Targeted therapy for the P2X7 receptor

Abstract

The present invention relates to a method for treating cancer, particularly cancers that have developed resistance to chemotherapy, particularly a method for treating cancer in an individual who has not responded or no longer responds to chemotherapy, comprising the steps of: providing an individual who has not responded or no longer responds to chemotherapy; providing in the individual a whole antibody or a fragment thereof comprising a variable domain for binding to a P2X7 receptor expressed by the individual; wherein the P2X7 receptor has an impaired response to ATP and is therefore unable to form apoptotic pores under normal physiological conditions, thereby treating cancer in the individual. Regarding the method.

Description

[Technical Field]

[0001] The present invention relates to a method for treating cancer, specifically cancers that have developed resistance to chemotherapy agents. Cross-reference to prior applications This application claims priority from Australian Provisional Application No. 2019902672, which is incorporated in its entirety by reference. [Background technology]

[0002] Despite improvements in treatments for cancer, cancer mortality rates remain high worldwide, and strategies to prevent cancer recurrence are still needed. Current cancer treatment strategies frequently result in treatment failure, often due to the development of multiple malignancies and / or resistance to chemotherapy and radiation therapy.

[0003] The development of chemotherapy resistance is a persistent problem in the course of chemotherapy treatment. For example, conventional treatment for acute myeloid leukemia (AML) includes the combined administration of cytarabine with anthracyclines such as daunorubicin. The 5-year overall survival rate is 40% in young adults and approximately 10% in elderly patients. The response rate changes dramatically with age, from 40% to 55% in patients over 60 years old, and from 24% to 33% in patients over 70 years old. These data highlight the need for novel approaches that reduce the dosage regimens of antitumor agents used to treat chemotherapy-sensitive tumors, while also circumventing the resistance of chemotherapy-resistant tumors to antitumor agents.

[0004] Various hypotheses have been proposed to explain the phenomenon of chemotherapy resistance. These hypotheses include altered drug transport across the plasma membrane, gene responses, enhanced DNA repair, changes in target molecules, access to target cells, metabolic effects, and growth factors. Recently, small pumps on the surface of cancer cells that actively transport chemotherapy drugs from the inside to the outside have been identified. Research on p-glycoprotein and other such chemotherapy efflux pumps is currently underway. Drug therapies that inhibit the function of p-glycoprotein have been explored to enhance the efficacy of chemotherapy. However, this approach has failed in the course of clinical evaluation. Nevertheless, it is increasingly recognized that the causes of chemotherapy resistance and relapse lie in a small number of cells that undergo further mutations as part of the transformation process acting in cancer.

[0005] Recent resurgence in our understanding of the role of clonal evolution in tumorigenesis has shed light on the challenge of acquired resistance, leading to two main models of tumorigenesis: the cancer stem cell model and the clonal evolution model. These can be considered complementary rather than mutually exclusive. Both indicate that the main obstacle to persistent therapies is the heterogeneity of cancer. However, current anticancer therapies largely fail to explain either model. While recent advances in targeted therapies are promising, the development of resistance is common because cancer heterogeneity and evolution present multiple mobile targets. Unfortunately, there has been little progress in targeting chemotherapy-resistant cells, which are the cause of recurrence.

[0006] New and / or improved cancer treatment regimens are needed, particularly those that overcome chemotherapy resistance in cancer cells or improve the sensitivity of cancer cells to non-targeted therapies such as chemotherapy and / or radiotherapy.

[0007] No prior art reference in this specification is acknowledged or implied in any jurisdiction that such prior art is part of common sense, nor is it acknowledged or implied that such prior art will be understood, deemed to be related to, and / or combined with other prior art by a person skilled in the art. [Overview of the Initiative] [Means for solving the problem]

[0008] In one aspect, the present invention is A method for treating cancer in individuals who have not responded to, or no longer respond to, chemotherapy and / or radiotherapy, - A step of preparing individuals that did not respond to, or no longer respond to, chemotherapy and / or radiotherapy; - A process for administering P2X7 receptor targeted therapy to an individual, Includes, Here, the P2X7 receptor has a impaired response to ATP and therefore cannot form an apoptotic pore under normal physiological conditions. This allows for the treatment of cancer in an individual. We will provide a method.

