Chimeric antigen receptor and its use

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

[0002]

[0002] The present invention relates to a chimeric antigen receptor, T cells expressing a chimeric antigen receptor, and a method for using a chimeric antigen receptor for the prevention and / or treatment of cancer. [Background technology]

[0003]

[0003] The immune system has highly evolved and specific mechanisms to protect against a variety of pathogens. Among these pathogens, in particular, is the detection and elimination of undesirable pathogens such as bacterial infections, virally infected cells, and, importantly, mutated cells that can cause malignant neoplasms (cancer). The immune system's ability to prevent the formation and growth of cancer depends on the immune system's ability to distinguish between "healthy" cells and "disease-state" (e.g., neoplasms or preneoplasms) cells. This is achieved by the recognition of cellular 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 immunotherapeutic approaches to treat cancer by manipulating or directing the immune system to target cells that express cancer cell antigens. Immunotherapeutic approaches have primarily utilized the humoral immune system using isolated or manipulated antibodies, or, more recently, have focused on the cellular arm of the immune system.

[0005]

[0005] Early attempts to use cellular immunotherapy for cancer treatment have involved using T lymphocytes isolated from tumors and grown ex vivo. While this approach initially showed some promise in early investigations, it is associated with numerous technical problems. The ability to isolate T cell populations and grow them to clinically viable numbers is a technical challenge, and the inability to properly control the nature of proliferation means that the final T cell population 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 shortcomings associated with the use of ex vivo-grown tumor-isolated T cells, chimeric antigen receptors (CARs or artificial T cell receptors) began to be developed in the late 1980s. Chimeric antigen receptors are created by linking an extracellular domain specific to a desired antigen to a signaling domain, resulting in an antigen-specific receptor that can induce T cell function.

[0007]

[0007] By transforming isolated T cells with CAR, a population of T cells specific to a given antigen can be obtained. As a result, a large population of antigen-specific T cells can be generated and used in immunotherapy.

[0008]

[0008] Early clinical trials of CAR-transformed T cells, which are specific to tumor-associated antigens, were promising. However, the efficacy of CAR-transformed T cells resulted in significant hypercytokinemia, which ultimately led to death in some patients. These side effects are thought to be induced primarily by the target-matching but tumor-missing activity of CAR-transformed T cells, which is induced as a result of endogenous expression of alloantigens of CAR in healthy, non-cancerous cell populations.

[0009]

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

[0010]

[0010] Considerations relating to documents, operations, materials, devices, articles, etc., are included herein solely for the purpose of providing the context of the present invention. It is not implied or shown that any or all of these issues existed prior to the priority date of each claim of this application, and therefore formed part of the foundation of the prior art, or were common general knowledge in the art relating to the present invention. [Overview of the Initiative]

[0011]

[0011] The present invention is based in part on the recognition that there is a need to develop CARs and genetically modified cells expressing them that target markers specifically associated with various neoplastic (cancerous) or preneoplastic (precancerous) cells, due to the remarkable "target hit" but "tumor miss" activity of immune cells expressing CARs. The inventors recognize that the dysfunctional P2X7 receptor is a suitable marker to be targeted 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 to ATP at its ATP binding site compared to the ATP binding ability of the wild-type (functional) P2X7 receptor. In some embodiments, the dysfunctional P2X7 receptor is unable to bind to ATP at its ATP binding site.

[0014]

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

[0015]

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

[0016]

[0016] The antigen-recognition domain of the CAR can be any suitable molecule that can interact with and 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 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-stranded variable fragment (scFv) or a polyvalent scFv that binds to a dysfunctional P2X7 receptor. In some embodiments, the polyvalent scFv is a divalent 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 a dysfunctional P2X7 receptor.

[0019] In some embodiments, the antigen recognition domain comprises a conjugating peptide having amino acid sequence homology to one or more CDR regions of an antibody that binds to a dysfunctional P2X7 receptor. In some embodiments, the conjugating peptide comprises a V of an antibody that binds to a dysfunctional P2X7 receptor. H Chain and / or V L This includes amino acid sequence homology to the CDR1, 2, and 3 domains of the chain. In some embodiments, the antigen recognition domain includes one or more amino acid sequences that are at least 50%, 60%, 70%, 80%, 90%, or 94% identical to any one of the regions extending from positions 30 to 35, 50 to 67, or 98 to 108 of the sequences described in SEQ ID NOs. 10, 32, 33, or 34. In some embodiments, the antigen recognition domain includes one or more sequences extending from positions 30 to 35, 50 to 67, or 98 to 108 of the sequences described in SEQ ID NOs. 10, 32, 33, or 34. In some embodiments, the antigen recognition domain includes one or more sequences described in SEQ ID NOs. 10, 32, 33, or 34.

[0019]

[0020] In some embodiments, the signaling domain includes 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 includes a portion derived from a costimulatory receptor. In some embodiments, the signaling domain includes 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 transcriptional control sequences. 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 transcription initiation 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 the 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 CAR having a signaling domain including a portion derived from an activating receptor, and a second CAR having a signaling domain including a portion derived from a co-stimulatory receptor. In some embodiments, the activating receptor is a member of the CD3 co-receptor complex or an Fc receptor. In some embodiments, the co-stimulatory 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 cells are further modified to constitutively express a costimulatory receptor. In some embodiments, the cells are further modified to express a ligand for the costimulatory receptor, thereby promoting cellular autostimulation.

[0028]

[0031] In some embodiments of the fourth and fifth aspects of the present invention, the cells are further modified to secrete cytokines. In some embodiments, the cytokines are 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 cells are leukocytes. In some embodiments, the cells are peripheral blood mononuclear cells (PBMCs), lymphocytes, T cells (including CD4+ T cells or CD8+ T cells), natural killer cells, or natural killer T cells.

[0030]

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

[0031]

[0034] In some embodiments of the sixth aspect of the present invention, the CAR either directly recognizes the dysfunctional P2X7 receptor or recognizes the 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 aptamer. In some embodiments, the probe includes a tag, and the CAR recognizes the tag.

[0032]

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

[0033]

[0036] In some embodiments of the sixth and seventh aspects of the present invention, cells expressing a dysfunctional P2X7 receptor are exposed to genetically modified cells along with exogenous cytokines. In some embodiments, the genetically modified cells are self-referential to cells expressing the dysfunctional P2X7 receptor.

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

[0036]

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

[0037]

[0039] In a ninth aspect, the present invention provides a method for growing genetically modified cells according to a fourth or fifth aspect of the present invention in vitro, comprising the steps of exposing the cells to a CAR antigen and simultaneously exposing the cells to cytokines.

[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, interferon subfamily, IL-10 subfamily, IL-1 subfamily, IL-17 subfamily, or 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 combinations thereof.

[0040]

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

[0041]

[0043] In an eleventh embodiment, the present invention provides a pharmaceutical composition comprising genetically modified cells according to a fourth or fifth embodiment of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a preferred adjuvant, which may consist of cytokines. In some embodiments, the pharmaceutical composition may also comprise an intermediary as described herein.

[0042]

[0044] For a further understanding of the aspects and advantages of the present invention, refer to the following detailed description in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0043] [Figure 1]

[0045] This is a schematic diagram showing the arrangement of an anti-nonfunctional (nf)P2X7 receptor chimeric antigen receptor (CAR) according to an embodiment of the present invention. [Figure 2]

[0046] Figure 1 is a schematic diagram showing the BLIV plasmid used for the expression of the anti-nf P2X7 receptor CAR. [Figure 3]

[0047] This is an electrophoresis gel showing restriction enzyme-treated fragments derived from DNA isolated from E. coli clones transformed with a BLIV plasmid and treated with BamHI restriction enzymes. [Figure 4]

[0048] This is an electrophoresis gel showing restriction enzyme-treated fragments derived from DNA isolated from selected E. coli clones transduced with BLIV plasmids and treated with restriction enzymes EcoRI, BamHI, and PstI. [Figure 5]

[0049] This figure shows 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 transfected with the supernatant containing the lentiviral vector. [Figure 6]

[0050] This figure shows 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 transfected with the supernatant containing the lentiviral vector. [Figure 7]

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

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

[0053] This graph illustrates the percentage of dye-labeled target cells that disappeared after 48 hours of co-culture of CD8+ T cells transduced with lentiviral vectors containing BLIV-CAR-short hinge plasmid and BLIV-CAR-long hinge plasmid, compared to CD8+ T cells that were not transduced and CD8+ T cells transduced with an empty BLIV plasmid. [Figure 10]

[0054] This shows the alignment of PEP2-2-1-1, PEP2-472-2, and PEP2-2-12 conjugated peptides with antibodies directed to the nf-P2X7 receptor. [Figure 11]

[0055] This is 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] Figure 11 is a schematic diagram showing the pCDH plasmid used for the expression of the anti-nf P2X7 receptor CAR. [Figure 13]

[0057] This is an electrophoresis gel diagram showing restriction enzyme-treated fragments derived from restriction enzyme-treated DNA isolated from selected Sure 2 clones transformed with the pCDH plasmid, using EcoRI and Not I. [Figure 14]

[0058] This is a FACS analysis of the transfection efficiency of HEK293T cells. [Figure 15]

[0059] This is a typical histogram of FACS analysis of lentiviral transduction efficiency. [Figure 16]

[0060] This is a FACS analysis of the percentage of transformed CD8 cells expressing GFP. [Figure 17]

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

[0062] This is an electrophoresis gel diagram showing restriction enzyme-treated fragments derived from DNA isolated from selected E. cloni® 10G clones transformed with EXD2_K193A or EXD2_WT containing the pDONR-107 vector, and treated with Bam HI and PmeI. [Figure 19]

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

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

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

[0066] This graph illustrates the killing of nfP2X7-expressing HEK target cells and 231 breast cancer cells by T cells expressing PEP2-2-1-1 and PEP2-472-2 CAR. [Modes for carrying out the invention]

[0044]

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

[0045] [Table 1-1]

[0046] [Table 1-2]

[0047]

[0068] The inventors recognize the need to develop CARs and genetically modified cells expressing them that target markers specifically associated with neoplastic (cancerous) or pre-neoplastic (precancerous) cells, due to the marked "target hit" but "tumor miss" activity of immune cells expressing chimeric antigen receptors (CARs). The inventors believe that dysfunctional P2X7 receptors are used by immune cells expressing CARs in various cancers. We recognize that it is a suitable marker for targeting.

