claim 33 or 35, wherein two or more different chimeric antigen receptors have different signaling domains. [Embodiment 37] The genetically modified cell of any one of Claims 32 to 36, comprising a first chimeric antigen receptor having a signaling domain comprising a portion derived from an activating receptor, and a second chimeric antigen receptor having a signaling domain comprising a portion derived from a costimulatory receptor. [Embodiment 38] The genetically modified cell of claim 37, wherein the activating receptor is a member of the CD3 co-receptor complex or an Fc receptor. [Embodiment 39] The genetically modified cell of claim 37 or 38, wherein the costimulatory receptor is selected from the group consisting of CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS. [Aspect 40] A genetically modified cell described in any one of claims 32 to 36, which is further modified to constitutively express a costimulatory receptor. [Aspect 41] The genetically modified cell of claim 40, which is further modified to express a ligand for a costimulatory receptor, thereby promoting self-stimulation of the cell. [Aspect 42] A genetically modified cell described in any one of claims 32 to 41, which is further modified to secrete a cytokine. [Aspect 43] The genetically modified cell of claim 42, wherein the cytokine is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-17, and IL-21, or a combination thereof. [Aspect 44] A genetically modified cell described in any one of claims 32 to 43, which is a white blood cell. [Aspect 45] A genetically modified cell described in any one of claims 32 to 44, which is a peripheral blood mononuclear cell (PBMC). [Aspect 46] A genetically modified cell described in any one of claims 32 to 45, which is a lymphocyte. [Aspect 47] A genetically modified cell described in any one of claims 32 to 46, which is a T cell. [Aspect 48] The genetically modified cell of claim 47, wherein the T cell is a CD4+ T cell. [Aspect 49] The genetically modified cell of claim 47, wherein the T cell is a CD8+ T cell. [Embodiment 50] A genetically modified cell described in any one of claims 32 to 46, which is a natural killer cell. [Aspect 51] A genetically modified cell described in any one of claims 32 to 46, which is a natural killer T cell. [Embodiment 52] A method for killing cells expressing a dysfunctional P2X7 receptor, comprising exposing the cells expressing the dysfunctional P2X7 receptor to genetically modified cells having a chimeric antigen receptor, wherein the chimeric antigen receptor is directed against the dysfunctional P2X7 receptor. [Embodiment 53] The method of claim 52, wherein the chimeric antigen receptor directly recognizes the dysfunctional P2X7 receptor. [Embodiment 54] The method of claim 52, wherein the chimeric antigen receptor recognizes the dysfunctional P2X7 receptor via an intermediary. [Embodiment 55] The method of claim 54, wherein the intermediary is a probe that binds to a dysfunctional P2X7 receptor, and the chimeric antigen receptor recognizes the probe. [Embodiment 56] The method of claim 52, further comprising exposing the cells expressing a dysfunctional P2X7 receptor to a probe. [Embodiment 57] The method described in claim 55 or 56, wherein the probe is an antibody or an aptamer. [Embodiment 58] The method of any one of claims 55 to 57, wherein the probe comprises a tag and the chimeric antigen receptor recognizes the tag. [Embodiment 59] A method for killing cells expressing a dysfunctional P2X7 receptor, comprising exposing the cells expressing the dysfunctional P2X7 receptor to a genetically modified cell according to any one of claims 32 to 51. [Embodiment 60] The method of any one of claims 52 to 59, further comprising exposing the cells expressing a dysfunctional P2X7 receptor to an exogenous cytokine. [Embodiment 61] The method of any one of claims 52 to 60, wherein the genetically modified cell is a genetically modified cell that is autologous to the cell expressing the dysfunctional P2X7 receptor. [Aspect 62] A method according to any one of claims 52 to 61, wherein the cells expressing a dysfunctional P2X7 receptor are present in the subject's body. [Aspect 63] A method according to any one of claims 52 to 62, wherein the cell expressing a dysfunctional P2X7 receptor is a cancer cell. [Embodiment 64] The method of claim 63, wherein the cancer cells are selected from one or more of brain cancer, esophageal cancer, oral cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cervical cancer, epithelial cell cancer, skin cancer, leukemia, lymphoma, myeloma, breast cancer, ovarian cancer, endometrial cancer, and testicular cancer. [Embodiment 65] The method of claim 63, wherein the cancer cells are selected from one or more of lung cancer, esophageal cancer, gastric cancer, colon cancer, prostate cancer, bladder cancer, cervical cancer, vaginal cancer, epithelial cell cancer, skin cancer, blood-related cancer, breast cancer, endometrial cancer, uterine cancer, and testicular cancer. [Aspect 66] A method described in any one of claims 63 to 65, wherein the cancer is metastatic. [Aspect 67] A method described in any one of claims 63 to 66, wherein the cancer is stage III cancer. [Aspect 68] A method described in any one of claims 63 to 66, wherein the cancer is stage IV cancer. [Aspect 69] A method for growing in vitro a genetically modified cell described in any one of claims 32 to 51, the method comprising a step of exposing the cell to an antigen of the chimeric antigen receptor. [Aspect 70] The method of claim 69, further comprising exposing the cells to a cytokine. [Aspect 71] A method for growing in vitro a genetically modified cell described in any one of claims 32 to 51, the method comprising the steps of exposing the cell to an antigen of a chimeric antigen receptor and simultaneously exposing the cell to a cytokine. [Embodiment 72] The method of claim 70 or 71, wherein the cytokine is a member of the IL-2 subfamily, the interferon subfamily, the IL-10 subfamily, the IL-1 subfamily, the IL-17 subfamily, or the TGF-β subfamily. [Embodiment 73] The method of claim 70 or 71, wherein the cytokine is selected from the group consisting of IFN-γ, IL-2, IL-5, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, TNF-α, TGF-β1, TGF-β2, TGF-β3, and GM-CSF, or a combination thereof. [Aspect 74] A method for growing in vitro a genetically modified cell described in any one of claims 32 to 51, the method comprising a step of exposing the cell to immobilized anti-CD3 antibody and anti-CD28 antibody. [Aspect 75] A pharmaceutical composition comprising a genetically modified cell described in any one of claims 32 to 51 and a pharmaceutically acceptable carrier. [Aspect 76] The pharmaceutical composition of claim 75, further comprising a cytokine. [Aspect 77] The pharmaceutical composition of claim 76, wherein the cytokine is a member of the IL-2 subfamily, the interferon subfamily, the IL-10 subfamily, the IL-1 subfamily, the IL-17 subfamily, or the TGF-β subfamily. [Aspect 78] The pharmaceutical composition of claim 76, wherein the cytokine is selected from the group consisting of IFN-γ, IL-2, IL-5, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, TNF-α, TGF-β1, TGF-β2, TGF-β3, and GM-CSF, or a combination thereof. [Aspect 79] A pharmaceutical composition described in any one of claims 75 to 78, further comprising an intermediary. [Embodiment 80] The pharmaceutical composition of claim 79, wherein the intermediary is a probe that binds to a dysfunctional P2X7 receptor, and the chimeric antigen receptor recognizes the probe. [Aspect 81] The pharmaceutical composition of claim 80, wherein the probe is an antibody or an aptamer. [Embodiment 82] The pharmaceutical composition of claim 80 or 81, wherein the probe comprises a tag and the chimeric antigen receptor recognizes the tag.