[0009] In any embodiment of the present invention, an individual no longer responds to one or more chemotherapeutic agents selected from the group consisting of: oxazaphosphorine, topoisomerase I inhibitor, topoisomerase II inhibitor, thymidylate synthase inhibitor, proteasome inhibitor, folate antimetabolites, nitrogen mustard, ethyleneimine derivatives, alkyl sulfonates, nitrosourea, triazenes, folate analogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs, purine antagonists, antimetabolites, antibiotics, epipodophyllotoxin, platinum-based agents, ribonucleotide reductase inhibitors, vinca alkaloids, substituted ureas, hydrazine derivatives, adrenal cortical depressants, endostatins, camptothecin, oxaliplatin, doxorubicin and doxorubicin analogs, antibiotics, L-asparaginase, tyrosine kinase inhibitors, or derivatives or variants thereof.

[0010] Preferably, one or more chemotherapeutic agents are selected from the group consisting of: doxorubicin, cisplatin, vincristine, dacarbazine (DTIC), cyclophosphamide, CPT-11, oxaliplatin, gemcitabine, and 5-fluorouracil / leucovorin.

[0011] Alternatively, one or more chemotherapeutic agents may be selected from the group consisting of: 5FU, folinic acid, bleomycin, etoposide, cisplatin, capecitabine, oxaliplatin, dacarbazine, cyclophosphamide, vincristine, doxorubicin, irinotecan, gemcitabine, mitomycin C, gemcitabine, carboplatin, paclitaxel, pemetrexed, hydroxyethyl-chloroethylnitrosourea (HeCNU), tamoxifen, methotrexate, epirubicin, vindesine, erlotinib, bevacizumab, and cetuximab.

[0012] In any aspect of the present invention, the individual has any cancer described herein. Preferably, the cancer is selected from the group consisting of colorectal cancer, testicular cancer, sarcoma, melanoma, bladder cancer, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, and breast cancer.

[0013] In any aspect of the present invention, the individual has colon cancer. In any aspect of the present invention, the individual has ovarian cancer. In any aspect, the targeted therapy of the P2X7 receptor results in a decrease in the survival rate of cancer cells that express a P2X7 receptor that has a response defect to ATP and thus cannot form an apoptotic pore under normal physiological conditions. The targeted therapy of the P2X7 receptor may be a direct or indirect inhibitor of the activity or expression level of the P2X7 receptor, where the P2X7 receptor has a response defect to ATP and thus cannot form an apoptotic pore under normal physiological conditions. For example, the inhibitor can be interfering RNA that can reduce the level of the P2X7 receptor in cancer cells.

[0014] The inhibitor of the P2X7 receptor can be selected from the group consisting of small molecules, antibodies, peptides, or interfering RNA. In any aspect, the inhibitor of the P2X7 receptor can be a molecule that induces an immune response of the individual to the P2X7 receptor expressed by the individual, preferably the P2X7 receptor expressed on cancer cells.

[0015] In another aspect, the present invention is a method of treating cancer in an individual who has not responded or no longer responds to chemotherapy and / or radiotherapy, comprising: - providing an individual who has not responded or no longer responds to a chemotherapeutic agent and / or radiotherapy; - supplying to the individual a whole antibody or a fragment thereof comprising a variable domain for binding to the P2X7 receptor expressed by the individual. Here, the P2X7 receptor has a impaired response to ATP and therefore cannot form an apoptotic pore under normal physiological conditions. This allows for the treatment of cancer in an individual. Provide a method.

[0016] In any embodiment, the step of administering a P2X7 receptor targeted therapy to an individual includes the step of supplying the individual with a whole antibody or a fragment thereof containing a variable domain for binding to a P2X7 receptor expressed by the individual, wherein the P2X7 receptor has a impaired response to ATP and therefore cannot form an apoptotic pore under normal physiological conditions.