[0048]

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

[0049]

[0070] Chimeric antigen receptors (CARs) are artificially constructed proteins that, when expressed on the surface of a cell, can induce an antigen-specific cellular response. A CAR comprises at least two domains: a first domain which is an antigen recognition domain or more specifically the epitope portion of the antigen that specifically recognizes the antigen, and a second domain which is a signaling domain that can induce or participate in the induction of an intracellular signaling pathway.

[0050]

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

[0051]

[0072] In addition to the antigen recognition domain and signaling domain, CARs may further include additional components or parts. For example, a CAR may include a transmembrane domain that may contain or be associated with a portion of the signaling domain of the CAR. The transmembrane domain is typically one or more hydrophobic helices that extend into the cellular lipid bilayer and embed the CAR within the cell membrane. The transmembrane domain of a CAR may be one of the determinants of the CAR's expression pattern when bound to a cell. For example, using a CD3 coreceptor-related transmembrane domain can enable CAR expression in naive T cells, while using a CD4 coreceptor-derived transmembrane domain can induce CAR expression in T helper cells but not in cytotoxic T cells.

[0052]

[0073] A further component or part of a CAR may be a linker domain. The linker domain (also known as a spacer or hinge domain) extends from the extracellular side of the transmembrane domain to the antigen-recognition domain, thereby allowing the antigen-recognition domain to be linked to the transmembrane domain. In some cases, a linker domain is not necessary for a functional CAR (i.e., the antigen-recognition domain can be directly connected to the transmembrane domain), but in some situations, the use of a linker domain can enhance the effectiveness of the CAR. Linker domains can have a variety of functionalities, including enabling the mobility of the CAR in such a way that it allows for the orientation of the antigen-recognition domain necessary for binding to an antigen. 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 with amino acid sequence homology to the hinge region of an IgG antibody, e.g., the IgG1 hinge region. Another example is an amino acid sequence with sequence homology to the CH2CH3 region of an antibody, or to a portion of the CD3 coreceptor complex, CD4 coreceptor, or CD8 coreceptor.

[0053]

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

[0054]

[0075] The mRNA, coding (cDNA), and amino acid sequences of the human P2X7 gene are described in Sequence IDs 1 through 3, respectively. The mRNA and amino acid sequences of the human P2X7 gene are also represented by GenBank accession numbers NM_002562.5 and NP_002553.3, respectively. The P2X7 gene is conserved in chimpanzees, rhesus monkeys, dogs, cattle, mice, rats, pigs, chickens, zebrafish, and frogs. Further details on the P2X7 gene in humans and other species can be found at the National Centre for Biotechnology. This information can be accessed from the GenBank database at NCBI Information (www.ncbi.nlm.nih.gov). For example, the Gene identifier number for human P2X7 is 5027, for chimpanzees it is 452318, for monkeys it is 699455, for dogs it is 448778, for cattle it is 286814, for mice it is 18439, for zebrafish it is 387298, and for frogs it is 398286. Furthermore, at least 73 species have orthologues of the human P2X7 gene.

[0055]

[0076] Further details regarding the P2X7 gene in humans and other species can also be found on the NCBI's 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, detailed nucleotide and amino acid sequences of the P2X7 gene can be accessed from the UniProt database (www.uniprot.org), where 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 human receptor, at least one of these monomers has the amino acid sequence described in Sequence ID No. 3. It should be understood that the “P2X7 receptor” as referred to herein also includes naturally occurring variants of this receptor, including splice variants, naturally occurring cleavage forms, and allele variants. The P2X7 receptor may also include subunits having modified amino acid sequences, such as those involving cleavage, deletion, or modification of the amino acids described in Sequence ID No. 3.

[0057]

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

[0058]

[0079] The P2X7 receptor is activated when ATP binds to the receptor's ATP binding site. This triggers a rapid opening of the channel (within a few milliseconds), which selectively allows small cations to move across the membrane. Shortly afterward (within a few seconds), a large pore is formed in the cell membrane, allowing molecules up to 900 Da in size to pass through the cell membrane. This pore formation ultimately leads to cell depolarization, often resulting in cytotoxicity and cell death. This role is attributed to the P2X7 receptor. This has led to the idea that the condition is involved in apoptosis in various cell types.

[0059]

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

[0060]

[0081] Accordingly, in a first aspect of the present invention, the CAR recognizes a dysfunctional P2X7 receptor. With respect to the P2X7 receptor, the term “dysfunctional” as used throughout this specification includes a reduction 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 reduced 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, meaning that the P2X7 receptor is unable to be induced to allow the passage of cations or other molecules across the cell membrane.

[0061]

[0082] Any alteration in the wild-type or native form of the receptor that results in a P2X7 dysfunctional receptor is incorporated herein. For example, a dysfunctional receptor may be the result of a mutation or modification in one or more amino acids of the receptor related to ATP binding to the receptor. In fact, a P2X7 receptor is dysfunctional if its ability to bind to ATP at the ATP binding site is reduced, or if it is unable to bind to ATP at all. 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 to ATP compared to the ATP-binding ability of the wild-type (functional) P2X7 receptor. In some embodiments, the dysfunctional P2X7 receptor is unable to bind to ATP.

[0062]

[0083] Modifications to one or more amino acids of the P2X7 receptor may include a change in the conformation of one or more amino acids of the receptor. Therefore, in some embodiments of the first aspect of the present invention, a chimeric antigen receptor binds to a dysfunctional P2X7 receptor having a conformational change that makes the receptor dysfunctional. Specifically, this conformational change may be a change from trans to cis configuration of one or more amino acids of the P2X7 receptor. In some embodiments, the proline at position 210 of the P2X7 receptor changes from trans to 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 extending 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 comprising the proline at position 210 of the dysfunctional P2X7 receptor and one or more amino acid residues ranging from glycine at amino acid position 200 to cysteine ​​at amino acid position 216 (including both ends) of the dysfunctional P2X7 receptor.

[0063]

[0084] I don't want to be constrained by theory, but the P2X7 receptor at position 210 As a result of a change in the stereochemistry of proline, the three-dimensional structure of the receptor may be altered. This alteration of the three-dimensional structure allows the antigen-recognition domain of CAR to bind to amino acids or epitopes that were previously inaccessible in the native three-dimensional structure of the P2X7 receptor. Thus, in some embodiments, CAR recognizes one or more epitopes of the P2X7 receptor that are exposed to the antigen-recognition domain as a result of a trans-to-cis stereochemistry of proline at position 210 of SEQ ID NO: 3. These epitopes may include one or more 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 epitopes including one or more 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” refers to the ability of an antigen-recognition domain to bind to a dysfunctional P2X7 receptor, a portion thereof, or its epitope. In some embodiments, the antigen-recognition domain may bind directly to the dysfunctional P2X7 receptor or its epitope. In other embodiments, the antigen-recognition domain may bind to a processed form of the dysfunctional P2X7 receptor. When used in this context, the term “processed form” refers to a form of the P2X7 receptor that has been cleaved or digested as a result of intracellular processing. Consequently, recognition of the “processed form” of the dysfunctional P2X7 receptor may result in it being presented bound to the major histocompatibility complex (MHC).

