Chimeric antigen receptors and uses thereof

Abstract

To provide chimeric antigen receptors, T cells expressing the chimeric antigen receptors, and methods of using the chimeric antigen receptors for prevention and / or treatment of cancer.SOLUTION: A chimeric antigen receptor comprises an antigen-recognition domain and a signalling domain, wherein the antigen-recognition domain recognizes a dysfunctional P2X7 receptor.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Claim of Priority

[0001] This application claims the priority of Australian Provisional Patent Application No. 2015903719, filed on September 11, 2015, the content of which is 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 for the prevention and / or treatment of cancer.

Background Art

[0003]

[0003] The immune system has highly evolved specific mechanisms to protect against various etiologies. Among these etiologies, it is especially to detect and eliminate unwanted pathogens such as bacterial infections, virus-infected cells, and importantly, mutant cells that can cause malignant neoplasms (cancer). The ability of the immune system to prevent the formation and growth of cancer depends on the ability of the cells of the immune system to distinguish between "healthy" cells and "disease-state" (e.g., neoplastic or preneoplastic) cells. This is achieved by recognizing cell markers (antigens) that indicate the transition of cells from a healthy state to a disease state.

[0004]

[0004] Numerous attempts have been made to develop immunotherapy approaches for treating cancer by manipulating or directing the immune system to target cells expressing cancer cell antigens. Immunotherapy approaches have mainly utilized the humoral immune system using isolated or engineered antibodies, or more recently, have focused on the cellular arm of the immune system.

[0005]

[0005] In the initial attempts to use adoptive cell transfer therapy for cancer treatment, T lymphocytes isolated from tumors and expanded ex vivo were used. This approach had some initial promise in early investigations, but there are a number of technical problems associated with this approach. The ability to isolate and expand a T cell population to a clinically relevant number is technically problematic, and the inability to adequately control the nature of the expansion results in a final T cell population that is clearly heterogeneous and may contain only a small number of cancer antigen-specific T cells. As a result, the effectiveness of this method is unpredictable and variable.

[0006]

[0006] To address some of the deficiencies associated with the use of ex vivo-expanded tumor-isolated T cells, chimeric antigen receptors (CARs or artificial T cell receptors) were first developed in the late 1980s. A chimeric antigen receptor is created by linking an extracellular domain specific for a desired antigen to a signaling domain, resulting in an antigen-specific receptor that can induce T cell function.

[0007]

[0007] By transducing isolated T cells with a CAR, a population of T cells specific for a given antigen can be obtained. As a result, multiple antigen-specific T cell populations can be generated and used for immunotherapy.

[0008]

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

[0009]

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

[0010]

[0010] Considerations regarding documents, actions, materials, devices, articles, etc. are included herein solely for the purpose of providing context for the present invention. It is not suggested, nor is it shown, that any or all of these issues existed prior to the date of priority of each of the patent claims of the present application and thus formed part of the state of the art or were common general knowledge in the field related to the present invention.

Summary of the Invention

[0011]

[0011] The present invention is partially based on the recognition that there is a need to develop chimeric antigen receptors (CARs) and genetically modified cells expressing the same that target markers specifically associated with various neoplastic (cancerous) or preneoplastic (precancerous) cells due to the significant "on-target, off-tumor" activity of immune cells expressing CARs. The inventors recognize that the dysfunctional P2X7 receptor is a suitable marker for targeting using CARs.

[0012]

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

[0013]

[0013] In some embodiments, the antigen recognition domain recognizes an epitope associated with the adenosine triphosphate (ATP) binding site of the dysfunctional P2X7 receptor. In some embodiments, the dysfunctional P2X7 receptor has a reduced ability to bind ATP at the ATP binding site as compared to the ATP binding ability of the wild-type (functional) P2X7 receptor. In some embodiments, the dysfunctional P2X7 receptor is unable to bind ATP at the ATP binding site.

[0014]

[0014] In some embodiments, the dysfunctional P2X7 receptor has a conformational change that renders the receptor dysfunctional. In some embodiments, the conformational change is a change from the trans configuration to the cis configuration of an amino acid. In some embodiments, the amino acid that changes from the trans configuration to the cis configuration is proline at position 210 of the amino acids of the dysfunctional P2X7 receptor.

[0015]

[0015] In some embodiments, the antigen recognition domain recognizes an epitope that includes proline at position 210 of the amino acids of the dysfunctional P2X7 receptor. In some embodiments, the antigen recognition domain recognizes an epitope that includes one or more amino acid residues spanning from glycine at position 200 to cysteine at position 216 (including both ends) of the amino acids of the dysfunctional P2X7 receptor.

[0016]

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

[0017]

[0017] In some embodiments, the antigen recognition domain includes amino acid sequence homology to the amino acid sequence of a single-chain variable fragment (scFv) or a multivalent scFv that binds to the 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 includes amino acid sequence homology to a single antibody domain (sdAb) that binds to the dysfunctional P2X7 receptor.

[0019] In some embodiments, the antigen recognition domain comprises a binding peptide that includes 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 amino acid sequence homology to the CDR1, 2, and 3 domains of the V H chain and / or the V L chain of an antibody that binds to a dysfunctional P2X7 receptor. 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 a region 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 coreceptor complex or an Fc receptor. In some embodiments, the portion derived from the CD3 coreceptor complex is CD3-ζ. In some embodiments, the portion derived from the 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 present invention.

[0023] In a third aspect, the present invention provides a nucleic acid construct comprising the 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 transcriptional control sequence. In some embodiments, the transcriptional control sequence can be a constitutive promoter or an inducible promoter.

[0022]

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

[0023]

[0025] In some embodiments of the third aspect of the present invention, the nucleic acid construct is a vector such as a viral vector, and this can be used to transform T cells to induce the 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 present invention. In some embodiments, the cell comprises two or more different CARs.

[0025]

[0027] In a fifth aspect, the present invention provides a genetically modified cell comprising the nucleic acid molecule according to the second aspect of the present invention, or the nucleic acid construct according to the third aspect of the present invention, or an integrated genomic 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 present 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 chimeric antigen receptor (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 present invention, the cell is further modified to constitutively express a costimulatory receptor. In some embodiments, the cell is further modified to express a ligand for the costimulatory receptor, thereby promoting autostimulation of the cell.

[0028]

[0031] In some embodiments of the fourth and fifth aspects of the present invention, the cell is further modified to secrete a cytokine. In 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 combinations thereof.

[0029]

[0032] In some embodiments of the fourth and fifth aspects of the present invention, the cell is a leukocyte. In some embodiments, the cell is a peripheral blood mononuclear cell (PBMC), lymphocyte, T cell (including CD4+ T cells or CD8+ T cells), natural killer cell, or 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, the CAR directly recognizes a dysfunctional P2X7 receptor or recognizes a dysfunctional P2X7 receptor via an intermediate. In some embodiments, the intermediate is a probe that binds to the dysfunctional P2X7 receptor, and the CAR recognizes the probe. In some embodiments, the probe is an antibody or an aptamer. In some embodiments, the probe includes a tag, and the CAR recognizes the tag.

[0032]

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

[0033]

[0036] In some embodiments of the sixth and seventh aspects of the present invention, cells that express a dysfunctional P2X7 receptor are exposed to the genetically modified cells together with an exogenous cytokine. In some embodiments, the genetically modified cells are genetically modified cells that are autologous to the cells that express a dysfunctional P2X7 receptor. In some embodiments, the cells that express a dysfunctional P2X7 receptor are cancer cells. In some embodiments, the cancer is selected from the group consisting of brain cancer, esophageal cancer, oral cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cervical cancer, epithelial cell cancer, skin cancer, leukemia, lymphoma, myeloma, breast cancer, ovarian cancer, endometrial cancer, and testicular cancer. In some embodiments, the cancer is selected from the group consisting of lung cancer, esophageal cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer, cervical cancer, vaginal cancer, epithelial cell cancer, skin cancer, blood-related cancer, breast cancer, endometrial cancer, uterine cancer, and testicular cancer.

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

[0035]

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

[0036]

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

[0037]

[0039] In a ninth aspect, the present invention provides a method for growing in vitro a genetically modified cell according to the fourth or fifth aspect of the present invention, the method comprising the steps of exposing the cell to the 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 present 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 present 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 in vitro expanding genetically modified cells according to the fourth or fifth aspect of the present invention, the method comprising exposing the cells to immobilized anti-CD3 antibody and anti-CD28 antibody. In some embodiments of the tenth aspect of the present invention, the antibody is immobilized on a bead substrate (e.g., "Human Activator" Dynabeads™). In some embodiments of the tenth aspect of the present invention, the antibody is 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 genetically modified cells 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 consist of a cytokine. In some embodiments, the pharmaceutical composition may also include an intermediate as described herein.

[0042]

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

Brief Description of the Drawings

[0043]

Figure 1

[0045] A schematic diagram showing the arrangement of an anti-non-functional (nf) P2X7 receptor chimeric antigen receptor (CAR) according to an embodiment of the present invention.

Figure 2

[0046] A schematic diagram showing the BLIV plasmid used for the expression of the anti-nf P2X7 receptor CAR of FIG. 1.

Figure 3

[0047] An electrophoresis gel showing a restriction enzyme-treated fragment derived from DNA restriction enzyme-treated with BamHI, isolated from an E. coli clone transformed with the BLIV plasmid.

Figure 4

[0048] An electrophoresis gel showing restriction enzyme fragments derived from DNA digested with EcoRI, BamHI, and PstI, isolated from selected E. coli clones transfected with the BLIV plasmid.

Figure 5

[0049] A diagram showing microscopic images of 293T cells transfected with the plasmid required for the construction of a lentiviral vector containing the BLIV-CAR-short hinge construct and 293T cells transduced with the supernatant containing the lentiviral vector.

Figure 6

[0050] A diagram showing microscopic images of 293T cells transfected with the plasmid required for the construction of a 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, including co-culture of CD8+ T cells and BT549 cells.

Figure 9

[0053] A graph showing the percentage of dye-labeled target cells that disappeared after 48 hours of co-culture of CD8+ T cells transduced with lentiviral vectors containing the BLIV-CAR-short hinge plasmid and the 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 the PEP2-2-1-1, PEP2-472-2, and PEP2-2-12 binding peptides with an antibody directed against the nf-P2X7 receptor.