[0017] In any aspect of the present invention, the antibody fragment is selected from the group consisting of dAb, Fab, Fd, Fv, F(ab')2, scFv, or any other antibody fragment format described herein.

[0018] In any aspect of the present invention, the antibody or fragment does not bind to a functional P2X7 receptor (i.e., a P2X7 receptor that does not have impaired response to ATP and is therefore capable of forming an apoptotic pore under normal physiological conditions). Preferably, the antibody or fragment binds to any one of SEQ ID NOs: 1 to 11, more preferably SEQ ID NOs: 2 to 5.

[0019] In any aspect of the present invention, the antibody or fragment thereof comprises the amino acid sequence of any antibody described below: PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or the corresponding U.S. Patents U.S. No. 7,326,415, U.S. No. 7,888,473, U.S. No. 7,531,171, U.S. No. 8,080,635, U.S. No. 8,399,617, U.S. No. (to any one of US Patent No. 8,709,425, US No. 9,663,584, or US No. 10,450,380), PCT / AU2007 / 001540 (or corresponding US Patent No. 8,067,550), PCT / AU2007 / 001541 (or corresponding US Publication No. 2010-0036101), PCT / AU2008 / 001364 (or corresponding (to any one of U.S. Patents U.S. 8,440,186, U.S. 9,181,320, U.S. 9,944,701 or U.S. 10,597,451), PCT / AU2008 / 001365 (or to any one of the corresponding U.S. Patents U.S. 8,293,491 or U.S. 8,658,385), PCT / AU2009 / 000869 (or to any one of the corresponding U.S. (to any one of U.S. Patents No. 8,597,643, No. 9,328,155, or No. 10,238,716) and PCT / AU2010 / 001070 (or the corresponding U.S. Patents No. 9,127,059, No. 9,688,771, or No. 10,053,508), the entire contents of which are incorporated herein by reference. Preferably, the antibody comprises a 2-2-1 CDR amino acid sequence described in PCT / AU2010 / 00170 (or any one of the corresponding U.S. Patent Nos. 9,127,059, 9,688,771, or 10,053,508), or BPM09 produced by hybridoma AB253 described in PCT / AU2007 / 001541 (or the corresponding U.S. Publication No. 2010-0036101) and deposited with the European Collection of Cultures (ECACC) under accession number 06080101.

[0020] In another embodiment, the present invention is A method for treating cancer in individuals who have not responded to, or no longer respond to, chemotherapy and / or radiotherapy, - A step of preparing individuals that did not respond to, or no longer respond to, chemotherapy and / or radiotherapy; - A step of supplying a cell therapy targeting cancer cells expressing the P2X7 receptor in an individual, - Here, the P2X7 receptor has a impaired response to ATP and therefore cannot form an apoptotic pore under normal physiological conditions. This allows for the treatment of cancer in an individual. Provide a method.

[0021] In any embodiment, cell therapy targeting cancer cells expressing the P2X7 receptor may be cytotoxic cells, such as CAR-T cells, that have the ability to bind to cancer cells expressing the P2X7 receptor.

[0022] In this embodiment, cytotoxic cells, preferably CAR-T cells, express a chimeric antigen receptor comprising an antigen recognition domain and a signaling domain, where the antigen recognition domain recognizes a dysfunctional or non-functional P2X7 receptor (i.e., the P2X7 receptor has a impaired response to ATP and is therefore unable to form an apoptotic pore under normal physiological conditions). Typically, a dysfunctional or non-functional P2X7 receptor has a reduced ability to bind ATP compared to the ATP-binding capacity of a wild-type (functional) P2X7 receptor. A dysfunctional or non-functional P2X7 receptor may have conformational changes that make the receptor dysfunctional or non-functional.

[0023] In this embodiment, the antigen recognition domain can recognize an epitope containing proline at amino acid position 210 of the P2X7 receptor. In this embodiment, the antigen recognition domain can recognize an epitope containing one or more amino acid residues spanning from glycine at amino acid position 200 to cysteine ​​at amino acid position 216 of the dysfunctional P2X7 receptor.