[0065]

[0086] The antigen-recognition domain may be any suitable domain capable of recognizing the dysfunctional P2X7 receptor or its epitope. As used throughout this specification, the term “antigen-recognition domain” refers to the portion of the CAR that provides specificity to the dysfunctional P2X7 receptor. The antigen-recognition domain may be all of the extracellular region of the CAR, or simply a portion thereof. Suitable antigen-recognition domains include, but are not limited to, polypeptides having sequence homology to the antigen-binding site of an antibody or fragment that binds to the dysfunctional P2X7 receptor. Therefore, 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 fragment that binds to the dysfunctional P2X7 receptor. In some embodiments, a portion of the antigen-recognition domain comprises an amino acid sequence having homology to an antibody or fragment that binds to the dysfunctional P2X7 receptor. The originating homologous antibody sequence may be any suitable sequence of an antibody having affinity for the P2X7 receptor. For example, the sequence may share sequence homology with antibodies originating from one or more of the following species: humans, non-human primates, mice, rats, rabbits, sheep, goats, ferrets, dogs, chickens, cats, guinea pigs, hamsters, horses, cattle, or pigs. The antigen-recognition domain may share sequence homology with monoclonal antibodies produced from hybridoma cell lines. If the species from which the homologous antibody sequence originates is not human, the antibody is preferably a humanized antibody. The homologous antibody sequence may also originate from non-mammalian species such as cartilaginous fish (see, e.g., shark IgNAR antibody, International Publication No. 2012 / 073048). Alternatively, the antigen-binding domain may include a modified protein backbone that provides similar functionality to a shark antibody, such as an i-body with a binding portion based on a shark IgNAR antibody (see International Publication No. 2005 / 118629). In addition, the antigen recognition domain may be any other suitable binding molecule or peptide capable of selectively interacting with the dysfunctional P2X7 receptor with sufficient affinity to activate the CAR signaling domain, may be derived from it, or may share sequence homology with it.Methods for identifying antigen-binding proteins, particularly panning of phage display libraries, protein affinity chromatography, co-immunoprecipitation, and yeast two-hybrid systems, are known in the art (Srinivasa Ra). See o, V. et al., Int J Proteomics, 2014; paper number 147648.

[0066]

[0087] In some embodiments, the antigen-recognition domain of the CAR includes 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 understood in the art, the Fab portion of an antibody consists of one constant region and one variable region in the heavy chain and light chain of the antibody, respectively. The Fab is the antigenic 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-strand variable fragment (scFv) that binds to a dysfunctional P2X7 receptor. As understood in the art, the scFv comprises two parts which may share homology with or may be identical to the variable heavy chain (VH) and variable light chain (VL) of an antibody, and which are linked together by a linker peptide to form a fusion protein. For example, the scFv may comprise the VH and VL amino acid sequences derived from an antibody that recognizes a dysfunctional P2X7 receptor. In this context, it is expected that the term “derived from” refers not to the origin of the polypeptide itself, but rather to the origin of the amino acid sequence that constitutes a portion of the antigen-binding region. Consequently, the term “derived from” includes polypeptides that share sequence identity with an antibody that binds to a dysfunctional P2X7 receptor, whether synthesized, artificially, or otherwise.

[0068]

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

[0069]

[0090] In some embodiments of the first aspect of the present invention, the antigen recognition domain has an 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 includes the amino acid sequence described in SEQ ID NO: 10, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34, or functional variants thereof.

[0070]

[0092] In some embodiments, the antigen recognition domain comprises a binding peptide comprising an amino acid sequence homologous to one or more CDR regions of an antibody that binds to a dysfunctional P2X7 receptor. In some embodiments, the binding peptide comprises an amino acid sequence homologous to the CDR region of an antibody that binds to a dysfunctional P2X7 receptor. H Chain and / or V LThe antigen recognition domain includes one or more regions having sequence homology to the CDR1, 2, and 3 domains of the chain. In some embodiments, the antigen recognition domain includes one or more sequences that are at least 50%, 60%, 70%, 80%, 90%, or 94% identical to one of the CDR regions extending from positions 30 to 35, 50 to 67, or 98 to 108 of the sequence described in SEQ ID NOs. 10, 32, 33, or 34. In some embodiments, the antigen recognition domain includes one or more sequences extending from positions 30 to 35, 50 to 67, or 98 to 108 of the sequence described in SEQ ID NOs. 10, 32, 33, or 34. Between the CDR regions of the antigen-binding peptide described in SEQ ID NOs. 10, 32, 33, or 34, there may be any public sequences that allow for the proper formation and placement of the CDR regions. In some embodiments, the antigen recognition domain includes a sequence that is 50%, 60%, 70%, 80%, or 90%, 95%, or 99% identical to one of the sequences described in SEQ ID NOs: 10, 32, 33, or 34.

[0071]

[0093] Antibodies directed to dysfunctional P2X7 receptors, from which suitable amino acid sequences may be derived, and methods for producing such antibodies have been described in the art (see, for example, International Publications 2001 / 020155, 2003 / 020762, 2008 / 043145, 2008 / 043146, 2009 / 033233, 2011 / 020155, and 2011 / 075789). Methods for generating polyclonal and monoclonal antibodies against specific epitopes (such as those mentioned above) are expected to be known to those skilled in the art. In summary, a desired epitope (such as a segment of a dysfunctional P2X7 receptor containing proline at position 210) is injected into a suitable host animal in the presence of a suitable immunogenic carrier protein and adjuvant. Next, serum can be collected from immunized animals, and antibodies can be isolated based on their antibody class or antigen specificity. After evaluating the compatibility 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. Suitable epitopes for the production of antibodies directed to dysfunctional P2X7 receptors are known in the art (see, for example, International Publications 2008 / 043146, 2010 / 000041, and 2009 / 033233).

[0072]

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

[0073]

[0095] As used throughout this specification, the term “segment” refers, when used in reference to activating receptors or co-stimulatory receptors, to any segment of the receptor that contains sequences responsible for or involved in the initiation / induction of intracellular signaling cascades following the interaction between the receptor and its alloantigen or ligand. An example of the initiation / induction of intracellular signaling cascades of T cell receptors (TCRs) via CD3 is outlined below.

[0074]

[0096] While not wishing to be constrained by theory, the extracellular portion of the TCR is primarily composed of heterodimers of either the clonal TCRα and TCRβ chains (TCRα / β receptor) or the TCRγ and TCRδ chains (TCRγδ receptor). These TCR heterodimers generally lack intrinsic signaling ability and therefore bind non-covalently to several signaling subunits of CD3 (primarily CD3-zeta, CD3-gamma, CD3-delta, and CD3-epsilon). Each of the CD3 gamma, delta, and epsilon chains has an intracellular (cytoplasmic) portion containing a single immunoreceptor-activated tyrosine motif (ITAM), while the CD3-zeta chain contains three tandem ITAMs. In the presence of MHC, binding of the TCR to its alloantigen and binding of essential coreceptors such as CD4 or CD8 initiates signal transduction, resulting in phosphorylation of two tyrosine residues within the intracellular ITAM of the CD3 chain by a tyrosine kinase (i.e., Lck). Consequently, a second tyrosine kinase (ZAP-70, which is itself activated by Lck phosphorylation) is recruited to biphosphorylate the ITAM. As a result, multiple The activation of several downstream target proteins ultimately leads to changes in intracellular spatial arrangement, calcium mobilization, and actin cytoskeleton rearrangement, which, when combined, ultimately result in the activation of transcription factors and the induction of a T cell immune response.

[0075]

[0097] As used throughout this specification, the term “activating receptor” refers to a receptor or co-receptor that forms components of a T cell receptor (TCR) complex or is involved in the formation of TCRs, or a receptor that is involved in the specific activation of immune cells as a result of the recognition of antigenic stimuli or other immunogenic stimuli.

[0076]

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

[0077]

[0099] The specific intracellular signaling regions of each CD3 chain are known in the art. For example, the intracellular cytoplasmic region of the CD3ζ chain spans amino acids 52 to 164 of the sequence described in SEQ ID NO: 4, and its three ITAM regions span amino acids 61 to 89, 100 to 128, and 131 to 159 of SEQ ID NO: 4. Furthermore, the intracellular portion of the CD3ε chain spans amino acids 153 to 207 of the sequence described in SEQ ID NO: 5, and its single ITAM region spans amino acids 178 to 205 of SEQ ID NO: 5. The intracellular portion of the CD3γ chain spans amino acids 138 to 182 of the sequence described in SEQ ID NO: 6, and its single ITAM region spans amino acids 149 to 177 of SEQ ID NO: 6. The intracellular portion of the CD3δ chain spans amino acids 127 to 171 of the sequence described in SEQ ID NO: 7, and its single ITAM region spans 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 includes a portion derived from either CD3 (CD3-ζ chain or a portion thereof) or an FC receptor (preferably FcεRI or FcγRI). In some embodiments, the CD3-ζ coreceptor complex portion includes the amino acid sequence or a functional variant thereof described in SEQ ID NO: 22.

[0079]

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

[0080]

[0102] Using various combinations of the activating receptor moieties, the transmembrane (TM) and intracellular (IC) moieties of CARs, for example, 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™ and CD3ζ IC (James SE et al., J Immunol. 2008;180:7028-38), CD8™ and CD3ζ IC (Patel SD et al., Gene Ther. 1999;6:412-9), and FcεRIγ™ 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 “costimulatory receptor” refers to a receptor or co-receptor that assists in the activation of immune cells when antigen-specific induction of an activating receptor occurs. As understood, a costimulatory receptor is not antigen-specific and does not require the presence of an antigen, but is typically one of two signals, the other being the activating signal required to induce an immune cell response. In the context of an immune response, costimulatory receptors are typically activated by the presence of their ligand, expressed on the surface of antigen-presenting cells (APCs), such as dendritic cells or macrophages. Specifically with respect to T cells, costimulation is necessary for cell activation, proliferation, differentiation, and survival (all of which are usually referred to as under the umbrella of T cell activation), but when an antigen is presented to a T cell in the absence of costimulation, anergy, clonal removal, and / or antigen-specific tolerance may occur. Importantly, costimulatory molecules can transmit T cell responses to antigens encountered simultaneously. Generally, antigens encountered in a "positive" co-stimulatory molecule environment are expected to lead to T cell activation and trigger a cellular immune response aimed at eliminating cells expressing that antigen. On the other hand, antigens encountered in a "negative" co-receptor environment are expected to induce tolerance to the antigen encountered simultaneously.