Figure 11

[0055] A schematic diagram showing the arrangement of the anti-nf P2X7 receptor CAR according to a further embodiment of the present invention.

Figure 12

[0056] Schematic diagram showing the pCDH plasmid used for the expression of the anti-nf P2X7 receptor CAR of FIG. 11.

Figure 13

[0057] Diagram of an electrophoresis gel showing restriction enzyme-treated fragments derived from DNA digested with EcoRI and Not I, isolated from selected Sure 2 clones transformed with the pCDH plasmid.

Figure 14

[0058] FACS analysis of the transfection efficiency of HEK293T cells.

Figure 15

[0059] Representative histogram of FACS analysis of lentiviral transduction efficiency.

Figure 16

[0060] FACS analysis of the proportion of transformed CD8 cells expressing GFP.

Figure 17

[0061] Diagram of the backbone of the fusion protein for the production of non-functional P2X7 receptor and functional P2X7 receptor.

Figure 18

[0062] Diagram of an electrophoresis gel showing restriction enzyme-treated fragments derived from DNA digested with Bam HI and PmeI, isolated from selected E.cloni® 10G clones transformed with EXD2_K193A or EXD2_WT containing the pDONR-107 vector.

Figure 19

[0063] Diagram of an electrophoresis gel showing restriction enzyme-treated fragments derived from DNA digested with Bam HI, 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 the 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 of either the pLV-416-EXD2_K193A construct or the pLV-416-EXD2_WT construct into HEK293.

Figure 22

[0066] Graph showing killing of HEK target cells expressing nfP2X7 and 231 breast cancer cells by T cells expressing PEP2-2-1-1, PEP2-472-2 CAR.

BEST MODE FOR CARRYING OUT THE INVENTION

[0044]

[0067] The nucleotide sequences and polypeptide sequences referred to in this specification are represented by sequence identifier numbers (SEQ ID NOs). An overview of the sequence identifiers is provided in Table 1. The sequence listing is further provided at the end of this specification.

[0045]

Table 1-1

[0046]

Table 1-2

[0047]

[0068] The inventors recognized the need to develop chimeric antigen receptors (CARs) and genetically modified cells expressing them that target markers specifically associated with neoplastic (cancerous) or preneoplastic (precancerous) cells, due to the significant "on-target, off-tumor" activity of immune cells expressing CARs. The inventors recognized that the dysfunctional P2X7 receptor is a suitable marker for targeting with immune cells expressing CARs in various cancers. in targeting.

[0048]

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

[0049]

[0070] A chimeric antigen receptor is an artificially constructed protein that, when expressed on the surface of a cell, can induce an antigen-specific cellular response. A CAR comprises at least two domains, namely, a first domain that specifically recognizes an antigen or more specifically an epitope portion of the antigen, which is the antigen recognition domain, and a second domain that can induce or be involved in inducing an intracellular signal transduction pathway, which is the signal transduction domain.

[0050]

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

[0051]

[0072] In addition to the antigen recognition domain and the signaling domain, the CAR may further include additional components or parts. For example, the CAR may include a transmembrane domain that may include or be associated with a portion of the signaling domain of the CAR. The transmembrane domain is typically one or more hydrophobic helices that span the lipid bilayer of the cell and embed the CAR within the cell membrane. The transmembrane domain of the CAR can be a determinant of the expression pattern of the CAR when bound to the cell. For example, by using a transmembrane domain associated with the CD3 co-receptor, expression of the CAR in naive T cells can be enabled, while use of a transmembrane domain derived from the CD4 co-receptor can induce expression of the CAR in T helper cells but not in cytotoxic T cells.

[0052]

[0073] A further component or part of the CAR can be a linker domain. The linker domain (also known as a spacer or hinge domain) spans from the extracellular side of the transmembrane domain to the antigen recognition domain, thereby enabling the antigen recognition domain to be linked 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, use of a linker domain can enhance the effectiveness of the CAR. The linker domain can have various functions, including allowing mobility of the CAR such that the orientation of the antigen recognition domain of the CAR necessary for binding to the antigen is permitted. 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, such as the IgG1 hinge region. Another example includes the CH2CH3 region of an antibody, or an amino acid sequence having sequence homology to a portion of the CD3 co-receptor complex, the CD4 co-receptor, or the CD8 co-receptor.

[0053]

[0074] The P2X7 receptor (purinergic receptor P2X, ligand-gated ion channel 7) is an ATP-gated ion channel expressed in several species, including humans. This receptor is encoded by a gene, and the official symbol of this gene 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 the purposes of the present disclosure, this gene and the encoded receptor are 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 chimpanzee, rhesus monkey, dog, cow, mouse, rat, pig, chicken, zebrafish, and frog. Further details of the P2X7 gene in humans and other species can be accessed from the GenBank database at the National Centre for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov). For example, the Gene identifier number for human P2X7 is 5027, for chimpanzee is 452318, for monkey is 699455, for dog is 448778, for cow is 286814, for mouse is 18439, for zebrafish is 387298, and for frog is 398286. Furthermore, at least 73 species have orthologs with the human P2X7 gene.

[0055]

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

[0056]

[0077] The P2X7 receptor is formed from three protein subunits (monomers), where in the native receptor in humans, at least one of these monomers has the amino acid sequence set forth in SEQ ID NO: 3. It is to be understood that the "P2X7 receptor" referred to herein includes naturally occurring variants of this receptor, including splice variants, naturally occurring cleavage forms and allelic variants of this receptor. The P2X7 receptor may also include subunits having modified amino acid sequences, for example, those including cleavage of the amino acids set forth in SEQ ID NO: 3, amino acid deletions, or modifications.

[0057]

[0078] A "variant" of the P2X7 gene or encoded protein can exhibit a nucleic acid sequence or amino acid sequence that is, for example, 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 the native P2X7 receptor.

[0058]

[0079] The P2X7 receptor is activated when ATP binds to the ATP-binding site of the receptor. This causes a rapid opening of the channel (within a few milliseconds), which selectively allows small cations to move across the membrane. After a short period (within a few seconds), large pores are formed in the cell membrane, which allows the cell membrane to be permeable to molecules up to 900 Da in size. This pore formation ultimately leads to depolarization of the cell and, in many cases, cytotoxicity and cell death. This role is linked to the idea that the P2X7 receptor is involved in apoptosis in various cell types.

[0059]

[0080] Similar to other molecules involved in apoptosis, such as Bcl2 and Bax, a decrease or loss of function of the P2X7 receptor can result in 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 mutant pre-cancerous or cancerous cells. As a result, the ability to target cells with a decreased or lost function of the P2X7 receptor provides a promising target for cancer therapy.

[0060]

[0081] Accordingly, in a first aspect of the present invention, the CAR recognizes a dysfunctional P2X7 receptor. As used throughout this specification, the term "dysfunctional" with respect to the P2X7 receptor includes a decrease in the function of the receptor compared to its corresponding function in normal non-tumor cells. In some embodiments, the function of the P2X7 receptor may be decreased by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99%. In some embodiments, the term "dysfunctional" may include a non-functional P2X7 receptor. This means that the P2X7 receptor cannot be induced to allow the permeation of cations or other molecules across the cell membrane.

[0061]

[0082] Any change in the wild-type or native form of the receptor that results in a P2X7 dysfunctional receptor is encompassed herein. For example, the dysfunctional receptor can be the result of a mutation or modification in one or more amino acids of the receptor that is associated with the binding of ATP to the receptor. Indeed, the P2X7 receptor is dysfunctional if it has a reduced ability to bind ATP at the ATP binding site or is unable to bind thereto. In this case, the antigen recognition domain of the chimeric antigen receptor is expected to recognize an epitope associated with the ATP binding site of the dysfunctional P2X7 receptor. As a result, in some embodiments of the first aspect of the present invention, the antigen recognition domain of the chimeric antigen receptor recognizes an epitope associated with the ATP binding site of the dysfunctional P2X7 receptor. In some embodiments, the dysfunctional P2X7 receptor has a reduced ability to bind ATP as compared to the ATP binding ability of the wild-type (functional) P2X7 receptor. In some embodiments, the dysfunctional P2X7 receptor is unable to bind ATP.

[0062]

[0083] Modifications in one or more amino acids of the P2X7 receptor may include a conformational change in one or more amino acids of this receptor. Thus, in some embodiments of the first aspect of the present invention, the chimeric antigen receptor binds to a dysfunctional P2X7 receptor having a conformational change that renders the receptor dysfunctional. Specifically, this conformational change may be a change in one or more amino acids of the P2X7 receptor from a trans configuration to a cis configuration. In some embodiments, the proline at position 210 of the P2X7 receptor changes from a trans configuration to a cis configuration. In this case, the antigen recognition domain of the CAR may recognize an epitope containing the proline at amino acid position 210 of the P2X7 receptor. In some embodiments of the first aspect of the present invention, the antigen recognition domain recognizes an epitope containing one or more amino acids spanning from glycine at amino acid position 200 to cysteine at amino acid position 216 (including both ends) of the dysfunctional P2X7 receptor. In some embodiments of the first aspect of the present invention, the antigen recognition domain recognizes an epitope containing the proline at position 210 of the dysfunctional P2X7 receptor and one or more of the amino acid residues spanning from glycine at amino acid position 200 to cysteine at amino acid position 216 (including both ends) of the dysfunctional P2X7 receptor.

[0063]

[0084] While not wishing to be bound by theory, position 210 of the P2X7 receptor As a result of the conformational change of the proline at position 210 of SEQ ID NO: 3, the three-dimensional structure of the receptor can be modified. This modification of the three-dimensional structure enables the antigen recognition domain of the CAR to bind to amino acids or epitopes that were inaccessible in the native three-dimensional structure of the P2X7 receptor. Thus, in some embodiments, the CAR recognizes one or more epitopes of the P2X7 receptor that are exposed to the antigen recognition domain as a result of the change in the conformation 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 from positions 200 to 210 or from positions 297 to 306 (including both ends) of the P2X7 receptor. Thus, in some embodiments of the first aspect of the present invention, the antigen recognition domain recognizes an epitope comprising one or more of the amino acids from positions 200 to 210 and / or from positions 297 to 306 of the P2X7 receptor.