[0024] In this embodiment, the antigen recognition domain may include an amino acid sequence homologous to the amino acid sequence of an antibody or fragment thereof, including any antibody or fragment thereof as described herein, that binds to a dysfunctional or non-functional P2X7 receptor.

[0025] In this embodiment, the antigen recognition domain may include an amino acid sequence homologous to the amino acid sequence of a fragment-antigen binding (Fab) portion, a single-chain variable fragment (scFv), or a single antibody domain (dAb) of an antibody that binds to a dysfunctional or non-functional P2X7 receptor.

[0026] In this embodiment, the antigen recognition domain may include an amino acid sequence homologous to the amino acid sequence of a multivalent single-chain variable fragment (scFv) that binds to a dysfunctional or non-functional P2X7 receptor. The multivalent single-chain variable fragment (scFv) may be a divalent scFv or a trivalent scFv.

[0027] In this embodiment, the signal transduction domain may include a portion derived from the activating receptor. Typically, the activating receptor is a member of the CD3 coreceptor complex. Preferably, the portion derived from the CD3 coreceptor complex is CD3-zeta. Alternatively, the activating receptor is an Fc receptor, and preferably, the portion derived from the Fc receptor is an Fc epsilon RI or an Fc gamma RI.

[0028] In this embodiment, the signal transduction domain may include a portion derived from the co-stimulatory receptor. In this embodiment, the signal transduction domain may include a portion derived from an activating receptor and a portion derived from a co-stimulatory receptor.

[0029] In this embodiment, the co-stimulatory receptor may be selected from the group consisting of CD27, CD28, CD30, CD40, DAP10, OX40, 4-1 BB (CD137), and ICOS. In this embodiment, cytotoxic cells are: ·White blood cells, ·Peripheral blood mononuclear cells (PBMC), Lymphocytes, ·T cells, ·CD4+ T cells, ·CD8+ T cells, • Natural killer cells, or • Natural killer T cells It is one of the following.

[0030] In another aspect, the present invention relates to a method for treating cancer in individuals who have not responded to, or no longer respond to, chemotherapy and / or radiotherapy, - A process of preparing individuals that have not responded to, or will no longer respond to, chemotherapy and / or radiotherapy; - A process of forming an individual's immune response to the P2X7 receptor expressed by the individual, Includes, Here, the P2X7 receptor has a impaired response to ATP and therefore cannot form an apoptotic pore under normal physiological conditions. This allows for the treatment of cancer in an individual. Provide a method.

[0031] In any aspect of the present invention, the immune response is formed by supplying an immunogen in an organism in the form of a P2X7 receptor or a fragment of a P2X7 receptor capable of inducing an immune response to the P2X7 receptor in the organism, wherein the P2X7 receptor has impaired response to ATP and therefore cannot form an apoptotic pore under normal physiological conditions. Preferably, the fragment of the P2X7 receptor has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 11. More preferably, SEQ ID NOs: 2 to 5.

[0032] The immunogen may contain at least one sequence that can be presented on a major histocompatibility complex class II molecule and / or can interact with a T cell receptor, a B cell receptor, or a B cell membrane-bound immunoglobulin.

[0033] According to the present invention, the individual is a human, in which case the immunogen is supplied in the form of a human P2X7 receptor, or a fragment thereof capable of inducing an immune response to the P2X7 receptor. Typically, the immune response formed in an individual is specific to P2X7 receptors that have impaired response to ATP and therefore cannot form apoptotic pores under normal physiological conditions. In this case, antibodies or cellular components are formed in the individual that are reactive to non-functional P2X7 receptors (i.e., to one or more sites that cannot bind to ATP) but not to functional P2X7 receptors (i.e., ATP-binding receptors).

[0034] In any aspect of the present invention, the immunogen is supplied to the individual during the initial administration, thereby forming a response in the individual that includes IgM production. In any aspect of the present invention, the immunogen is supplied in an initial dose to an individual, thereby forming a response including IgM production, and is subsequently supplied in a further dose compared to the initial dose, thereby forming a response including IgG production.