[0082]

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

[0084]

[0106] Various combinations of co-stimulatory receptor moieties can be used to form the transmembrane (TM) and intracellular (IC) portions of CARs. 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 for the activating and co-stimulatory receptors mentioned above is readily accessible in various databases. For example, embodiments of 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 co-stimulatory receptors, but those skilled in the art will expect to understand that homologous and orthologous forms of each receptor exist in the majority of mammalian and vertebrate species. Therefore, 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 present invention, as well as homologous and orthologous 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 present invention, the signaling domain includes a portion derived from an activating receptor and a portion derived from a co-stimulatory receptor. Although not desirable, in this context, antigen recognition by the antigen-recognition domain of the CAR is expected to simultaneously induce both intracellular activating and intracellular costimulatory signals. Consequently, this is expected to stimulate antigen presentation by APCs expressing the costimulatory ligand. Alternatively, the CAR may have a signaling domain that induces a portion derived from either the activating receptor or the costimulatory receptor. In this alternative form, the CAR induces only either the activating intracellular signaling cascade or the 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 comprising one portion derived from a single activating receptor and multiple portions derived from multiple co-stimulatory receptors. In some embodiments, the CAR is expected to have a signaling domain comprising multiple portions derived from multiple activating receptors and one portion derived from a single co-stimulatory receptor. In some embodiments, the CAR is expected to have a signaling domain comprising multiple portions derived from multiple activating receptors and multiple portions derived from multiple co-stimulatory receptors. In some embodiments, the CAR is expected to have a signaling domain comprising one portion derived from a single activating receptor and multiple portions derived from two co-stimulatory receptors. In some embodiments, the CAR is expected to have a signaling domain comprising one portion derived from a single activating receptor and multiple portions derived from three co-stimulatory receptors. In some embodiments, the CAR is expected to have a signaling domain comprising multiple portions derived from two activating receptors and one portion derived from one co-stimulatory receptor. In some embodiments, the CAR is expected to have a signaling domain comprising multiple portions derived from two activating receptors and multiple portions derived from two co-stimulatory receptors. As should be understood, there are further variations in the number of activating and co-stimulatory receptors from which the signaling domains may originate, and the examples described above are not intended 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 an amino acid sequence described 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 described 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" may include any amino acid sequence as long as it maintains the function of any one of the 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] Therefore, as long as the functional variant maintains the function of one of the following: SEQ ID NOs: 10, 18, 20, 22, 26, 27, 32, 33, 34, 52, 53, or 54, the functional variant is, for example, an insertion, deletion, or substitution of one or more amino acids into one of the following: SEQ ID NOs: 10, 18, 20, 22, 26, 27, 32, 33, 34, 52, 53, or 54; SEQ ID NOs: 10, 18, 20, 22, 26, 27, 32, 33, 34 ,

[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 receptors, including 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 intracellular signaling that leads to the activation of CAR-expressing T cells, without significant recognition of the functional P2X7 receptor. As will be understood by those skilled in the art, changes to the amino acid sequence of the chimeric antigen receptors described in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54 can be made without significant alteration of the recognition of the dysfunctional P2X7 receptor and / or the activation of CAR-expressing T cells. Such changes may include, but are not limited to, changes in the hinge region of the chimeric antigen receptor, changes in the transmembrane domain, and changes in the activating receptor and / or co-stimulatory receptor portions that constitute the intracellular domain of the chimeric antigen receptor.

[0094]

[0115] As shown above, functional variants may include individual amino acid substitutions, deletions, or insertions compared to one of SEQ ID NOs: 10, 18, 20, 22, 26, 27, 32, 33, 34, 52, 53, or 54. For example, a person skilled in the art would expect to recognize that any amino acid may be substituted with a chemically (functionally) similar amino acid while retaining the function of the polypeptide. Such conservative amino acid substitutions are well known in the art. The groups in Table 3 below each contain amino acids that are conservative substitutions of each other.

[0095] [Table 3]

[0096]

[0116] Furthermore, if desired, non-natural amino acids or chemical amino acid analogs may be introduced into the polypeptides incorporated herein by substitution or addition. 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-methylamino acids, Cα-methylamino acids, Nα-methylamino acids, and 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 contain an amino acid sequence that is at least 80% identical to any one of the 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 include 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 for 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, a comparison frame may be created over at least 20 amino acid residues, at least 50 amino acid residues, at least 75 amino acid residues, at least 100 amino acid residues, at least 200 amino acid residues, at least 300 amino acid residues, at least 400 amino acid residues, at least 500 amino acid residues, at least 600 amino acid residues, or the entire length of any one of SEQ ID NOs. 10, SEQ ID NOs. 18, SEQ ID NOs. 20, SEQ ID NOs. 22, SEQ ID NOs. 26, SEQ ID NOs. 27, SEQ ID NOs. 32, SEQ ID NOs. 33, SEQ ID NOs. 34, SEQ ID NOs. 52, SEQ ID NOs. 53, or SEQ ID NOs. 54. The comparison frame may include approximately 20% or less of additions or deletions (i.e., gaps) compared to a reference sequence (without additions or deletions) for optimal alignment of the two sequences. The optimal alignment of sequences for aligning comparison frames can be performed by computer implementations of algorithms such as the BLAST family of programs disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389-3402. Global alignment programs can also be used to align similar sequences of roughly equivalent size. An example of a global alignment program is the EMBOSS package (Rice P et al., 2000, Trends). NEEDLE (available at www.ebi.ac.uk / Tools / psa / emboss_needle / ), which is part of Genet., 16:276-277, and the GGSEARCH program (fasta.bioch.virginia.edu / fa), which is part of the FASTA package (Pearson W and Lipman D, 1988, Proc. Natl. Acad. Sci. USA, 85:2444-2448). Examples include those available at sta_www2 / fasta_www.cgi?rm=compare&pgm=gnw. All of these programs are based on the Needleman-Wunsch algorithm, which is used to find the optimal alignment (including gaps) of two sequences along their entire length. A detailed discussion of sequence analysis can also be found in Unit 19.3 of Ausubel et al. ("Current Protocols in Molecular Biology," John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998).

[0099]

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

[0100]

[0120] In some embodiments of a second aspect of the present invention, the nucleic acid molecule comprises a nucleotide sequence that encodes the amino acid sequence described 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.

[0101]

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

[0102]

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

[0103]

[0123] Those skilled in the art will understand that the present invention aims to encode any nucleotide sequence of an amino acid sequence described in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37, or any nucleotide sequence encoding a chimeric antigen receptor having 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. For example, a variant of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37 is intended, which comprises 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 encodes the same amino acid sequence. Due to the degeneracy of genetic coding, many nucleic acids can encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at all positions in SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37 where alanine is specified by a codon, the codon may be modified to any of the corresponding codons described without altering the encoded polypeptide. Therefore, any nucleotide sequences herein that encode the amino acid sequences described in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37, or any nucleotide sequences herein that encode a chimeric antigen receptor having 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, represent any possible silent changes to the nucleotide sequence. Those skilled in the art will expect to understand that functionally identical molecules can be obtained by modifying each codon of a nucleic acid (excluding AUG, which is usually the sole codon of methionine, and TGG, which is usually the sole codon of tryptophan). Thus, any silent changes to the nucleotide sequences encoding a polypeptide are implicit in each described sequence.

[0104]

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

[0105]

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

[0106]

[0126] A “selectable marker gene” comprises any nucleotide sequence that, when expressed by transformed cells, confers a phenotype to the cells that facilitates the identification and / or selection of these transformed cells. A variety of nucleotide sequences encoding suitable selectable markers are known in the art (e.g., Mortesen, RM. and Kingston RE. Curr Protoc Mol Biol, 2009; Unit 9.5). Exemplary nucleotide sequences encoding selectable markers include the adenosine deaminase (ADA) gene; the cytosine deaminase (CDA) gene; the dihydrofolate reductase (DHFR) gene; the histidinol dehydrogenase (hisD) gene; the puromycin-N-acetyltransferase (PAC) gene; the thymidine kinase (TK) gene; the xanthine-guanine phosphoribosyltransferase (XGPRT) gene; or antibiotic resistance genes, such as ampicillin resistance genes, puromycin resistance genes, bleomycin resistance genes, hygromycin resistance genes, kanamycin resistance genes, and ampicillin resistance genes; fluorescent reporter genes, such as green, red, yellow, or blue fluorescent protein coding genes; and, in particular, luminescence-based reporter genes, such as luciferase genes, which enable the optical selection of cells using techniques such as fluorescence-activated cell classification (FACS).

[0107]

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

[0108]

[0128] As described above, a nucleic acid construct may also include one or more transcriptional regulatory sequences. The term “transcriptional regulatory sequence” should be understood to include any nucleic acid sequence that performs transcription of an operablely connected nucleic acid. Transcriptional regulatory sequences may include, for example, a leader, a polyadenylation sequence, a promoter, an enhancer or upstream activator sequence, and a transcription termination factor. Typically, a transcriptional regulatory 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 transcriptional control sequence is a second embodiment of the present invention. A transcriptional regulatory sequence is operably connected to a given nucleic acid molecule. For the purposes of this specification, a transcriptional regulatory sequence is said to be "operably connected" to a given nucleic acid molecule if it can promote, inhibit, or otherwise modulate the transcription of the nucleic acid molecule. Thus, in some embodiments, the nucleic acid molecule is under the control of a transcriptional regulatory sequence, such as a constitutive promoter or an inductive promoter.