[0064]

[0085] As used throughout this specification, the term "recognize" relates to the ability of the antigen recognition domain to bind to a dysfunctional P2X7 receptor, a portion thereof, or an epitope thereof. In some embodiments, the antigen recognition domain may bind directly to a dysfunctional P2X7 receptor or an epitope thereof. In other embodiments, the antigen recognition domain may bind to a processed form of the dysfunctional P2X7 receptor. As used in this context, the term "processed form" relates to a form of the P2X7 receptor that has been cleaved or digested as a result of intracellular processing. As a result, the recognition of the "processed form" of the dysfunctional P2X7 receptor may be the result of binding to and presentation by the major histocompatibility complex (MHC).

[0065]

[0086] The antigen recognition domain can be any suitable domain that can recognize a dysfunctional P2X7 receptor or an epitope thereof. As used throughout this specification, the term "antigen recognition domain" refers to the portion of the CAR that provides specificity for the dysfunctional P2X7 receptor of the CAR. The antigen recognition domain may be all or merely a part of the extracellular region of the CAR. Suitable antigen recognition domains include, but are not limited to, polypeptides having sequence homology to the antigen-binding site of an antibody or a fragment thereof that binds to a dysfunctional P2X7 receptor. Thus, in some embodiments of the first aspect of the present invention, the antigen recognition domain comprises an amino acid sequence having homology to an antibody or a fragment thereof that binds to a dysfunctional P2X7 receptor. In some embodiments, a portion of the antigen recognition domain comprises an amino acid sequence having homology to an antibody or a fragment thereof that binds to a dysfunctional P2X7 receptor. The originating homologous antibody sequence can be any suitable sequence of an antibody having affinity for the P2X7 receptor. For example, the sequence can share sequence homology with an antibody 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 can share sequence homology with the sequence of a monoclonal antibody produced from a hybridoma cell line. When the species from which the homologous antibody sequence originates is non-human, the antibody is preferably a humanized antibody. The homologous antibody sequence may also be derived from non-mammalian species such as cartilaginous fish (see, for example, shark IgNAR antibodies, WO 2012 / 073048). Alternatively, the antigen-binding domain can include a modified protein scaffold that provides functionality similar to that of a shark antibody, such as an iBody having a binding portion based on a shark IgNAR antibody (see WO 2005 / 118629). In addition, the antigen recognition domain can be any other suitable binding molecule or peptide that can selectively interact with a dysfunctional P2X7 receptor with sufficient affinity to activate the CAR signaling domain, can be derived therefrom, or can share sequence homology therewith.Methods for identifying antigen-binding proteins, such as, among others, panning of phage display libraries, protein affinity chromatography, co-immunoprecipitation, and yeast two-hybrid systems, are known in the art (Srinivasa Ra. o, V. et al., Int J Proteomics, 2014; see article number 147648).

[0066]

[0087] In some embodiments, the antigen recognition domain of the CAR comprises amino acid sequence homology to the amino acid sequence of the antigen-binding fragment (Fab) portion of an antibody that binds to a dysfunctional P2X7 receptor. As is understood in the art, the Fab portion of an antibody is composed of one constant region and one variable region of each of the heavy and light chains of the antibody. Fab is the antigen determinant region of the antibody and can be generated by enzymatically cleaving the Fc region from the 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 a single-chain variable fragment (scFv) that binds to a dysfunctional P2X7 receptor. As is understood in the art, scFv comprises two portions that can share homology with or be identical to the variable heavy chain (VH) and variable light chain (VL) of an antibody, and these two portions are a fusion protein connected together by a linker peptide. For example, scFv can comprise VH and VL amino acid sequences derived from an antibody that recognizes a dysfunctional P2X7 receptor. In this context, the term "derived from" is not intended to refer to the origin of the polypeptide itself, but rather is expected to be understood to refer to the origin of the amino acid sequence that constitutes a portion of the antigen-binding region. As a result, the term "derived from" includes polypeptides made synthetically, artificially, or otherwise that share sequence identity with an antibody that binds to a 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 a 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 is the V H chain and / or V LIt comprises one or more regions having sequence homology with the CDR1, 2, and 3 domains of the lock. In some embodiments, the antigen recognition domain is 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 sequences set forth in SEQ ID NO: 10, 32, 33, or 34. 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 sequences set forth in SEQ ID NO: 10, 32, 33, or 34. Between the CDR regions of the antigen-binding peptide set forth in SEQ ID NO: 10, 32, 33, or 34, any public sequence that allows for proper formation and arrangement of the CDR regions may be present. 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] Antibodies directed against dysfunctional P2X7 receptors from which suitable amino acid sequences may be derived, and methods for producing such antibodies, are described in the art (see, e.g., WO 2001 / 020155, WO 2003 / 020762, WO 2008 / 043145, WO 2008 / 043146, WO 2009 / 033233, WO 2011 / 020155, and WO 2011 / 075789). Methods for generating polyclonal and monoclonal antibodies against specific epitopes (such as those described above) are expected to be known to those of skill in the art. Briefly, the desired epitope (such as a segment of the dysfunctional P2X7 receptor containing proline at position 210) is injected into a suitable host animal in the presence of an appropriate immunogenic carrier protein and adjuvant. Serum is then collected from the immunized animal, and the antibodies can be isolated based on their antibody class or antigen specificity. After evaluation of the suitability and specificity of the purified antibodies, the antibodies can be further processed to isolate antigen-binding fragments or sequenced to identify the relevant VH and VL domains. Epitopes suitable for the production of antibodies directed against dysfunctional P2X7 receptors are known in the art (see, for example, WO 2008 / 043146, WO 2010 / 000041, and WO 2009 / 033233).

[0072]

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

[0073]

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

[0074]

[0096] Although not wishing to be bound by theory, the extracellular portion of the TCR is mainly composed of a heterodimer of either the clonotypic TCRα and TCRβ chains (TCRα / β receptor) or the TCRγ and TCRδ chains (TCRγδ receptor). These TCR heterodimers generally lack essential signaling capabilities and thus bind non-covalently to multiple signaling subunits of CD3 (mainly CD3-zeta, CD3-gamma, CD3-delta, and CD3-epsilon). Each of the gamma, delta, and epsilon chains of CD3 has an intracellular (cytoplasmic) portion containing a single immunoreceptor activation tyrosine motif (ITAM), while the CD3-zeta chain contains three tandem ITAMs. When binding of the TCR to its cognate antigen in the presence of MHC and binding of an essential co-receptor such as CD4 or CD8 occur, signaling is initiated, resulting in phosphorylation of two tyrosine residues within the intracellular ITAMs of the CD3 chains by a tyrosine kinase (i.e., Lck). As a result, a second tyrosine kinase (ZAP-70, which itself is activated by Lck phosphorylation) is recruited to biphosphorylate the ITAMs. As a result, multiple downstream target proteins are activated, which ultimately cause changes in intracellular conformation, calcium mobilization, and actin cytoskeleton rearrangement, which, when combined, ultimately cause activation of transcription factors and induction of the T cell immune response. Multiple downstream target proteins are activated, which ultimately cause changes in intracellular conformation, calcium mobilization, and actin cytoskeleton rearrangement, which, when combined, ultimately cause activation of transcription factors and induction of the T cell immune response.

[0075]

[0097] As used throughout this specification, the term "activating receptor" relates to a receptor or co-receptor that forms a component of the T cell receptor (TCR) complex or is involved in the formation of the TCR, or a receptor involved in the specific activation of immune cells as a result of the 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), the CD4 co-receptor, the CD8 co-receptor, the Fc receptor, or natural killer (NK) cell-related activating receptors such as LY-49 (KLRA1), natural cytotoxic receptors (NCR, preferably NKp46, NKp44, NKp30, or NKG2, or the CD94 / NKG2 heterodimer). As a result, in some embodiments of the first aspect of the present invention, the signaling domain comprises a portion derived from any one or more of the members of the CD3 co-receptor complex (preferably, the CD3-zeta chain or a portion thereof), the CD4 co-receptor, the CD8 co-receptor, the Fc receptor (FcR) (preferably, FcεRI or FcγRI), or an NK-related receptor such as LY-49.

[0077]

[0099] The specific intracellular signaling portions of each of the CD3 chains are known in the art. For example, the intracellular cytoplasmic region of the CD3ζ chain spans from amino acid 52 to amino acid 164 of the sequence set forth in SEQ ID NO: 4, and the three ITAM regions span from amino acids 61 to 89, 100 to 128, and 131 to 159 of SEQ ID NO: 4. Further, the intracellular portion of the CD3ε chain spans from amino acid 153 to 207 of the sequence set forth in SEQ ID NO: 5, and the single ITAM region spans from amino acids 178 to 205 of SEQ ID NO: 5. The intracellular portion of the CD3γ chain spans from amino acid 138 to 182 of the sequence set forth in SEQ ID NO: 6, and the single ITAM region spans from amino acids 149 to 177 of SEQ ID NO: 6. The intracellular portion of CD3δ spans from amino acid 127 to 171 of the sequence set forth in SEQ ID NO: 7, and the single ITAM region spans from amino acids 138 to 166 of SEQ ID NO: 7.

[0078]

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

[0079]

[0101] The intracellular portion of the Fc receptor is 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. Further, the intracellular portion of FcγRI spans amino acids 314 to 374 of the sequence set forth in SEQ ID NO: 9.