[0035] The immune response can be a humoral and / or cellular response. Humoral responses may include transformation of B cells into antibody-secreting plasma cells, Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation of B cells and / or memory cell generation.

[0036] Cellular responses may include activation of antigen-specific cytotoxic T lymphocytes, activation of macrophages and natural killer cells, and / or stimulation of cytokine-secreting cells.

[0037] Humoral and / or cellular responses formed within an individual can treat or improve cancer in that individual, or minimize the progression of cancer in that individual. In another aspect, the present invention also provides a method for treating cancer in an individual, - The process of administering chemotherapeutic agents and / or radiotherapy to an individual whose cancer is to be treated; - The process includes administering to an individual a whole antibody or a fragment thereof containing a variable domain for binding to the P2X7 receptor expressed by the individual. Here, the P2X7 receptor has a impaired response to ATP and therefore cannot form an apoptotic pore under normal physiological conditions. This allows for the treatment of cancer in an individual. Provide a method.

[0038] In another aspect, the present invention also provides a method for treating cancer in an individual, - A step of preparing individuals that have responded to chemotherapy and / or radiotherapy; - The process includes administering to an individual a whole antibody or a fragment thereof containing a variable domain for binding to the P2X7 receptor expressed by the individual. Here, the P2X7 receptor has a impaired response to ATP and therefore cannot form an apoptotic pore under normal physiological conditions. This allows for the treatment of cancer in an individual. Provide a method.

[0039] In this embodiment, chemotherapeutic agents and / or radiotherapy may be administered simultaneously with antibodies or fragments thereof. In one embodiment, chemotherapy and antibodies or fragments thereof are administered simultaneously. In another embodiment, radiotherapy and antibodies or fragments thereof are administered simultaneously.

[0040] In this embodiment, chemotherapeutic agents and / or radiotherapy may be administered following the antibody or a fragment thereof. In this embodiment, the chemotherapeutic agent may be administered prior to the antibody or a fragment thereof.

[0041] In this embodiment, the chemotherapeutic agent may be any of those described herein. Preferably, the chemotherapeutic agent is selected from the group consisting of: oxazaphosphorine, topoisomerase I inhibitor, topoisomerase II inhibitor, thymidylate synthase inhibitor, proteasome inhibitor, folic acid antimetabolites, nitrogen mustard, ethyleneimine derivatives, alkyl sulfonates, nitrosourea, triazenes, folic acid analogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs, purine antagonists, antimetabolites, antibiotics, epipodophyllotoxin, platinum-based drugs, ribonucleotide reductase inhibitors, vinca alkaloids, substituted ureas, hydrazine derivatives, adrenal cortical depressants, endostatins, camptothecin, oxaliplatin, doxorubicin and doxorubicin analogs, antibiotics, L-asparaginase, tyrosine kinase inhibitors, or derivatives or variants thereof.

[0042] Preferably, the chemotherapeutic agent is selected from the group consisting of: doxorubicin, cisplatin, vincristine, dacarbazine (DTIC), cyclophosphamide, CPT-11, oxaliplatin, gemcitabine, and 5-fluorouracil / leucovorin.

[0043] Alternatively, the chemotherapeutic agent may be selected from the following group: 5FU, folinic acid, bleomycin, etoposide, cisplatin, capecitabine, oxaliplatin, dacarbazine, cyclophosphamide, vincristine, doxorubicin, irinotecan, gemcitabine, mitomycin C, gemcitabine, carboplatin, paclitaxel, pemetrexed, hydroxyethyl-chloroethylnitrosourea (HeCNU), tamoxifen, methotrexate, epirubicin, vindesine, erlotinib, bevacizumab, cetuximab.

[0044] In another aspect, the present invention also provides a method for treating cancer in an individual, - The process of administering chemotherapeutic agents and / or radiotherapy to an individual whose cancer is to be treated; - A process of forming an individual's immune response to the P2X7 receptor expressed by the individual, Includes, Here, the P2X7 receptor has a impaired response to ATP and therefore cannot form an apoptotic pore under normal physiological conditions. This allows for the treatment of cancer in an individual. Provide a method.