[0110]

[0130] A "nucleic acid construct" can be any preferred form, such as a plasmid, phage, transposon, cosmid, chromosome, or vector, which is replicable when bound to appropriate regulatory elements and allows the gene sequences contained within the construct to be moved between cells. Therefore, this 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 that is operably connected to a cell, tissue, or organ in which expression occurs. Therefore, promoters may include, for example, constitutive promoters or inductive promoters. A “constitutive promoter” is a promoter that is active under most environmental and physiological conditions. An “inductive promoter” is a promoter that is active under specific environmental and physiological conditions. The present invention intends to utilize any promoter that is active in the target cell. Therefore, a wide range of promoters is expected to be readily identifiable by those skilled in the art.

[0112]

[0132] Examples of constitutive promoters in mammals 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 β-actin (CAGG).

[0113]

[0133] Inducible promoters include, but are not limited to, chemically inducible promoters and physically inducible promoters. Chemically inducible promoters include promoters whose activity is controlled by a compound, such as alcohols, antibiotics, steroids, metal ions, or other compounds. Examples of chemically inducible promoters include, in particular, tetracycline-controlled promoters (see, e.g., U.S. Patent Nos. 5,851,796 and 5,464,758); steroid-responsive promoters, such as the glucocorticoid receptor promoter (see, e.g., U.S. Patent No. 5,512,483) and the ecdysone receptor promoter (see, e.g., U.S. Patent No. 6,379,945); and metal-responsive promoters, such as the metallothionein promoter (see, e.g., U.S. Patents Nos. 4,940,661, 4,579,821, and 4,601,978).

[0114]

[0134] As mentioned above, regulatory sequences may also include termination factors. The term “termination factor” refers to the DNA sequence at the end of a transcription unit that signals the termination of transcription. Termination factors are generally 3' untranslated DNA sequences containing polyadenylation signals, which facilitate the addition of polyadenylation sequences to the 3' end of the primary transcript. Like promoter sequences, termination factors can be any termination factor sequence that is operative in the cell, tissue, or organ in which they are intended to be used. Suitable termination factors are expected to be known to those skilled in the art.

[0115]

[0135] As can be understood, the nucleic acid construct of the third aspect of the present invention includes an additional sequence, For example, these may further include sequences that enable enhancement of expression, cytoplasmic or membrane transport, and location signaling. A non-limiting specific example is an internal ribosome entry site (IRES).

[0116]

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

[0117]

[0137] Methods for exploring the transfection of exogenous genetic material into eukaryotic cells, such as nucleic acid constructs according to a third aspect of the present invention, are known in the art. As is understood, 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 efficiency of the desired transfection, and, if desired or necessary, the final viability of the transfected cell. Non-limiting examples of such methods include chemical transfection of 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; and particle-based methods, such as "gene gun" delivery, magnetofection, or impalefection, or viral transfection.

[0118]

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

[0119]

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

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

[0120]

[0141] In a fifth aspect, the present invention provides genetically modified cells comprising a nucleic acid molecule according to a second aspect of the present invention, or a nucleic acid construct according to a third aspect of the present invention, or a genome 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 two or more nucleic acid molecules or nucleic acid constructs, each encoding a different CAR.

[0122]

[0143] When referred to herein, “genetically modified cells” means “cells enclosed by the present invention.” This includes any cells containing nucleic acid molecules or nucleic acid constructs that are not naturally occurring and / or introduced. Introduced nucleic acid molecules or nucleic acid constructs may be maintained as distinct DNA molecules within the cell or may be incorporated into the cell's genomic DNA.

[0123]

[0144] The genomic DNA of a cell should be understood in a broad sense, encompassing all endogenous DNA that constitutes the cell's genetic complementarity. Therefore, the genomic DNA of a cell should be understood to include chromosomes, mitochondrial DNA, etc. Thus, the term "genomic integration" refers to chromosomal integration, mitochondrial DNA integration, etc. The "genomic integration form" of a construct may be all or part of the construct. However, in some embodiments, the genomic integration form of a construct includes at least a nucleic acid molecule according to 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 embodiment, “different CARs” include two CARs having the same antigen-recognition domain (for example, both CARs may recognize a dysfunctional P2X7 receptor) but having different signaling domains, such as one CAR having a signaling domain with a portion of an activating receptor and the other CAR having a signaling domain with a portion of a co-stimulatory receptor. As understood, at least one of the two or more CARs in this embodiment has an antigen-recognition domain that recognizes a dysfunctional P2X7 receptor, and the other CARs may take any preferred form and may be directed to any preferred antigen.

[0125]

[0146] Accordingly, in some embodiments of the fourth and fifth aspects of the present invention, two or more different CARs may have different signaling domains and the same 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 comprising a portion derived from an activating receptor, and a second chimeric antigen receptor having a signaling domain comprising 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 portion 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 co-stimulatory receptor (from which a portion of the signaling moiety is derived) is selected from the group consisting of CD27, CD28, CD-30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS.

[0128]

[0149] In some embodiments of the fourth or fifth aspect of the present invention, the co-stimulatory receptor (from which a portion of the signaling partial 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 cells are further modified to constitutively express a co-stimulatory receptor.

[0151] As described above, the immune response of cells is typically induced only when an activating signal (typically in response to an antigen) and a co-stimulatory signal are experienced simultaneously. Therefore, the above-described implementation involves two or more CARs that combine to provide both intracellular activating and intracellular co-stimulatory signals. By having genetically modified cells with a portion of the state, it is ensured that a sufficient immune response can be induced in response to the recognition of an alloantigen 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 may constitutively express a costimulatory receptor, thereby increasing the likelihood that costimulation will occur simultaneously when the CAR is activated. Alternatively, the genetically modified cells may be further modified to constitutively express both the costimulatory receptor and its ligand. In this case, the cells receive continuous costimulation, and only activation of a CAR having a signaling domain containing a portion derived from the activating receptor is required for the immune activation of the cells.

[0130]

[0152] Accordingly, in some embodiments of the fourth or fifth aspect of the present invention, the genetically modified cells are further modified to constitutively express costimulatory receptors. In further embodiments, the genetically modified cells are further modified to express ligands for costimulatory receptors, thereby promoting cellular autostimulation. Examples of CAR-expressing T cells that express both costimulatory receptors and their alloligans (to induce autostimulation) are known in the art, in particular by Stephen MT. et al., Nat Examples include those disclosed in Med, 2007;13:1440-9.

[0131]

[0153] The capabilities of genetically modified cells containing CARs can be enhanced by further modifying the cells to secrete cytokines, preferably pro-inflammatory cytokines or pro-proliferative cytokines. This cytokine secretion both provides autosecretory support for the CAR-expressing cells and alters the local environment surrounding the CAR-expressing cells so that other cells of the immune system are recruited and activated. As a result, in some embodiments of the fourth or fifth aspects of the present invention, the genetically modified cells are further modified to secrete cytokines. This secretion may be constitutive or induced upon recognition of the CAR's alloligand antigen.

[0132]

[0154] Any one or more cytokines may be selected depending on 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 present invention may be any suitable immune cells or a homogeneous or heterogeneous cell population. In some embodiments, the cells are leukocytes, peripheral blood mononuclear cells (PBMCs), lymphocytes, T cells, CD4+ T cells, CD8+ T cells, natural killer cells, or natural killer T cells.

[0134]

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

[0135]

[0157] Therefore, 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.

[0136]

[0158] As used herein, the term “directly recognize” includes the direct binding of the antigen-recognizing domain of a CAR to a dysfunctional P2X7 receptor or its epitope, if such an epitope is present in its native form. In another non-limiting example, antigen recognition The recognition domain may directly bind to a processed form of the dysfunctional P2X7 receptor, which may be presented by antigen-presenting molecules such as major histocompatibility complex (MHC).

[0137]

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

[0138]

[0160] As a result, in certain embodiments of the sixth aspect of the present invention, the chimeric antigen receptor recognizes the dysfunctional P2X7 receptor via an intermediary. The intermediary may 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, antibody Fabs, scFvs, soluble modified TCRs, or aptamers. The CAR may directly recognize the probe, or the probe may have a tag that is recognized by the CAR. In either case, the probe provides specificity to target cells (i.e., cells having the dysfunctional P2X7 receptor), while genetically modified cells having the CAR provide efficacy and direct the immune response to the target cells. Alternatively, the intermediary may be an intracellular marker that is associated with the dysfunctional P2X7 receptor or whose expression correlates with the dysfunctional P2X7 receptor. Abnormal regulation of the marker may result in or cause dysfunction of the P2X7 receptor.

[0139]

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

[0140]

[0162] In some embodiments of the sixth aspect of the present invention, the intermediary is a probe that binds to a 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 oligonucleotide, polynucleotide, peptide, or polypeptide that specifically binds to or preferentially complexes with a target (specifically, a dysfunctional P2X7 receptor).

[0142]

[0164] In some embodiments of a 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 an intermediary are known in the art, for example, European Patent Application No. 2651442.