[0080]

[0102] Using various combinations of portions of the activating receptor, the transmembrane (TM) and intracellular (IC) portions of the CAR, such as 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] As used throughout this specification, the term "co-stimulatory receptor" relates to receptors or co-receptors that assist in the activation of immune cells when antigen-specific induction of activating receptors occurs. As will be understood, co-stimulatory receptors do not require the presence of an antigen and are not antigen-specific, but are typically one of two signals, the other being the activating signal required for the induction of an immune cell response. In the context of an immune response, co-stimulatory 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, co-stimulation is required to effect cell activation, proliferation, differentiation, and survival (all of which are typically subsumed under the umbrella of T cell activation), but when an antigen is presented to a T cell in the absence of co-stimulation, anergy, clonal deletion, and / or the development of antigen-specific tolerance can occur. Importantly, co-stimulatory molecules can communicate a T cell response to an antigen encountered simultaneously. Generally, an antigen encountered in the context of a "positive" co-stimulatory molecule is expected to result in T cell activation and elicit a cell-mediated immune response aimed at eliminating the cells expressing that antigen. On the other hand, an antigen encountered in the context of a "negative" co-receptor is expected to result in the induction of a state of tolerance to the antigen encountered simultaneously.

[0082]

[0104] Non-limiting examples of T cell co-stimulatory receptors include CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), ICOS. Specifically, CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS all represent "positive" co-stimulatory molecules that enhance the activation of T cell responses. Thus, in some embodiments of the first aspect of the present 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 the CD28, OX40, or 4-1BB co-stimulatory receptor. In some embodiments, the signaling domain comprises a portion of the CD28 co-stimulatory receptor. In some embodiments, the signaling domain comprises a portion of the OX40 co-stimulatory receptor. In some embodiments, the portion of the OX40 co-stimulatory receptor comprises the amino acid sequence set forth in SEQ ID NO: 20 or a functional variant thereof.

[0084]

[0106] Various combinations of portions of co-stimulatory receptors can be used to form the transmembrane (TM) and intracellular (IC) portions of the CAR. For example, 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] The sequence information of the activating and co-stimulatory receptors mentioned above is readily accessible in various databases. For example, embodiments of the human amino acid, gene, and mRNA sequences for these receptors are provided in Table 2.

[0086]

Table 2

[0087]

[0108] Table 2 is provided in relation to human activating receptors and costimulatory receptors, and it is expected that those skilled in the art will understand that homolog and ortholog forms of each receptor are present in most mammalian and vertebrate species. Accordingly, the sequences mentioned above are provided only as non-limiting examples of receptor sequences that may be included in the CARs of the first aspect of the invention, as well as homolog and ortholog sequences from any desired species that may be used to generate CARs suitable for a given species.

[0088]

[0109] In some embodiments of the first aspect of the invention, the signaling domain comprises a portion derived from an activating receptor and a portion derived from a costimulatory receptor. Without being bound by theory although not wishing to be so bound, in this context, recognition of an antigen by the antigen recognition domain of the CAR is expected to simultaneously induce both an intracellular activating signal and an intracellular costimulatory signal. As a result, it is expected that this will stimulate antigen presentation by APCs expressing the costimulatory ligand. Alternatively, the CAR may have a signaling 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 signaling cascade or a costimulatory intracellular signaling cascade.

[0089]

[0110] In some embodiments of the first aspect of the present invention, the CAR is expected to have a signaling domain that includes one part derived from a single activating receptor and multiple parts derived from multiple co-stimulatory receptors. In some embodiments, the CAR is expected to have a signaling domain that includes multiple parts derived from multiple activating receptors and one part derived from a single co-stimulatory receptor. In some embodiments, the CAR is expected to have a signaling domain that includes multiple parts derived from multiple activating receptors and multiple parts derived from multiple co-stimulatory receptors. In some embodiments, the CAR is expected to have a signaling domain that includes one part derived from a single activating receptor and multiple parts derived from two co-stimulatory receptors. In some embodiments, the CAR is expected to have a signaling domain that includes one part derived from a single activating receptor and multiple parts derived from three co-stimulatory receptors. In some embodiments, the CAR is expected to have a signaling domain that includes multiple parts derived from two activating receptors and one part derived from a single co-stimulatory receptor. In some embodiments, the CAR is expected to have a signaling domain that includes multiple parts derived from two activating receptors and multiple parts derived from two co-stimulatory receptors. As will be appreciated, there are further variations in the number of activating receptors and co-stimulatory receptors from which the signaling domain can be derived, and the examples above are not considered to limit the possible combinations included herein.

[0090]

[0111] In some embodiments of the first aspect of the present 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 shown above, the present invention includes any one of the functional variants 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" can include any amino acid sequence as 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, as long as a 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, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54, the 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 , one of the mutant forms or allelic variants of SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54; one ortholog 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; one homeologue 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; one analog 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, etc. may be included.

[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 a dysfunctional P2X7 receptor and induce an intracellular signal that results in the activation of T cells expressing the CAR without significant recognition of a functional P2X7 receptor. As will be understood by those skilled in the art, changes to portions of the amino acid sequence of the chimeric antigen receptor set forth 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 modification of the recognition of the dysfunctional P2X7 receptor and / or the activation of T cells expressing the CAR. Such changes can include, but are not limited to, changes in the hinge region of the chimeric antigen receptor, changes in the transmembrane domain, and changes in portions of the activating receptor and / or co-stimulatory receptor that make up the intracellular domain of the chimeric antigen receptor.

[0094]

[0115] As shown above, a functional variant 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, one of ordinary skill in the art would be expected to recognize that any amino acid can be substituted with a chemically (functionally) similar amino acid and the function of the polypeptide can be retained. 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 one another.

[0095]

Table 3

[0096]

[0116] Furthermore, if desired, non-natural amino acids or chemical amino acid analogs can be introduced into the polypeptides encompassed herein as substitutions or additions. Such amino acids include, but are 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-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, as well as common amino acid analogs.

[0097]

[0117] As described above, any one of the functional variants 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, the functional variant may have 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 frame determined by the length of the polypeptide. For example, 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 full length comparison frame 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 is utilized. The comparison frame may include an addition or deletion of about 20% or less (i.e., a gap) compared to the reference sequence (without addition or deletion) for optimal alignment of the two sequences. The optimal alignment of the sequences for aligning the comparison frame can be performed, for example, by a computer implementation of an algorithm such as the programs of the BLAST family disclosed by Altschul et al., 1997, Nucl. Acids Res. 25: 3389 - 3402. Global alignment programs can also be used to align similar sequences of approximately equal size. Examples of global alignment programs include NEEDLE (available at www.ebi.ac.uk / Tools / psa / emboss_needle / ), which is part of the EMBOSS package (Rice P et al., 2000, Trends Genet., 16: 276 - 277), and the GGSEARCH program (fasta.bioch.virginia.edu / fa Those available at sta_www2 / fasta_www.cgi?rm=compare&pgm=gnw can be mentioned. All of these programs are based on the Needleman-Wunsch algorithm used to find the optimal alignment (including gaps) of two sequences along their entire lengths. A detailed consideration 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 present invention. In some embodiments, the nucleic acid molecule is a non-natural nucleic acid molecule.

[0100]

[0120] In some embodiments of the second aspect of the present invention, the nucleic acid molecule comprises a nucleotide sequence encoding 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 encoding 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] A nucleic acid molecule can comprise any polynucleotide or polydeoxynucleotide, which can be RNA or DNA, whether unmodified or modified. 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, hybrid molecules comprising DNA and RNA, which may be single-stranded, more typically may be double-stranded, or may be a mixture of single-stranded and double-stranded regions. In addition, the nucleic acid molecule may include a triple-stranded region, comprising RNA or DNA, or both RNA and DNA. The nucleic acid molecule may also include one or more modified bases, or a DNA or RNA backbone that has been modified for stability or other reasons. A variety of modifications can be made to DNA and RNA, and thus the term "nucleic acid molecule" encompasses chemically, enzymatically, or metabolically modified forms.

[0102]

[0122] In some embodiments of the second aspect of the present 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] 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, as would be understood by those skilled in the art. 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 different 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 any position where alanine in SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37 is specified by a codon, the codon may be modified to any of the corresponding codons described without modifying the encoded polypeptide. Thus , any 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, encompasses any possible silent changes in the nucleotide sequence. It is expected that those skilled in the art will understand that each codon of a nucleic acid (except for AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Thus, any silent changes in the nucleotide sequence encoding a polypeptide are implicitly included in the respective described sequences.

[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 present invention. The nucleic acid construct may further comprise one or more origins of replication of one or more hosts, one or more selectable marker genes active in one or more hosts, and / or one or more of one or more transcriptional control sequences.

[0105]

[0125] As used herein, the term "selectable marker gene" includes any gene that confers a phenotype on a cell in which the gene is expressed so as to facilitate the identification and / or selection of cells transfected or transformed with the construct.

[0106]

[0126] "Selectable marker gene" includes any nucleotide sequence that, when expressed by a cell transformed with a construct, confers on the cell a phenotype that facilitates the identification and / or selection of these transformed cells. Various nucleotide sequences encoding suitable selectable markers are known in the art (e.g., Mortesen, RM. and Kingston RE. Curr Protoc Mol Biol, 2009; Unit 9.5). Exemplary nucleotide sequences encoding selectable markers include the 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 an antibiotic resistance gene, e.g., 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, a fluorescent reporter gene, e.g., a gene encoding a green, red, yellow, or blue fluorescent protein; and, among others, a luminescence-based reporter gene, e.g., the luciferase gene, that enables the optical selection of cells using techniques such as fluorescence-activated cell sorting (FACS).

[0107]

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

[0108]

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

[0109]

[0129] In some embodiments, at least one transcription control sequence is operably linked to a nucleic acid molecule of the second aspect of the present invention. For the purposes of this specification, a transcription control sequence is said to be "operably linked" to a given nucleic acid molecule if it can promote, inhibit, or otherwise regulate the transcription of the nucleic acid molecule. Thus, in some embodiments, the nucleic acid molecule is under the control of a transcription control sequence such as a constitutive promoter or an inducible promoter.

[0110]

[0110]

[0130] A "nucleic acid construct" can be in any suitable form, such as a plasmid, phage, transposon, cosmid, chromosome, vector, etc., which is capable of replication when combined with appropriate control elements and can transfer the gene sequences contained within the construct between cells. Thus, the term includes cloning vehicles 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 a nucleic acid molecule operably linked to a cell, tissue, or organ in which expression occurs. Therefore, the 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, a wide range of promoters are expected to be readily identified by those skilled in the art.