[0045] In another aspect, the present invention also provides a method for treating cancer in an individual, - A step of preparing individuals that have responded to chemotherapy and / or radiotherapy; - A process of forming an individual's immune response to the P2X7 receptor expressed by the individual, Includes, Here, the P2X7 receptor has a impaired response to ATP and therefore cannot form an apoptotic pore under normal physiological conditions. This allows for the treatment of cancer in an individual. Provide a method.

[0046] In any aspect of the present invention, the immune response is formed by supplying an immunogen in an organism in the form of a P2X7 receptor or a fragment of a P2X7 receptor that can induce an immune response to the P2X7 receptor in the organism, wherein the P2X7 receptor has impaired response to ATP and therefore cannot form an apoptotic pore under normal physiological conditions. Preferably, the fragment of the P2X7 receptor has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 11, more preferably SEQ ID NOs: 2 to 5.

[0047] In this embodiment, the chemotherapeutic agent and the immunogen may be administered simultaneously. In this embodiment, the chemotherapeutic agent and immunogen may be administered sequentially. In this embodiment, the chemotherapeutic agent may be administered prior to the immunogen.

[0048] In this embodiment, the chemotherapeutic agent may be any of those described herein. Preferably, the chemotherapeutic agent is selected from the group consisting of: nitrogen mustard, ethyleneimine derivatives, alkyl sulfonates, nitrosourea, triazenes, folic acid analogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs, antimetabolites, antibiotics, epipodophyllotoxin, platinum coordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenal cortical inhibitors, endostatins, taxol, camptothecin, oxaliplatin, doxorubicin, and doxorubicin analogs. Preferably, the chemotherapeutic agent is selected from the group consisting of: doxorubicin, cisplatin, vincristine, dacarbazine (DTIC), cyclophosphamide, CPT-11, oxaliplatin, gemcitabine, and 5-fluorouracil / leucovorin.

[0049] In any embodiment, the cancer may be a blood-derived cancer or a solid tumor. In any embodiment, cancer may be any of those described herein, including but not limited to those 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, neuroblastoma, leukemia, lymphoma, myeloma, breast cancer, ovarian cancer, endometrial cancer, and testicular cancer.

[0050] As used herein, unless the context requires otherwise, the term “contains” and its variations, such as “contains,” “contains,” and “contained,” are not intended to exclude any further additions, components, integers, or processes.

[0051] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description given as examples and with reference to the accompanying drawings. [Brief explanation of the drawing]

[0052] [Figure 1] This figure shows the effects of chemotherapy on myeloma RPMI-8226 cell lines and neuroblastoma Kelly cell lines. A) Figure showing normalized ethidium inflow in response to 0.5 mM BzATP stimulation in myeloma RPMI-8226 cell lines and neuroblastoma Kelly cell lines. Mean of 3 independent experiments is shown. B) Figure showing the effect of dose-increasing doxorubicin on RPMI-8226 cell viability as measured using the CellTitle-Blue (CTB) assay. C) Figure showing the effect of dose-increasing doxorubicin on Kelly cell viability as measured using the CTB assay. D) Figure showing the effect of dose-increasing 5 Fu on Kelly cell viability as measured using the CTB assay. [Figure 2A] This figure shows the effects of chemotherapy treatment and induction of nfP2X7 detected by BPM09 in functional myeloma RPMI-8226 cell line and non-functional neuroblastoma Kelly cell line. A) This figure shows the effect of increasing the dose of doxorubicin (0.0625 μM to 0.25 μM) on the binding of nfP2X7 antibody (here, BPM09) to living RPMI-8226 cells by flow cytometry. [Figure 2B] A) Figure showing the induction of nfP2X7 detected by chemotherapy and BPM09 in functional myeloma RPMI-8226 cell line and non-functional neuroblastoma Kelly cell line. B) Figure showing the effect of increasing the dose of doxorubicin (0.0625 μM~0.25 μM) on the binding of nfP2X7 antibody (here, BPM09) to living Kelly cells by flow cytometry. [Figure 2C] This figure shows the effects of chemotherapy treatment and the induction of nfP2X7 detected by BPM09 in functional myeloma RPMI-8226 cell line and non-functional neuroblastoma Kelly cell line. C) This figure shows the effect of increasing the dose of 5Fu (1 μM to 8 μM) on the binding of nfP2X7 antibody to living Kelly cells by flow cytometry. [Figure 3]This figure shows the changes in living ovarian A2780 parental cells and the nfP2X7 antibody (here, BPM09) that binds to A2780 cells, exhibiting acquired resistance to doxorubicin and cisplatin. [Figure 4] This figure shows BPM09 membranes scored after immunohistochemistry in a series of 200 patient-derived xenograft models, some previously treated with chemotherapy or radiotherapy, and some not. A similar analysis is performed on a colorectal dataset (37 samples). [Figure 5] This figure shows the effect of 5Fu on complement-dependent cytotoxicity mediated by the nfP2X7 antibody (here, a polyclonal mouse antibody) in Kelly cells. [Figure 6] A) Figure showing data demonstrating that HMGB1 can drive the increase of nfP2X7. B) Figure showing that nfP2X7 induction in response to conditioned medium from doxorubicin-treated cells is not blocked by a P2X7 inhibitor. Conditioned medium derived from doxorubicin-treated RPMI-8226 does not act via ATP. [Modes for carrying out the invention]