[0143]

[0165] In some embodiments of the sixth aspect of the present invention, the cells having a dysfunctional P2X7 receptor are present in the body of a subject. In some embodiments, the subject is human. In some embodiments, the method further includes the step of exposing cells expressing a dysfunctional P2X7 receptor to a genetically modified organism together with exogenous cytokines.

[0144]

[0166] In some embodiments of the sixth aspect of the present invention, the genetically modified cells are genetically modified cells that are self to cells expressing a 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 present 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 treats cancer in a subject or The present invention provides a method for prevention, comprising the step of providing a subject with genetically modified cells having a chimeric antigen receptor, wherein the chimeric antigen receptor is directed to target cells having a dysfunctional P2X7 receptor.

[0147]

[0169] When the terms “to treat,” “to treat,” or “treatment” are used herein, it should be understood that their scope includes one or more of the following results: (i) inhibiting to some extent the growth of a primary tumor in a subject (including delayed and complete cessation of growth, and including reduction of primary tumor growth after resection); (ii) inhibiting to some extent the growth and formation of one or more secondary tumors in a subject; (iii) reducing the number of tumor cells in a subject; (iv) reducing the size of a tumor in a subject; (v) inhibiting the invasion of tumor cells into peripheral organs (i.e., including reduction, delay, or complete cessation); (vi) inhibiting metastasis (i.e., including reduction, delay, or complete cessation); (vii) improving the life expectancy of a subject compared to an untreated state; (viii) improving the quality of life of a subject compared to an untreated state; (ix) alleviating, reducing, or mitigating at least one symptom of cancer in a subject; (x) resulting in regression or remission of cancer in a subject; (xi) alleviating a condition in a subject caused by cancer; and (xii) cessating a symptom associated with cancer in a subject.

[0148]

[0170] When used herein, the terms “prevent” or “prevent” should be understood to include, within their scope, inhibiting the formation of a primary tumor in a subject, inhibiting the formation of one or more secondary tumors in a subject, or reducing or eliminating cancer recurrence in a subject in remission.

[0149]

[0171] When used herein, the term “inhibit” means a reduction or reduction in the growth of cancer, cancerous cells, or tumors compared to the growth in a control, such as untreated cells or subjects. In some embodiments, the growth may be reduced or reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to an untreated control.

[0150]

[0172] The inhibition of cancer, tumor, or cancerous cell growth can be evaluated by a wide range 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 assesses the degree of tritiated thymidine integration into cellular DNA over a given period. For tumor 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] The term “subject” as used herein may refer to any animal that is susceptible to cancer. Subjects of particular interest are humans, as well as scientifically related species such as mice, rats, ferrets, guinea pigs, hamsters, non-human primates, dogs, pigs, and sheep, or economically related animals such as horses, dogs, cats, and cattle. In a preferred embodiment of a sixth aspect of the present invention, the subject is humans.

[0152]

[0174] The reference to "to provide a subject with..." relates to administering genetically modified cells to a subject. Alternatively, genetically modified cells may be generated within the subject. For example, genetically modified cells may be generated in vivo such that the subject has an endogenous population of genetically modified cells. Preferred means for such in vivo generation are known in the art and include gene therapy of the subject.

[0153]

[0175] Throughout this specification, targets having dysfunctional P2X7 receptors are used. The reference to "directed" CARs implies the intention to selectively target an immune response to certain cells based on the presence of a dysfunctional P2X7 receptor. Importantly, such targeting is not limited to the direct recognition of the dysfunctional P2X7 receptor by the CAR. That is, the CAR itself does not need to directly recognize or bind to the dysfunctional P2X7 receptor, but simply needs to be able to selectively recognize and thereby activate cells expressing the dysfunctional P2X7 receptor.

[0154]

[0176] Therefore, 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 direct binding of the antigen-recognizing domain of a CAR to a dysfunctional P2X7 receptor or its epitope, if such a receptor exists in its native form. In another non-limiting example, the antigen-recognizing domain may directly bind to a processed form of the dysfunctional P2X7 receptor, which may be presented by an antigen-presenting molecule such as a major histocompatibility complex (MHC).

[0156]

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

[0157]

[0179] As a result, in certain embodiments of the sixth aspect of the present invention, the chimeric antigen receptor recognizes the dysfunctional P2X7 receptor via an intermediary. The intermediary may 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, antibody Fabs, scFvs, soluble modified TCRs, or aptamers. The CAR may directly recognize the probe, or the probe may have a tag that is recognized by the CAR. In either case, the probe provides specificity to target cells (i.e., cells having the dysfunctional P2X7 receptor), while genetically modified cells having the CAR provide efficacy and direct the immune response to the target cells. Alternatively, the intermediary may be an intracellular marker that is associated with the dysfunctional P2X7 receptor or whose expression correlates with the dysfunctional P2X7 receptor. Abnormal regulation of the marker may result in or cause dysfunction of the P2X7 receptor.

[0158]

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

[0181] In some embodiments of the sixth aspect of the present invention, the intermediary is a probe that binds to a 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 oligonucleotide, polynucleotide, peptide, or polypeptide that specifically binds to or preferentially complexes with a target (specifically, a dysfunctional P2X7 receptor).

[0160]

[0183] In some embodiments of a 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 an intermediary are known in the art, for example, European Patent Application No. 2651442.

[0161]

[0184] In a seventh embodiment, the present invention provides a method for treating or preventing cancer in a subject, comprising administering to the subject genetically modified cells according to a fourth or fifth embodiment of the present invention. Provide a method that includes steps.

[0162]

[0185] While providing genetically modified cells expressing CARs directed to target cells with dysfunctional P2X7 receptors may be sufficient to provide effective immunotherapy against precancerous or cancerous cells, providing adjuvants along with the genetically modified cells can further enhance the induction of the immune response and supplement the immunotherapy. Cytokines, preferably pro-inflammatory cytokines, are particularly suitable adjuvants to be provided to the target along with the genetically modified cells containing the CARs.

[0163]

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

[0164]

[0187] Cytokines suitable for administration with genetically modified cells include IL-2, IL-4, IL-6, IL-7, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, IFNα, IFNβ, IFNγ, GM-CSF, TGFβ, and TNFα. Preferred cytokines include IL-2 and IFNα. Furthermore, cytokines can be administered via delivery systems such as protein fusions, delivered in recombinant form, in their natural form, expressed in genetically modified cells, or as nucleic acid sequences conjugated with polymers such as polyethylene glycol (PEG).

[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 self cells, which are cells that are self to cells expressing a dysfunctional P2X7 receptor. Advantageously, self cells are not recognized as "non-self" by the target immune system and are therefore expected to be tolerated by the target. However, in some forms of cancer, suitable self cells may not be readily available. Therefore, in some embodiments of the present invention, the cells to be genetically modified are allogeneic or heterogeneic cells.

[0166]

[0189] P2X7 dysfunction is a common molecular modification 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 a partial form of the sixth or seventh aspect of the present invention, the method is used for the prevention or treatment of one or more cancers selected from among brain cancer, esophageal cancer, oral cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cervical cancer, epithelial cell carcinoma, skin cancer, leukemia, lymphoma, myeloma, breast cancer, ovarian cancer, endometrial cancer, and testicular cancer. Preferably, the cancer is lung cancer, One or more of the following cancers are selected: esophageal cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer, cervical cancer, vaginal cancer, epithelial cell carcinoma, skin cancer, blood-related cancers, breast cancer, endometrial cancer, uterine cancer, and testicular cancer.

[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 a fourth or fifth aspect of the present invention, it may be desirable to grow the cell population in vitro to increase the total number of cells available for therapeutic use. This can be done by using a step of exposing the cells to the antigen of the CAR. Accordingly, in an eighth aspect, the present invention provides a method for growing genetically modified cells according to a fourth or fifth aspect of the present invention in vitro, comprising the step of exposing the cells to the antigen of the CAR. In some embodiments, the method includes a further step of exposing the cells to cytokines.

[0169]

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

[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 embodiment, the present invention provides a method for growing genetically modified cells according to a fourth or fifth embodiment of the present invention in vitro, comprising the step of exposing the cells to immobilized anti-CD3 antibodies and anti-CD28 antibodies. In some embodiments of the tenth embodiment of the present invention, the antibodies are immobilized on a bead substrate (e.g., "Human Activator" Dynabeads®). In some embodiments of the tenth embodiment of the present invention, the antibodies are immobilized on an alternative surface, such as the surface of a tissue culture vessel, culture flask, plate, or bioreactor.

[0172]

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

[0173]

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

[0174]

[0198] Methods for providing cells containing CARs for immunotherapy are known in the art (e.g., Kershaw, MH. et al., Clin Cancer Res. 2020). See 06;12(20):6106-15;Parker LL. et al., Hum Gene Ther 2000;11:2337-87). Furthermore, protocols and methods for the preparation, proliferation, and evaluation of mammalian CAR-expressing cells are publicly 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 include one or more pharmaceutically acceptable additives, including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients, and bulking agents, taking into consideration the specific physical and chemical characteristics of the cells to be administered. In some embodiments, the pharmaceutical composition includes a suspension of genetically modified cells according to a fourth or fifth aspect of the present invention in a suitable medium such as isotonic saline. In some embodiments, the pharmaceutical composition may include a suitable adjuvant, such as one or more cytokines as described above. In some embodiments, the pharmaceutical composition may also include the intermediaries described above.