Claims

1. A pharmaceutical composition comprising a genetically modified cell and a mediator, The genetically modified cells are an antigen recognition domain that recognizes the intermediary; a transmembrane domain; a signaling domain comprising the intracellular signaling portion of an activating receptor and / or the intracellular signaling portion of a costimulatory receptor; a chimeric antigen receptor comprising The mediator is a dysfunctional P2X ７ an antigen recognition domain for binding to a receptor, the antigen recognition domain of the mediator comprising three CDRs of a variable region having an amino acid sequence set forth in any one of SEQ ID NOs: 10, 32, 33, or 34; ７ The receptor is wild-type (functional) P2X ７ a dysfunctional P2X receptor having a reduced ability to bind ATP compared to the ATP binding ability of the receptor; ７ The receptor has a conformational change that makes the receptor dysfunctional, and the conformational change is P2X ７ A pharmaceutical composition, wherein the proline at amino acid position 210 of the receptor is changed from a trans to a cis configuration.

2. 2. The pharmaceutical composition of claim 1, wherein the genetically modified cell comprises a nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor.

3. The pharmaceutical composition of claim 2 , wherein the genetically modified cell comprises a viral vector comprising the nucleic acid molecule.

4. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the genetically modified cells are leukocytes, peripheral blood mononuclear cells (PBMCs), lymphocytes, T cells, CD4+ T cells, CD8+ T cells, natural killer cells or natural killer T cells.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the intermediary is in the form of an antibody.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the antigen recognition domain of the intermediary is an sdAb.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the antigen recognition domain of the intermediary is multivalent.

8. The pharmaceutical composition of claim 7 , wherein the antigen recognition domain of the intermediary is bivalent or trivalent.

9. The antigen recognition domain of the intermediary - CDR1 comprising the amino acid sequence of residues 30 to 35 of SEQ ID NO: 10; a CDR2 comprising the amino acid sequence of residues 50 to 67 of SEQ ID NO: 10, and CDR3 comprising the amino acid sequence of residues 98 to 108 of SEQ ID NO: 10 The pharmaceutical composition according to any one of claims 1 to 8, comprising:

10. The antigen recognition domain of the intermediary - CDR1 comprising the amino acid sequence of residues 30 to 35 of SEQ ID NO: 32; - a CDR2 comprising the amino acid sequence of residues 50 to 67 of SEQ ID NO: 32, and CDR3 comprising the amino acid sequence of residues 98 to 108 of SEQ ID NO: 32 The pharmaceutical composition according to any one of claims 1 to 9, comprising:

11. The antigen recognition domain of the intermediary - CDR1 comprising the amino acid sequence of residues 30 to 35 of SEQ ID NO: 33; - a CDR2 comprising the amino acid sequence of residues 50 to 67 of SEQ ID NO: 33, and CDR3 comprising the amino acid sequence of residues 98 to 108 of SEQ ID NO: 33 The pharmaceutical composition according to any one of claims 1 to 10, comprising:

12. The antigen recognition domain of the intermediary - CDR1 comprising the amino acid sequence of residues 30 to 35 of SEQ ID NO: 34; - a CDR2 comprising the amino acid sequence of residues 50 to 67 of SEQ ID NO: 34, and CDR3 comprising the amino acid sequence of residues 98 to 108 of SEQ ID NO: 34 The pharmaceutical composition according to any one of claims 1 to 11, comprising:

13. The pharmaceutical composition of any one of claims 1 to 12, wherein the activating receptor of the chimeric antigen receptor is a member of the CD3 co-receptor complex and / or an Fc receptor, and / or the costimulatory receptor is selected from the group consisting of CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS.

14. The pharmaceutical composition of any one of claims 1 to 13, further comprising a pharmaceutically acceptable carrier.

15. Dysfunctional P2X ７ In vitro method for killing cells expressing a dysfunctional P2X receptor ７ Cells expressing the receptor are exposed to a pharmaceutical composition according to any one of claims 1 to 14, thereby inhibiting dysfunctional P2X receptors. ７ killing cells that express the dysfunctional P2X receptor; ７ The receptor is wild-type (functional) P2X ７ a dysfunctional P2X receptor having a reduced ability to bind ATP compared to the ATP binding ability of the receptor; ７ The receptor has a conformational change that renders the receptor dysfunctional, and the conformational change is P2X ７ A method in which the proline at amino acid position 210 of the receptor is changed from a trans to a cis configuration.

16. Dysfunctional P2X ７ Use of a pharmaceutical composition according to any one of claims 1 to 14 in the manufacture of a medicament for killing cells expressing the dysfunctional P2X receptor. ７ The receptor is wild-type (functional) P2X ７ a dysfunctional P2X receptor having a reduced ability to bind ATP compared to the ATP binding ability of the receptor; ７ The receptor has a conformational change that renders the receptor dysfunctional, and the conformational change is P2X ７ Use, which is a change from trans to cis configuration of the proline at amino acid position 210 of the receptor.

17. Dysfunctional P2X in a subject ７ Dysfunctional P2X in the manufacture of a medicament for the treatment of cancers that express the receptor ７ Use of an intermediary comprising an antigen recognition domain for binding to a receptor, wherein the antigen recognition domain of the intermediary comprises three CDRs of a variable region having an amino acid sequence set forth in any one of SEQ ID NOs: 10, 32, 33 or 34; The subject is an antigen recognition domain that recognizes the intermediary; a transmembrane domain; a signaling domain comprising the intracellular signaling portion of an activating receptor and / or the intracellular signaling portion of a costimulatory receptor; and a genetically modified cell comprising a chimeric antigen receptor comprising said dysfunctional P2X ７ The receptor is wild-type (functional) P2X ７ a dysfunctional P2X receptor having a reduced ability to bind ATP compared to the ATP binding ability of the receptor; ７ The receptor has a conformational change that makes the receptor dysfunctional, and the conformational change is P2X ７ Use, which is a change from trans to cis configuration of the proline at amino acid position 210 of the receptor.

18. Dysfunctional P2X in a subject ７ 1. Use of genetically modified cells containing a chimeric antigen receptor in the manufacture of a medicament for the treatment of cancer expressing the receptor, wherein the subject has a dysfunctional P2X ７ receiving an intermediary containing an antigen recognition domain for binding to the receptor, The antigen recognition domain of the intermediary comprises three CDRs of a variable region having an amino acid sequence set forth in any one of SEQ ID NOs: 10, 32, 33, or 34; The chimeric antigen receptor an antigen recognition domain that recognizes the intermediary; a transmembrane domain; a signaling domain comprising the intracellular signaling portion of an activating receptor and / or the intracellular signaling portion of a costimulatory receptor; and the dysfunctional P2X ７ The receptor is wild-type (functional) P2X ７ a dysfunctional P2X receptor having a reduced ability to bind ATP compared to the ATP binding ability of the receptor; ７ The receptor has a conformational change that makes the receptor dysfunctional, and the conformational change is P2X ７ Use, which is a change from trans to cis configuration of the proline at amino acid position 210 of the receptor.

19. Dysfunctional P2X ７ 17. The method or use of claim 15 or 16, wherein the cells expressing the receptor are cancer cells.

20. 20. The method or use of any one of claims 17 to 19, wherein the cancer is selected from one or more of brain cancer, esophageal cancer, oral cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cervical cancer, epithelial cell cancer, skin cancer, leukemia, lymphoma, myeloma, breast cancer, ovarian cancer, endometrial cancer, and testicular cancer.

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