[0112]

[0132] Examples of mammalian constitutive promoters 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 include, but are not limited to, chemical inducible promoters and physical inducible promoters. Chemical inducible promoters include promoters having activity controlled by a compound, such as alcohol, antibiotics, steroids, metal ions, or other compounds. Examples of chemical inducible promoters include, inter alia, tetracycline-regulated promoters (see, for example, U.S. Patent Nos. 5,851,796 and 5,464,758); steroid-responsive promoters, such as the glucocorticoid receptor promoter (see, for example, U.S. Patent No. 5,512,483), the ecdysone receptor promoter (see, for example, U.S. Patent No. 6,379,945), etc.; and metal-responsive promoters, such as the metallothionein promoter (see, for example, U.S. Patent Nos. 4,940,661, 4,579,821, and 4,601,978).

[0114]

[0134] As described above, the control array may also include a terminator. The term "terminator" refers to a DNA sequence at the end of a transcription unit that signals the termination of transcription. Terminators are generally 3' untranslated DNA sequences that include a polyadenylation signal, which facilitates the addition of a polyadenylation sequence to the 3' end of the primary transcript. Similar to promoter sequences, terminators can be any terminator sequence that is operative in the cell, tissue, or organ in which it is intended to be used. Suitable terminators are expected to be known to those of skill in the art.

[0115]

[0135] As will be appreciated, the nucleic acid construct of the third aspect of the invention may further comprise additional sequences, for example, sequences that enable enhancement of expression, cytoplasmic or membrane transport, and localization signals. Non-limiting specific examples include internal ribosome entry sites (IRES).

[0116]

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

[0117]

[0137] Methods for examining the introduction (transfection / transduction) of exogenous gene materials, such as nucleic acid constructs of the third aspect of the present invention, into eukaryotic cells are known in the art. As will be appreciated, the most suitable method for introducing a nucleic acid construct into a desired host cell depends on a number of factors, such as the size of the nucleic acid construct, the type of host cell, the desired efficiency of transfection / transduction, and, if desired or necessary, the ultimate viability of the transfected / transduced cells. Non-limiting examples of such methods include chemical transfection with chemicals such as cationic polymers, calcium phosphate, or structures such as liposomes and dendrimers; non-chemical methods, such as electroporation, 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 is expected to be selected according to 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 a host cell is viral transduction. Methods of using viral transduction to induce CAR expression in PBMC (Parker, LL. et al., Hum Gene Ther. 2000;11:2377-87), and more generally, methods of using retroviral systems for the 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 present invention.

[0140] In some embodiments of the fourth aspect of the present invention, the genetically modified cell comprises 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 present invention, or a nucleic acid construct according to the third aspect of the present invention, or a genomic integration form of the nucleic acid construct.

[0121]

[0142] In some embodiments of the fifth aspect of the present invention, the genetically modified cell comprises a nucleic acid molecule or a nucleic acid construct encoding two or more different CARs. In some embodiments of the fifth aspect of the present 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 used herein, "genetically modified cell" includes any cell comprising a nucleic acid molecule or nucleic acid construct that is non-naturally occurring and / or introduced and encompassed by the present invention. The introduced nucleic acid molecule or nucleic acid construct can be maintained as a separate DNA molecule in the cell or integrated into the genomic DNA of the cell.

[0123]

[0123]

[0144] The genomic DNA of a cell should be understood in a broad sense to include all endogenous DNA that constitutes the genetic complementarity of the cell. Therefore, the genomic DNA of a cell should be understood to include chromosomes, mitochondrial DNA, and the like. Accordingly, the term "genomic integration" contemplates chromosomal integration, mitochondrial DNA integration, and the like. The "genomic integration form" of a construct can be all or part of the construct. However, in some embodiments, the genomic integration form of a construct includes at least the nucleic acid molecule of the second aspect of the present invention.

[0124]

[0145] As used herein, the terms "different CARs" or "different chimeric antigen receptors" refer to any two or more CARs having either non-identical antigen recognition domains and / or non-identical signaling domains. In one example, "different CARs" include two CARs having different signaling domains, such as having the same antigen recognition domain (e.g., both CARs can recognize a dysfunctional P2X7 receptor), where one CAR has a signaling domain that is part of an activating receptor and the other CAR has a signaling domain that is part of a co-stimulatory receptor. As will be appreciated, at least one of the two or more CARs within this embodiment has an antigen recognition domain that recognizes a dysfunctional P2X7 receptor, and the other CARs may take any suitable form and may be directed against any suitable antigen.

[0125]

[0146] Accordingly, in some embodiments of the fourth and fifth aspects of the present invention, two or more different CARs have different signaling domains and may have identical or different antigen recognition domains. Specifically, a genetically modified cell according to the fourth or fifth aspect of the present invention may include a first chimeric antigen receptor having a signaling domain that includes a portion derived from an activating receptor and a second chimeric antigen receptor having a signaling domain that includes a portion derived from a co-stimulatory receptor.

[0126]

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

[0127]

[0148] In some embodiments of the fourth or fifth aspect of the present invention, the costimulatory receptor (from which a part of the signaling portion 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 present invention, the costimulatory receptor (from which a part of the signaling portion domain 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 present invention, the genetically modified cell is further modified to constitutively express a costimulatory receptor.

[0151] As described above, the immune response of a cell is typically induced only when an activating signal (typically in response to an antigen) and a costimulatory signal are experienced simultaneously. Thus, two or more CARs are combined to provide two or more CARs that provide both an intracellular activating signal and an intracellular costimulatory signal, in the above-described embodiments By having genetically modified cells by a part of the state, ensure that a sufficient immune response can be induced in response to the recognition of allogeneic antigens by the CAR. Alternatively, the genetically modified cells may contain only one CAR having an antigen recognition domain that recognizes a dysfunctional P2X7 receptor, and constitutively express a costimulatory receptor, thereby increasing the possibility that costimulation is provided simultaneously when the CAR is activated. Alternatively, the genetically modified cells may be further modified to constitutively express both a costimulatory receptor and its ligand. In this case, the cells are continuously stimulated by costimulation, and for the immune activation of the cells, only the activation of the CAR having a signaling domain containing a portion derived from the activating receptor is required.

[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 for the costimulatory receptor, thereby promoting self-stimulation of the cells. Examples of CAR-expressing T cells that express both a costimulatory receptor and their allogeneic ligand (so as to be able to induce self-stimulation) are known in the art, and in particular, those disclosed in Stephen MT. et al., Nat Med, 2007; 13: 1440-9 are included.

[0131]

[0153] The ability of genetically modified cells comprising a CAR can be enhanced by further modifying the cells to secrete a cytokine, preferably a pro-inflammatory cytokine or a growth-promoting cytokine. Secretion of this cytokine serves both to provide autocrine support for cells expressing the CAR and to alter the local environment surrounding the CAR-expressing cells such 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 invention, the genetically modified cells are further modified to secrete a cytokine. This secretion may be constitutive or inducible upon recognition of its cognate ligand antigen by the CAR.

[0132]

[0154] Any one or more cytokines can be selected according to the desired immune response, but 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 aspect of the invention can be any suitable immune cells 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 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 to 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. In other embodiments, the CAR indirectly recognizes the dysfunctional P2X7 receptor.

[0136]

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

[0137]

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

[0138]

[0160] As a result, in some embodiments of the sixth aspect of the present invention, the chimeric antigen receptor recognizes a dysfunctional P2X7 receptor via a mediator. The mediator can be a molecule such as a probe that directly binds to or interacts with the dysfunctional P2X7 receptor. Non-limiting examples of such probes include antibodies, Fab of antibodies, scFv, soluble engineered TCR, or aptamers. The CAR may directly recognize the probe, or the probe may have a tag that is recognized by the CAR. In any case, the probe provides specificity for the target cell (i.e., the cell having a dysfunctional P2X7 receptor), while the genetically modified cell having the CAR provides efficacy and directs the immune response to the target cell. Alternatively, the mediator may be an intracellular endogenous marker that is associated with or whose expression correlates with the dysfunctional P2X7 receptor. Dysregulation of the marker may be a result or a cause of the dysfunction of the P2X7 receptor.

[0139]

[0161] In some embodiments of the sixth aspect of the present invention, the method of killing cells having a dysfunctional P2X7 receptor further comprises the step of exposing the cells having the dysfunctional P2X7 receptor to a mediator.

[0140]

[0162] In some embodiments of the sixth aspect of the present invention, the mediator 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] As used throughout this specification, the term "aptamer" refers to any oligonucleic acid, polynucleic acid, peptide, or polypeptide that specifically binds to or preferentially forms a complex with a target (specifically, a dysfunctional P2X7 receptor).

[0142]

[0164] In some embodiments of the sixth aspect of the present invention, the probe includes a tag, and the chimeric antigen receptor recognizes the tag. Examples of CARs that recognize cells using a mediator 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 cells having the dysfunctional P2X7 receptor are in the body of a subject. In some embodiments, the subject is a human. In some embodiments, the method further comprises the step of exposing cells expressing the dysfunctional P2X7 receptor to a genetically modified cell together with an exogenous cytokine.

[0144]

[0166] In some embodiments of the sixth aspect of the present invention, the genetically modified cell is a genetically modified cell that is 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 cells expressing the dysfunctional P2X7 receptor are in the body of the subject. In some embodiments of the sixth aspect of the present invention, the cells expressing the dysfunctional P2X7 receptor are cancer cells.

[0146]

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

[0147]

[0169] As used herein, the terms "treat," "treating," or "treatment" are to 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 the subject (including delaying and completely stopping growth, and 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 the subject, (iii) reducing the number of tumor cells in the subject, (iv) reducing the size of a tumor in the subject, (v) inhibiting the invasion of tumor cells into peripheral organs (i.e., including reducing, delaying, or completely stopping), (vi) inhibiting metastasis (i.e., including reducing, delaying, or completely stopping), (vii) improving the life expectancy of the subject as compared to an untreated state, (viii) improving the quality of life of the subject as compared to an untreated state, (ix) alleviating, attenuating, or reducing at least one symptom of cancer in the subject, (x) causing regression or remission of cancer in the subject, (xi) alleviating the condition in the subject caused by cancer, and (xii) stopping symptoms associated with cancer in the subject.