[0176]

[0200] The 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 cancerous mass.

[0177]

[0201] Throughout this specification, unless the context requires otherwise, it is expected that the words “comprise,” or variations such as “comprises,” or “comprising,” mean the inclusion of the element or integer, or group of elements or integers, as indicated, but not the exclusion of any other element or integer, or group of elements or integers.

[0178]

[0202] Finally, references to standard textbooks in molecular biology are provided, including methods for performing the fundamental techniques encompassed by this invention. For example, Green MR and Sambrook J, Molecular Cloning: A Laboratory Please refer to the Manual (4th edition), Cold Spring Harbor Laboratory Press, 2012.

[0179]

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

[0180]

[0204] The present invention is further illustrated in the following embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope to the foregoing description. [Examples]

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

[0205] An exemplary protocol illustrating the design and expression process of the anti-nonfunctional (nf)P2X7 receptor CAR according to embodiments 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 shown in Figure 1.

[0183]

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

[0184]

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

[0185]

[0209] The antigen-recognition domain 1 of the CAR was linked to the transmembrane domain 3 via one of two hinge regions, referred to as long hinge 4 and short hinge 5. Providing long hinge 4 may enable the mobility of the antigen-recognition domain, which may be required for the antigen-recognition domain to interact with its allogeneic ligand (dysfunctional P2X7). The amino acid and nucleotide sequences of long hinge 4 are described in SEQ ID NOs. 14 and 15, respectively. The amino acid and nucleotide sequences of short hinge 5 are described in SEQ ID NOs. 16 and 17, respectively.

[0186]

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

[0187]

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

[0188] Lentiviral vector design and assembly

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

[0189]

[0213] Sequences homologous to the upstream and downstream sequences of the NheI restriction site of the BLIV vector were added to the 5' and 3' ends of the designed CAR, resulting in Sequence ID No. 2. The final nucleotide sequences described in 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 anti-nf P2X7CAR into the BLIV vector using Gibson cloning.

[0190]

[0214] The nucleotide sequences of anti-nf P2X7CAR-long hinge and anti-nf P2X7CAR-short hinge were constructed using gene block technology (gBlock®, Gene Fragments-Integrated DNA Technologies, Iowa, USA) and assembled using the Gibson Assembly Cloning Kit (New England Biolabs inc., Ipswich MA, USA - catalog number E5510S) according to the manufacturer's instructions.

[0191]

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

[0216] New England Biolabs' 5-alpha competent E. coli cells (provided in the Gibson Assembly Cloning Kit) were transformed with the generated BLIV-CAR vector according to the manufacturer's instructions. Summary: - Tubes of NEB 5-alpha competent E. coli cells were thawed on ice for 10 minutes. - 1 to 5 μl containing 1 pg to 100 ng of BLIV-CAR plasmid DNA was added to the cell mixture and mixed by shaking the tube from side to side 4 to 5 times. - The E. coli and plasmid mixture was left undisturbed on ice for 30 minutes. - The cell and plasmid mixture was subjected to a heat shock at 42°C for 30 seconds, then left undisturbed on ice for 5 minutes. - 950 μl of SOC was added to the mixture, then heated to 37°C for 60 minutes and shaken vigorously. - A selection plate was prepared and heated to 37°C. - 10-fold serial dilutions of cells were prepared in SOC solution. - 50-100 μl of each dilution was spread onto a selection plate and incubated overnight at 37°C.

[0192]

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

[0193]

[0218] Clones 2 through 4 (L2 through 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 restriction enzymes EcoRI, BamHI, and PstI. All colonies showed restriction enzyme-treated fragments of the predicted lengths, as shown in Table 4 and Figure 4.

[0194] [Table 4]

[0195] Construction and validation of lentiviral vectors

[0219] Lentiviruses were packaged using 293T cells with a 3 plasmid protocol according to the following method. Day 1: 293T cells were seeded in 35 ml of DMEM medium containing 10% serum in a T-225 flask so that they would reach 90-95% confluence the following day. Day 2: 30 ug of either the generated BLIV-CAR plasmid (or unmodified BLIV plasmid), 30 ug of gag-pol plasmid delta 8.2, and 15 ug of VSV-G plasmid (pMD2.G) were added to OptiMEM medium to a final volume of 750 ul and mixed. 300 ul of PEI solution was added and incubated at room temperature for at least 20 minutes. The mixture was then added to confluent 293T cells and incubated at 37°C. Day 3: 24 hours after adding the plasmid mixture, the supernatant was decanted from the 293T cells and stored at 4°C. The decanted mixture was replaced with 35 ml of fresh medium and further incubated at 37°C. Day 4: 48 hours after adding the plasmid mixture, the culture medium was removed and combined with the supernatant collected at 24 hours. The combined supernatant was spun at 1500g for 15 minutes to remove all remaining cell debris. The supernatant was filtered through a 0.45um filter and then spun in a WX ultracentrifuge at 17,000 rpm for 1 hour. After centrifugation, the supernatant was manually decanted, leaving 50-200ul in the tube. The centrifuge tube was placed in a 50ml screw-cap tube to prevent contamination and evaporation, and the virus was resuspended overnight at 4°C. Day 5: The virus was resuspended from the bottom of the centrifuge tube and transferred to a new 1.5 ml tube. The resuspended virus was spun in a microcentrifuge tube at 5000 rpm for 5 minutes to remove all remaining debris.

[0196]

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

[0197] Screening for CAR T cell function

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

[0198]

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

[0199]

[0223] Target cells expressing the non-functional P2X7 receptor were obtained using the mammalian cancer cell line BT549 (ATCC HTB-122). These cells were labeled with the fluorescent insertion 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 PBS at room temperature. - A 10 μM solution of the cell proliferation dye eFluor(registered trademark) 670 was prepared in PBS at room temperature. - An equal volume of 10 μM dye solution was added to the prepared BT549 cells to obtain a final dye solution of 5 μM. - BT549 cells in dye solution were incubated in the dark at 37°C for 10 minutes, then labeling was stopped by adding four times the volume of cold culture medium containing 10% serum, and finally incubated on ice in the dark for 5 minutes. - Finally, the cells were washed three times in the culture medium and then resuspended in the culture medium at the desired concentration.

[0200]

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

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

[0201]

[0226] As can be seen in Figure 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, the target cells transformed into non-transduced cells or controls. Compared to co-culture with transduced (unmodified BLIV vector) CD8 T cells, there was an increase in the number of eliminated (killed) BT549 target cells.

[0202]

[0227] Considering the results presented in Figure 9, it is clear that CD8+ T cells transduced with anti-nfP2X7CAR receptors (with short or long hinges) exhibit increased levels 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 an alternative anti-nfP2X7 chimeric antigen receptor

[0228] Further exemplary protocols illustrating the design and expression process of the anti-nonfunctional (nf)P2X7 receptor CAR in T cells according to embodiments of the present invention are described below in detail.

[0204]

[0229] Anti-nfP2X7 CARs were designed using three anti-nonfunctional P2X7 binding peptides. Specifically, the CARs were designed to contain an antigen recognition domain with sequence homology to peptides PEP2-2-1-1, PEP2-472-2, or PEP2-2-12 (each having the amino acid sequences described in SEQ ID NOs. 32, 33, and 34, respectively). These binding peptides have been shown to bind to nonfunctional P2X7 receptors (Barden, JA, Sluyter, R., Gu, BJ & Wiley, JS2003. Specific detection of non-functional human P2X(7) receptors in HEK293 cells and B-lymphocytes.FEBS Lett 538,159-162).

[0205]

[0230] Figure 10 shows the alignment of the aforementioned binding peptide with the heavy chain variable region of the antibody that recognizes the non-functional P2X7 receptor. The alignment of the complementarity-determining regions (CDR1 to CDR3) is indicated by squares.

[0206]

[0231] A specific example of constructing a CAR containing the PEP2-2-1-1 sequence is detailed below. The same CAR structure and sequence were used in CARs having the PEP2-472-2 sequence or the PEP2-2-12 sequence as the binding peptide, as an alternative binding peptide to 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 produce a CAR having the structure described below.

[0233] Referring to Figure 11, the antigen recognition domain was prepared by ligating 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) 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).

[0208]

[0234] The antigen recognition domain was then ligated to the transmembrane domain via a modified IgG4 hinge-CH2-CH4 13 having the long hinge sequence 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] An extracellular domain containing a CD8 reader sequence 11 and a PEP2-2-1 binding peptide 12 was ligated to a transmembrane domain 14 provided by a portion 15 of human CD28 (having the amino acid sequence described in SEQ ID NO: 18 and the nucleotide sequence described in SEQ ID NO: 19), which also includes a portion 16 of the CD28 cytoplasmic domain.

[0210]

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

[0211] Lentiviral vector design and assembly

[0237] The designed nucleotide sequences of PEP2-2-1-1, PEP2-472-2, and PEP2-2-12 CAR were constructed using gene block technology (gBlock®, Gene Fragments-Integrated DNA Technologies, Iowa, USA) and the Gibson Assembly Cloning Kit (New The nucleotide constructs for PEP2-2-1-1, PEP2-472-2, or PEP2-2-12 CAR for incorporation into cloning vectors (including restriction sites) are described in SEQ ID NOs. 35, 36, and 37, respectively.