[0148]

[0170] As used herein, the term "prevent" or "preventing" includes 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 cancer recurrence in a subject in remission.

[0149]

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

[0150]

[0172] Inhibition of the growth of cancer, tumors, or cancerous cells can be evaluated by a wide variety of methods known in the art. For example, for cancerous cells in vitro, cell growth can be determined by a suitable proliferation assay or by a method that evaluates the extent of incorporation of tritiated thymidine into cellular DNA over a given period of time. For tumor cells or cancerous cells present in vivo, tumor or cell growth can be determined, for example, by a suitable imaging method known in the art.

[0151]

[0173] As used herein, the term "subject" can refer to any animal that can be afflicted with cancer. Particular subjects of interest are humans, as well as scientific relevant species such as mice, rats, ferrets, guinea pigs, hamsters, non-human primates, dogs, pigs, and sheep, or economic relevant animals such as horses, dogs, cats, and cattle. In a preferred embodiment of the sixth aspect of the present invention, the subject is a human.

[0152]

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

[0153]

[0175] As used throughout this specification, reference to a "directed" CAR to a target having a dysfunctional P2X7 receptor is intended to selectively define a cell as a target of an immune response based on cells having a dysfunctional P2X7 receptor. Importantly, such targeting is not limited to direct recognition of the dysfunctional P2X7 receptor by the CAR. That is, the CAR itself need not directly recognize or bind to the dysfunctional P2X7 receptor, but simply be able to selectively recognize and thereby be activated by cells expressing the dysfunctional P2X7 receptor.

[0154]

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

[0155]

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

[0156]

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

[0157]

[0179] As a result, in some embodiments of the sixth aspect of the present invention, the chimeric antigen receptor recognizes a dysfunctional P2X7 receptor via a mediator. The mediator can be a molecule such as a probe that binds directly to or interacts with the dysfunctional P2X7 receptor. Non-limiting examples of such probes include antibodies, Fab of antibodies, scFv, soluble engineered TCR, or aptamers. The CAR may directly recognize the probe, or the probe may have a tag that is recognized by the CAR. In any case, the probe provides specificity for the target cell (i.e., the cell having a dysfunctional P2X7 receptor), while the genetically modified cell having the CAR provides efficacy and directs the immune response to the target cell. Alternatively, the mediator may be an endogenous cellular marker that is associated with or whose expression correlates with the dysfunctional P2X7 receptor. Dysregulation of the marker can be a result or a cause of the dysfunction of the P2X7 receptor.

[0158]

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

[0181] In some embodiments of the sixth aspect of the present invention, the mediator 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] As used throughout this specification, the term "aptamer" refers to any oligonucleic acid, polynucleic acid, peptide, or polypeptide that specifically binds to or preferentially forms a complex with a target (specifically, a dysfunctional P2X7 receptor).

[0160]

[0183] In some embodiments of the sixth aspect of the present invention, the probe includes a tag, and the chimeric antigen receptor recognizes the tag. Examples of CARs that recognize cells using a mediator 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, the method comprising administering to the subject genetically modified cells according to the fourth or fifth aspect of the present invention. step.

[0162]

[0185] Although the provision of genetically modified cells expressing a CAR directed to target cells having a dysfunctional P2X7 receptor may be sufficient to provide an effective immunotherapy against pre-cancerous or cancerous cells, by providing an adjuvant together with the genetically modified cells, the induction of an immune response can be further enhanced and the immunotherapy can be supplemented. Cytokines, preferably pro-inflammatory cytokines, are particularly suitable adjuvants to be provided to the subject together with the genetically modified cells having a CAR.

[0163]

[0186] Thus, in some embodiments of the sixth and seventh aspects of the present invention, the genetically modified cells are administered to a subject together with a cytokine. As used throughout this specification, the term "together with" is to be understood to include that the genetically modified cells are administered simultaneously with the cytokine or administered in combination with the cytokine. As a result, when administered together with the cytokine, this can be considered to include combination therapy, in which case the immunotherapy of the subject includes both treatment with the cytokine and treatment with the genetically modified cells having a CAR directed to target cells expressing a dysfunctional P2X7 receptor. In some forms, the cytokine is administered on a different day (more than 24 hours) from the administration of the genetically modified cells. In other forms, the cytokine is administered on the same day (within 24 hours) as the genetically modified cells. In further forms, the cytokine and the genetically modified cells are administered within 18 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 2 minutes, or 1 minute of each other.

[0164]

[0187] Cytokines suitable for administration together with the genetically modified cells include IL-2, IL-4, IL-6, IL-7, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, IFNα, IFNβ, IFNγ, GM-CSF, TGFβ, and TNFα. Preferred cytokines include IL-2 and IFNα. Further, the cytokine can be administered via a delivery system such as a protein, as a recombinant form, as a natural form, or as a nucleic acid sequence expressed in the genetically modified cells or conjugated to a polymer such as polyethylene glycol (PEG), such as a fusion with a protein.

[0165]

[0188] The cells 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 cells to be genetically modified are autologous cells, which are cells that are autologous to cells expressing a dysfunctional P2X7 receptor. Advantageously, autologous cells are not recognized as "non-self" by the immune system of the subject and are thus expected to be tolerated by the subject. However, in some forms of cancer, suitable autologous cells may not be readily available. Thus, in some embodiments of the present invention, the cells to be genetically modified are allogeneic or xenogeneic cells.

[0166]

[0189] P2X7 dysfunction is a common molecular alteration in various cancers. As a result, the methods 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 present invention, the method is used for the prevention or treatment of cancer selected from one or more of brain cancer, esophageal cancer, oral cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cervical cancer, epithelial cell cancer, skin cancer, leukemia, lymphoma, myeloma, breast cancer, ovarian cancer, endometrial cancer, testicular cancer. Preferably, the cancer is 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, testicular cancer.

[0168]

[0191] In some embodiments of the sixth or seventh aspect of the present invention, the cancer is metastatic cancer, for example, 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 so as to increase the total number of cells available for treatment. This can be done using the step of exposing the cells to the antigen of the CAR. Thus, in an eighth aspect, the present invention provides a method for expanding in vitro genetically modified cells according to the fourth or fifth aspect of the present invention, the method comprising the step of exposing the cells to the antigen of the CAR. In some embodiments, the method further comprises the step of exposing the cells to a cytokine.

[0169]

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

[0170]

[0194] Preferred cytokines used in the eighth or ninth aspect of the present invention may include members of the IL-2 subfamily, interferon subfamily, IL-10 subfamily, IL-1 subfamily, IL-17 subfamily, or TGF-β subfamily. In some embodiments of the eighth or ninth aspect of the present 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 combinations thereof.

[0171]

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

[0172]

[0196] As will be appreciated by those skilled in the art, depending on the signaling domain of the CAR, recognition of its cognate antigen by the CAR causes intracellular signaling, which can ultimately cause cell proliferation. Thus, even a small number of cells, or even individual cells, can proliferate (or in the case of a single cell, clonally expand) to form a therapeutically significant number. This process can be further enhanced by the provision of cytokines.

[0173]

[0197] Delivery or administration of a genetically modified cell according to the fourth or fifth aspect of the present invention may be delivery or administration of the cell alone, or delivery or administration of the cell formulated in a suitable pharmaceutical composition. Thus, 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.

[0174]

[0198] Methods for providing cells comprising a CAR for immunotherapy are known in the art (e.g., Kershaw, MH. et al., Clin Cancer Res. 20 06;12(20):6106-15; see Parker LL. et al., Hum Gene Ther 2000;11:2337-87). Further, protocols and methods for the preparation, expansion, 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 following examples.

[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 diluents, 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 of the cytokines described above. In some embodiments, the pharmaceutical composition may also contain the mediators 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 into the site of a tumor or cancer mass.

[0177]

[0201] Throughout this specification, unless the context requires otherwise, variations such as "comprise", "comprises", or "comprising" are to be understood to imply the inclusion of the 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 of molecular biology, including methods for performing the basic techniques encompassed by the present invention. For example, see Green MR and Sambrook J, Molecular Cloning: A Laboratory Manual (4th Edition), 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 will be apparent to those skilled in the art that various modifications and changes can be made to the embodiments and methods described herein without departing from the scope of the inventive concepts disclosed herein.

[0180]

[0204] The present invention is further illustrated in the following examples. These examples are for the purpose of illustrating specific embodiments only and are not intended to limit the foregoing description.

Examples

[0181] Example 1 Protocol for the Design and Expression of the PEP2-2-3 Binding Peptide Chimeric Antigen Receptor (CAR)

[0205] An exemplary protocol detailing the process of designing and expressing an anti-non-functional (nf) P2X7 receptor CAR according to an embodiment of the present invention is described in detail below.

[0182] Design of the PEP2-2-3 (anti-nf P2x7) Chimeric Antigen Receptor

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

[0183]

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

[0184]

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

[0185]

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

[0186]

[0210] A portion of the transmembrane domain 3 and the intracellular domain 6 of the CAR is provided by a portion 7 of the CD28 co-stimulatory receptor (the amino acid sequence is described in SEQ ID NO: 18 and the nucleotide sequence is described in SEQ ID NO: 19). The intracellular domain further includes a portion 8 of the co-stimulatory receptor OX40 (the amino acid sequence is described in SEQ ID NO: 20 and the nucleotide sequence is described in SEQ ID NO: 21), and a portion 9 of the activating receptor CD3 zeta (the amino acid sequence is described in SEQ ID NO: 22 and the nucleotide sequence is described in SEQ ID NO: 23).

[0187]

[0211] The P2A sequence 10 (the amino acid sequence is described in SEQ ID NO: 24 and the nucleotide sequence is described in SEQ ID NO: 25) was added to the C-terminus of the CAR, enabling post-translational excision of any peptide sequence added to the C-terminus of the CAR. The amino acid sequences of the constructed anti-nfP2X7 CAR-long hinge and anti-nfP2X7 CAR-short hinge are described in SEQ ID NOs: 26 and 27, respectively.

[0188] Design and assembly of the lentiviral vector

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

[0189]

[0213] Sequences having homology to the sequences upstream and downstream of the NheI restriction site of the BLIV vector were added to the 5'-end and 3'-end of the designed CAR, SEQ ID NO: 2 The final nucleotide sequences described in SEQ ID NO: 8 (CAR-long hinge) and SEQ ID NO: 29 (CAR-short hinge) were obtained. By including the 5' and 3' sequences, it became possible to incorporate the anti-nf P2X7 CAR into the BLIV vector using Gibson cloning.

[0190]

[0214] The nucleotide sequences of anti-nf P2X7 CAR-long hinge and anti-nf P2X7 CAR-short hinge were constructed using gene block technology (gBlock™ Gene Fragments - Integrated DNA Technologies, Iowa, USA) and assembled using the 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 treated with a restriction enzyme at the NheI cloning site, and the anti-nf P2X7 CAR coding sequence was incorporated using Gibson assembly. Cloning and evaluation of the BLIV-CAR vector

[0216] The generated 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. Summary: - Tubes of NEB's 5-alpha competent E. coli cells were 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 gently swirling the tube 4 to 5 times. - The mixture of E. coli and plasmid was left on ice for 30 minutes without mixing. - The cell and plasmid mixture 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, then heated at 37°C for 60 minutes with vigorous shaking. - The selection plate was prepared and heated to 37°C. - Serial 10-fold dilutions of the cells were prepared in SOC solution. - 50 - 100 μl of each dilution was spread on the selection plate and incubated overnight at 37°C.

[0192]

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

[0193]

[0218] Clones 2 to 4 (L2 to L4) of bacteria containing the BLIV-CAR-long hinge plasmid and clone 4 (S4) of bacteria 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 showed restriction enzyme-digested fragments of the predicted lengths as described in Table 4 and Figure 4.

[0194]

Table 4

[0195] Construction and verification of the lentiviral vector

[0219] Lentiviruses were packaged by the three-plasmid protocol using 293T cells according to the following method. Day 1: Cells were seeded into a T-225 flask with 35 ml of DMEM medium containing 10% serum so that the 293T cells would reach 90 - 95% confluence the next day. Day 2: Add any 30 μg of the generated BLIV-CAR plasmid (or unmodified BLIV plasmid), 30 μg of the gag-pol plasmid delta 8.2, and 15 μg of the VSV-G plasmid (pMD2.G) to OptiMEM medium to make a final volume of 750 μl and mix. Add 300 μl of the PEI solution and incubate at room temperature for at least 20 minutes. Then add the mixture to the confluent 293T cells and incubate 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. After replacing the decanted mixture with 35 ml of fresh medium, it was further incubated at 37°C. Day 4: 48 hours after adding the plasmid mixture, the medium was removed and combined with the supernatant collected over 24 hours. The combined supernatant was centrifuged at 1500 g for 15 minutes to remove all remaining cell debris. The supernatant was filtered through a 0.45 μm filter and then centrifuged at 17,000 rpm for 1 hour in a WX ultracentrifuge. After centrifugation, the supernatant was manually decanted, leaving 50 - 200 μl in the tube. The centrifuge tube was placed in a 50 ml screw-cap tube to prevent contamination and evaporation, and the virus was resuspended at 4°C overnight. 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 centrifuged at 5000 rpm for 5 minutes in a microcentrifuge tube to remove all remaining debris.

[0196]

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

[0197] Screening for CAR T cell function

[0221] 10 8 Ten 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. Analysis of purity showed that 76.6% of the purified cells were CD8+ as shown in FIG. 7.

[0198]

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

[0199]

[0223] Target cells expressing a non-functional P2X7 receptor were obtained with the mammalian cancer cell line BT549 (ATCC HTB-122). These cells were labeled with the fluorescent membrane-inserting dye eFluor™ 670 (affymetrix eBioscience) according to the manufacturer's instructions. Summary: - BT549 cells were prepared as a single cell suspension and washed twice in PBS to remove all residual serum. - The 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 the 10 μM dye solution was added to the prepared BT549 cells to obtain a dye solution with a final concentration of 5 μM. - The BT549 cells in the dye solution were incubated at 37 °C for 10 minutes in the dark, and then labeling was stopped by adding four volumes of cold culture medium containing 10% serum. The cells were then incubated 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 the 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, the cells were harvested and analyzed using fluorescence-activated cell sorting (FACS). The number of target cells containing the membrane-inserted dye was quantified to evaluate whether the co-cultured T cells had caused the death or arrest of cell proliferation of the target cells. 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 after co-culturing control CD8+ T cells and BT549 targets for 24 hours. Figure 8D illustrates the gating and histogram analysis after co-culturing CD8+ T cells transduced with BLIV-CAR-long hinge and BT549 target cells for 24 hours. Figure 8E illustrates the gating and histogram analysis after co-culturing CD8+ T cells transduced with BLIC-CAR-short hinge and BT549 target cells for 24 hours.

[0201]

[0226] As can be seen in FIG. 9, when target cells were co-cultured with CD8 T cells transduced with a lentivirus containing either BLIV-CAR-long hinge or BLIV-CAR-short hinge, there was an increase in the number of BT549 target cells that disappeared (were killed) compared to co-culture with non-transduced cells or cells transduced with a control (unmodified BLIV vector) CD8 T cell. There was an increase in the number of BT549 target cells that disappeared (were killed) compared to co-culture with non-transduced cells or cells transduced with a control (unmodified BLIV vector) CD8 T cell.

[0202]

[0227] Considering the results presented in FIG. 9, it is clear that CD8+ T cells transduced with an anti-nfP2X7 CAR receptor (having a short or long hinge) show an increased level of cytotoxic activity against non-functional P2X7-expressing target cells, demonstrating the ability of CAR-T cells to kill cancer cell targets.

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

[0228] A further exemplary protocol detailing the design and expression process of an anti-non-functional (nf) P2X7 receptor CAR according to an embodiment of the present invention in T cells is described in detail below.

[0204]

[0229] Three anti-non-functional P2X7 binding peptides were used to design an anti-nfP2X7 CAR. Specifically, the CAR was designed to include an antigen recognition domain having sequence homology to peptide 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 the non-functional P2X7 receptor (Barden, J.A., Sluyter, R., Gu, B.J. & Wiley, J.S. 2003. Specific detection of non-functional human P2X(7) receptors in HEK293 cells and B-lymphocytes. FEBS Lett 538, 159-162).

[0205]

[0230] An alignment of the above binding peptide and the heavy chain variable region of an antibody that recognizes a non-functional P2X7 receptor is shown in FIG. 10. The alignment of the sequences of the complementarity determining regions (CDR1 to 3) is indicated by squares.

[0206]

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

[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 having the structure described below.

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

[0208]

[0234] The antigen recognition domain was then ligated 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 described in SEQ ID NO: 14 and the nucleotide sequence described in SEQ ID NO: 15).

[0209]

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

[0210]

[0236] The intracellular portion 17 of the CAR was provided by linking the cytoplasmic domain 18 of a portion 14 of the human CD28 molecule described above and the 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] Design and Assembly of Lentiviral Vectors

[0237] The nucleotide sequences of the designed PEP2-2-1-1, PEP2-472-2, and PEP2-2-12 CARs were constructed using gene block technology (gBlock™ Gene Fragments - Integrated DNA Technologies, Iowa, USA) and assembled using the Gibson Assembly Cloning Kit (New 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 CAR for incorporation into a cloning vector (containing restriction sites) are set forth in SEQ ID NO: 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) illustrated in Figure 11. This vector contains the green fluorescent protein (GFP), a fluorescent reporter protein. The pCDH vector further contains a T2A coding sequence between the cloning site and GFP, enabling post-translational separation of the cloned CAR and the GFP protein.

[0213]

[0239] To incorporate the nucleotide constructs of PEP2-2-12 and PEP2-472-2 CAR into the pCDG vector, the pCDH vector was digested with restriction enzymes EcoR1 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 restriction enzymes. The digested gBlock fragments were then purified using the QIAquick PCR Purification Kit according to the manufacturer's instructions. The digested vector was ligated to the digested CAR construct at a molar ratio of insert to vector of 3:1. 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 thus incorporated into the pCDH vector in a different manner from the PEP2-2-12 and PEP2-472-2 CAR nucleotide constructs. The pCDH vector was digested with EcoR1, and the resulting 5'-overhangs were filled in with T4 DNA polymerase in the presence of 100 μM dNTP (15 minutes at 12 °C). The reaction was terminated (20 minutes at 75 °C in the presence of 10 mM 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 and then digested with NotI (both at 25 °C). The digested gBlock fragment was purified using the QIAquick PCR purification kit according to the manufacturer's instructions. The digested vector

[0215] Cloning and evaluation of the pCDH-CAR vector

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

[0216]

[0242] After incubating the transformed (SURE2) cells, for each of the PEP2 - 2 - 1 - 1, PEP2 - 472 - 2, or PEP2 - 2 - 12 binding peptides, 5 - 6 colonies of cells transformed with pCDH - CAR were isolated and further incubated overnight. Plasmid DNA was isolated from each of the cultured colonies using the Quicklyse MiniPrep Kit (QIAGEN) and digested with the EcoRI / Not I restriction enzyme. The digested DNA was analyzed by gel electrophoresis of restriction enzyme - treated fragments of the appropriate size.

[0217]

[0243] As shown in Figure 13, colonies 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 restriction enzyme - treated fragments of the appropriate size.

[0218]

[0244] Sequencing was performed on each of the selected clones, and using appropriate primers selected from Table 5, the integration of CAR was confirmed.

[0219]

Table 5

[0220]

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

[0221] Construction and verification of viral vectors

[0246] In transiently transfected Hek293T cells, lentivirus packaging was performed using Lipofectamine 2000 reagent (Invitrogen) according to standard laboratory protocols (Brown, C.Y. et al., 2010. Robust, reversible gene knockdown using a single lentiviral short hairpin RNA vector. Hum Gene Ther 21, 1005-1017). Summary: - 12.5 μg of lentiviral vector DNA was mixed in a T75 cm flask at 3.75 μg of pMD2.g (VSV-G envelope expression vector), 6.25 μg of pRSV-Rev, and 7.5 μg of pCMVdelta8.2 per transfection, and 75 μl of Lipofectin was used according to the manufacturer's protocol and incubated overnight. - The next morning, the medium was replaced and the supernatant containing the virus 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. - Virus particles from the filtered supernatant were concentrated by ultracentrifugation (68,000×g for 90 minutes at 4°C, Beckman SW32 rotor). The supernatant was removed, and the virus pellet was slowly resuspended in DMEM on ice. - 100 ul aliquots of the virus were stored at -70°C until needed.