[0212]

[0238] The CAR nucleotide construct was incorporated into the pCDH-CMV-MCS-T2A(pCDH) vector (System Biosciences, California, USA, catalog number CD524A-1) shown in Figure 11, which contains a fluorescent reporter protein, green fluorescent protein (GFP). The pCDH vector further includes 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 PEP2-2-12 and PEP2-472-2 CAR nucleotide constructs into a pCDG vector, the pCDH vector was restriction-treated with 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 digestive enzymes. The restriction-treated gBlock fragments were then purified using the QIAquick PCR purification kit according to the manufacturer's instructions. The restriction-treated vector was ligated into the restriction-treated CAR constructs at an insert-to-vector molar ratio 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 therefore incorporated into the pCDH vector in a different manner than the PEP2-2-12 and PEP2-472-2 CAR nucleotide constructs. The pCDH vector was restricted with EcoR1, and the resulting 5'-overhang was packed with T4 DNA polymerase in the presence of 100 μM dNTPs (15 minutes at 12°C). The reaction was terminated (20 minutes at 75°C in the presence of 10 mM EDTA), and the restriction-treated vector was column purified (QIAquick PCR purification kit, QIAGEN). The purified vector was then further restricted with NotI and gel purified (QIAquick gel extraction kit, QIAGEN). The PEP2-2-1-1 CAR construct fragment was first restricted with SmaI and then digested with NotI (both at 25°C). Restriction enzyme-treated gBlock fragments are processed using QIAquick. Restriction enzyme-treated vectors were purified using a PCR purification kit according to the manufacturer's instructions. This was ligated with a CAR construct using an insert-to-vector molar ratio of 3:1.

[0215] Cloning and evaluation of pCDH-CAR vectors

[0241] The ligation mixes for each of the three CAR constructs described above were transformed into chemically competent SURE2 cells (Agilent) according to the manufacturer's instructions. Summary: - SURE2 cells were thawed on ice. After thawing, the cells were slowly mixed, and 100 μl of cell aliquots were placed in pre-cooled 14 ml round-bottom tubes. - 2 μl of β-mercaptoethanol was added to each cell aliquot. - The tubes were mixed and incubated on ice for 10 minutes, swirling slowly every 2 minutes. - 0.1 to 50 ng of each pCDH-CAR vector was added to cell aliquots. - After slowly mixing the aliquots, the mixture was incubated on ice for 30 minutes. - The tubes were subjected to a heat pulse treatment 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 while vigorously mixing at 225-250 rpm. - Up to 200 μl of the transformation mixture was placed in an LB agar plate containing antibiotics and incubated overnight at 37°C. - Colonies were collected and cultured overnight. Plasmid DNA was isolated from cultured clones using the Quicklyse Miniprep Kit (QIAGEN), digested by EcoRI / Not I digestion, and clones containing appropriately sized CAR-pCDH vectors were identified.

[0216]

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

[0217]

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

[0218]

[0244] Each selected clone was sequenced, and CAR incorporation was confirmed using appropriate primers selected from Table 5.

[0219] [Table 5]

[0220]

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

[0221] Construction and validation of viral vectors

[0246] Lentiviral packaging was performed in transiently transfected Hek293T cells using lipofectamine 2000 reagent (Invitrogen) according to a standard laboratory protocol (Brown, CY et al., 2010. Robust, reversible gene knockdown using a single lentiviral short hairpin RNA vector. Hum Gene Ther 21, 1005-1017). Abstract: - 12.5 ug of lentiviral vector DNA was mixed in a T75 cm flask with 3.75 ug of pMD2.g (VSV-G envelope expression vector), 6.25 ug of pRSV-Rev, and 7.5 ug of pCMVdelta8.2, along with 75 ug of lipofectin, per transfection, according to the manufacturer's protocol, and incubated overnight. - The culture medium was replaced the following morning, 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 µl of virus aliquots were stored at -70°C until needed.

[0222]

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

[0223]

[0248] Viral titers were calculated by transducing a known number of Hek293T cells with serial dilutions (1:50 and 1:100) of concentrated LV stock. Transduction was performed overnight in the presence of 8 ug / ml of polybren (hexadimethrin bromide). The following day, the medium containing the virus and polybren was replaced with fresh medium, and cells were collected after 24 hours. The percentage of GFP-positive cells was determined by flow cytometry. Viral titers were calculated using the formula: transduction units / ml (TU) = (F x C / V) x D (where F = frequency of GFP+ cells (%GFP+ / 100), C = number of cells at the time of virus addition, V = transduction volume in mL, and D = dilution factor). Representative flow data for LV-PEP2-472-2 transduction are shown in Figure 15. The TUs for 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 Production 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 (MNC) isolated from Buffy Coats (Australian Red Cross Blood Service) from anonymous donors. The MNC were isolated using Ficoll-Paque™ density gradient medium. CD8 T cells were purified from the MNC using the Dynabeads® Untouched™ Human CD8 T Cell Kit (Invitrogen) according to the manufacturer's instructions. The purity of the isolated cells evaluated by flow cytometry was 85% or higher.

[0227]

[0251] 2×10 6 Individual purified cells were pre-incubated with CD3 / CD28 beads (bead-to-cell ratio of 3:1) and IL2 (500 U / ml) for 30 minutes, 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 of polybrene. After incubating the cells with the virus for 16 hours, the virus-containing medium was removed. The remaining cells and beads were incubated in fresh medium containing IL2 for 40 hours, and then the GFP fluorescence level was analyzed.

[0228]

[0252] As shown in Figure 16, the 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 overexpressing 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 NO: 47 and 48, respectively) were ordered from Integrated DNA technologies (IDT). The EXD2 domain was designed from pDisplay (Invitrogen, Figure 17) such that a DNA sequence encoding a fusion protein consisting of IgK-leader-HA-MYC-PDGFR transmembrane domain was expressed in-frame. These fusion proteins were designed to be surface-expressed. 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-flanked 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 using the BP recombination reaction according to the manufacturer's protocol. - The transformed cells were seeded on LB agar plates containing 50 μg / ml kanamycin (Sigma) and incubated overnight at 37°C. - Two clones were selected from each plate and microcultures (2 mL) were prepared in LB culture medium 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 digestion using diagnostic Bam H1-HF(NEB) and Pmel(NEB). After digestion with Bam H1 and Bam H1 / PmeI, gel electrophoresis (Figure 18) confirmed that both EXD2_K193A and EXD2_WT clones were correctly digested.

[0231]

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

[0232]

[0257] After selecting clones, LR recombination was performed to transfer each EXD2 insert from the pDONR-107 entry clone to the target pLV-416 vector to produce an expression vector. The final LR recombination was used to transform chemically competent E. cloni® 10G cells (Lucigen®) according to the manufacturer's protocol. Summary: - Transformed cells were seeded onto LB agar plates containing 100 ug / ml ampicillin (SIGMA) and incubated overnight at 37°C. - Select 6 clones from each plate and prepare microcultures (2 mL) in LB culture medium containing ampicillin (50 ug / ml). Incubate these at 37°C overnight with stirring. It was incubated. The following day, plasmid DNA was isolated and digested with Bam H1 to identify recombinant clones. Recombinant clones were identified by the presence of three appropriately sized bands (3431, 1056, and 5844 bp; see Figure 19). As can be seen in Figure 19, appropriately sized restriction enzyme-treated fragments were obtained from all six clones selected from each plate. - Two clones transduced with pLV-416 constructs containing EXD2_K193A or EXD2_WT were sequenced using the primers listed in Table 7 to confirm the appropriateness of the constructs.

[0233] [Table 7]

[0234]

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

[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 the flask containing the control GFP-expressing lentivirus (LV-411-GFP). HEK293T cells transfected 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 HA-epitope tags and MYC-epitope tags. 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 to either PEP2-2-1-1 or PEP2-472-2 CAR constructs (prepared as described above) were cultured in 96-well round-bottom plates in 1 × 10⁶ cells expressing the nf-P2X7 receptor. 4 The target cells (prepared as described above) and MDA-MB-231 breast cancer cells expressing the non-functional P2X7 receptor (231 P2X7 cells) were co-cultured in a 1:1 ratio for 4 hours.

[0241]

[0263] The rate of cytotoxicity was determined using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wisconsin, USA) according to the manufacturer's instructions. Summary: - 10 μl of lysis solution (10x dilution) was added to each well for 100 μl of target cells for 45 minutes prior to 4 hours. - After another 45 minutes, the plate was 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 for 30 minutes at room temperature. - After 30 minutes, 50 μl of stop solution was added to each well, and the absorbance at 490 nm was read from each well.

[0242]

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

[0243]

Num

[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 syntax 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] Titles used herein are included solely for the convenience of reader reference and are not intended to limit the subject matter found throughout this disclosure or the claims. Titles are not intended to be used to interpret the claims or limitations thereof.

[0248]

[0269] Those skilled in the art will expect to recognize that the invention described herein may be subject to variations and modifications other than those specifically described. It is important to understand 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 indicated herein, as well as any combination of any two or more of the steps or features.

[0249]

[0270] Furthermore, it should be noted that, as used herein, the singular forms "a," "an," and "the" include plural forms unless otherwise indicated by the context.

[0250]

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

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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