[0222]

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

[0223]

[0248] The virus titer was calculated by transducing serial dilutions (1:50 and 1:100) of the concentrated LV stock into a known number of Hek293T cells. Transduction was carried out overnight in the presence of 8 ug / ml polybrene (hexadimethrine bromide). The next day, the medium containing the virus and polybrene was replaced with fresh medium, and the cells were harvested 24 hours later. The proportion of GFP-positive cells was determined by flow cytometry. The virus titer was calculated using the formula: transduction units / ml (TU) = (FxC / V)xD, where F = frequency of GFP+ cells (%GFP+ / 100), C = number of cells at the time of virus addition, V = volume of transduction in mL, and D = dilution factor. Representative flow data for LV-PEP2-472-2 transduction are shown in Figure 15. The TUs of the PEP2-2-1-1, PEP2-12-2, and PEP2-472-2 CAR virus vectors are 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 Service) from anonymous donors. The MNCs were isolated using Ficoll-Paque™ density gradient medium. CD8 T cells were purified from the MNCs using the Dynabeads® Untouched™ Human CD8 T Cell Kit (Invitrogen) according to the manufacturer's instructions. The purity of the isolated cells, as evaluated by flow cytometry, was 85% or greater.

[0227]

[0251] 2×10 6 Individual purified cells were pre-incubated for 30 minutes with CD3 / CD28 beads (bead-to-cell ratio 3:1) and IL2 (500 U / ml), and then virus-containing LV-PEP2-2-1-1, LV-PEP2-472-2, or empty LV vector (GFP control virus) at a multiplicity of infection (MOI) of 1 to 2 units was added together with 8 μg / ml polybrene. The cells were incubated with the virus for 16 hours, after which the virus-containing medium was removed. The remaining cells and beads were incubated for 40 hours in fresh medium containing IL2, after which the GFP fluorescence level was analyzed.

[0228]

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

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

[0229]

[0254] 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 from pDisplay (Invitrogen, Figure 17) such that the DNA sequence encoding the fusion protein consisting of IgK-leader-HA-MYC-PDGFR-transmembrane domain was expressed in-frame. These fusion proteins were designed to be surface-expressed. 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 carried out according to the manufacturer's protocol. Overview: - First, a BP recombination reaction was performed 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 to generate entry clones. Chemically competent E.cloni® 10G cells (Lucigen®) were transformed according to the manufacturer's protocol using the BP recombination reaction. - The transformed cells were seeded onto LB agar plates containing 50 μg / ml kanamycin (Sigma) and incubated overnight at 37°C. - Two clones were selected from each plate and used to prepare microcultures (2 mL) in LB broth with kanamycin (SIGMA) (50 μg / ml). After incubation overnight at 37°C, agitation was performed. - The next day, plasmid DNA was extracted from the microcultures using the QIAGEN QuickLyse miniprep kit. - Recombinant clones were identified by performing diagnostic Bam H1-HF (NEB) and Pmel (NEB) digestions. After Bam H1 and Bam H1 / PmeI digestions, gel electrophoresis (Figure 18) was used to confirm that both the EXD2_K193A and EXD2_WT clones were digested correctly.

[0231]

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

[0232]

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

[0233]

Table 7

[0234]

[0258] The following protocol was used to produce viral particles for transfection 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 6 cells per flask). - HEK293T cells were transfected with a lentiviral packaging vector 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 containing 10% FCS). 10 ml of the medium was collected after 24 hours and stored at -80 °C in 2 ml aliquots until needed. Another 10 ml of fresh medium (DMEM containing 10% FCS) was added to the flask and this was collected after a further 24 hours. - Viral particles were isolated from the collected 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 for transfection of HEK293 cells.

[0235]

[0259] To evaluate the transfection efficiency, after removing the second 10 ml of medium, the cells were harvested 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 at efficiencies of 97% and 85%, respectively.

[0236]

[0260] To generate stable HEK293 cells that overexpress the extracellular domains of functional and non-functional P2X7 on the cell surface, the following protocol was used. - HEK293 cells were seeded into T25 flasks the day before transduction (one flask per 7×10 5 cells). - The next day, the medium was removed from each flask and fresh medium containing the virus particles generated according to the above protocol was added according to the ratios described in Table 8. - Polybrene was added to each flask to a final concentration of 8 μg / mL.

[0237]

Table 8

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

[0239]

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

[0240] Screening for CAR T cell function

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

[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. Overview: - 10 μl of lysis solution (10×) was added to each well for 100 μl of target cells 45 minutes to 4 hours before. - After an additional 45 minutes, the plates were centrifuged at 250 × g for 4 minutes. - 50 μl aliquots were 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 aliquots, and the plate was covered with foil and incubated at room temperature for 30 minutes. - 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 of each well were corrected according to the manufacturer's instructions, and the percentage of cytotoxicity was calculated using the following formula and normalized to T cells transfected with the empty vector 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 transfected with the 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 transfected with the empty vector.

[0245]

[0266] All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly inconsistent with the context. The use of any examples or exemplary language provided herein (e.g., "such as") is merely intended to better illustrate embodiments and does not impose a limitation on the scope of the claimed invention. No syntax in this specification 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 characteristics and features. It should be understood that one or more features of one embodiment may be combinable with one or more features of other embodiments. Additionally, a single feature or combination of features of an embodiment may constitute further embodiments.

[0247]

[0268] The headings used in this specification are included for ease of reference by the reader only and are not used to limit the subject matter found through the present disclosure or the claims. The headings are not used in the interpretation of the claims or limitations of the claims.

[0248]

[0269] Those skilled in the art would expect that the invention described herein is likely to receive changes and modifications other than those specifically described. It is to be understood that the invention includes such variations and modifications. The invention also includes, individually or collectively, all of the steps, features, compositions, and compounds referenced or shown herein, and any combination of any two or more of the steps or features.

[0249]

[0270] Further, it should be noted that as used herein, the singular forms “a,” “an,” and “the” include plural aspects unless the context clearly dictates otherwise.

[0250]

[0271] For example, future patent applications may be filed based on this application by claiming priority under this application, claiming divisional status, and / or claiming continuation status. It is understood that the following claims are not intended to limit the scope that may be claimed in any such future application.

Claims

1. An antigen recognition domain that recognizes a dysfunctional P2X 7 receptor but does not recognize a functional P2X 7 receptor, Transmembrane domain and A signaling domain including the intracellular signaling portion of an activating receptor and / or the intracellular signaling portion of a co-stimulatory receptor A chimeric antigen receptor containing, A chimeric antigen receptor in which the antigen recognition domain contains three CDRs of the amino acid sequence shown in SEQ ID NO:

34.

2. The antigen recognition domain is i) CDR1 containing the amino acid sequence from positions 30 to 35 of SEQ ID NO: 34, ii) CDR2 containing the amino acid sequence from positions 50 to 67 of SEQ ID NO: 34, iii) CDR3 containing the amino acid sequence from positions 98 to 108 of SEQ ID NO: 34 A chimeric antigen receptor according to claim 1, comprising:

3. The chimeric antigen receptor according to claim 1 or 2, wherein the antigen recognition domain contains an amino acid sequence that is 80%, 90%, 95%, or 99% identical to the sequence described in SEQ ID NO: 34, and there are no sequence changes in the CDR compared to SEQ ID NO:

34.

4. The chimeric antigen receptor according to claim 1 or 2, wherein the antigen recognition domain comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHDDMGWVRQAPGKGLEWVSAISGSGGGSTYYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTEFDYRSPGGTLVTVSS.

5. The chimeric antigen receptor according to any one of claims 1 to 4, wherein the antigen recognition domain recognizes an epitope comprising amino acids at positions 200 to 210 of a dysfunctional P2X 7 receptor.

6. The chimeric antigen receptor according to any one of claims 1 to 5, wherein the antigen recognition domain recognizes an epitope comprising amino acids at positions 297 to 306 of a dysfunctional P2X 7 receptor.

7. The chimeric antigen receptor according to any one of claims 1 to 6, wherein the antigen recognition domain is Fab, scFV, sdAb, or a peptide.

8. The chimeric antigen receptor according to any one of claims 1 to 7, wherein the antigen recognition domain is polyvalent.

9. The chimeric antigen receptor according to claim 8, wherein the polyvalent is bivalent or trivalent.

10. A nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor according to any one of claims 1 to 9.

11. A viral vector comprising the nucleic acid molecule according to claim 10 for viral transduction of host cells.

12. A genetically modified cell comprising a chimeric antigen receptor according to any one of claims 1 to 9, a nucleic acid molecule according to claim 10, or a viral vector according to claim 11.

13. The genetically modified cell according to claim 12, which is a leukocyte, peripheral blood mononuclear cell (PBMC), lymphocyte, T cell, CD4+ T cell, CD8+ T cell, natural killer cell, or natural killer T cell.

14. Use of a chimeric antigen receptor according to any one of claims 1 to 9, a nucleic acid molecule according to claim 10, a viral vector according to claim 11, or a genetically modified cell according to claim 12 or 13 in the manufacture of a pharmaceutical product for treating cancer.

15. The use according to claim 14, wherein the cancer is selected from brain cancer, esophageal cancer, oral cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cervical cancer, epithelial cell carcinoma, skin cancer, leukemia, lymphoma, myeloma, breast cancer, ovarian cancer, endometrial cancer, and testicular cancer, preferably selected from one or more of lung cancer, esophageal cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer, cervical cancer, vaginal cancer, epithelial cell carcinoma, and skin cancer.

16. A pharmaceutical composition comprising a chimeric antigen receptor according to any one of claims 1 to 9, a nucleic acid molecule according to claim 10, a viral vector according to claim 11, or a genetically modified cell according to claim 12 or 13, and a pharmaceutically acceptable carrier.

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