[0053] Next, we will refer in detail to certain embodiments of the present invention. While the present invention is described in relation to embodiments, it will be understood that the present invention is not limited to those embodiments. Rather, the present invention is intended to cover all substitutes, modifications, and equivalents, which may fall within the scope of the present invention as defined by the claims.

[0054] Those skilled in the art will understand that there are many methods and materials similar to or equivalent to those described herein that can be used in carrying out the present invention. The present invention is not limited in any way to the methods and materials described herein.

[0055] It will be understood that the present invention, as disclosed and defined herein, extends to all selective combinations of two or more of the individual features described or evident from the text or drawings. All of these various combinations constitute various selective embodiments of the present invention.

[0056] As used herein, unless the context requires otherwise, the term “contains” and its variations, such as “contains,” “contains,” and “contained,” are not intended to exclude any further additions, components, integers, or processes.

[0057] All patents and publications referenced herein are incorporated in their entirety by reference. For the purposes of interpreting this specification, the following definitions will apply throughout, and where appropriate, terms used in the singular will also include the plural, and vice versa. In the event of any conflict between any definition provided herein and any reference incorporated herein by reference, the definitions below shall prevail.

[0058] The inventors have surprisingly confirmed that chemotherapy or radiotherapy increases the level of P2X7 receptors that are unable to form apoptotic pores in living cells. Furthermore, the inventors have confirmed that the level of P2X7 receptors that are unable to form apoptotic pores increases on cells that have developed complete or partial resistance to chemotherapy. These findings are independent of the type of chemotherapy, as different structural classes of chemotherapeutic agents with different mechanisms of action; all result in an increase in the level of P2X7 receptors that are unable to form apoptotic pores. Moreover, these findings apply to a wide variety of cancers, regardless of tissue origin (e.g., blood-derived or solid). Finally, the inventors have shown that these chemotherapy-driven level increases occur in both cell lines derived from patient samples and primary cells.

[0059] The inventors also demonstrated that cancer cells pretreated with chemotherapy, and therefore cancer cells having elevated levels of P2X7 receptors that are unable to form apoptotic pores, are sensitive to various interventions targeting the P2X7 receptor.

[0060] While not bound by any particular theory or mode of action, it is surprising to see that the mechanism by which non-targeted therapies such as chemotherapy and radiotherapy increase the levels of P2X7 receptors, which are unable to form apoptotic pores on cells, does not appear to be mediated via ATP. Instead, it is more likely that DAMPs such as HMGB1 mediate the effect.

[0061] definition "Purine receptors" as a whole refer to receptors that use purines (for example, ATP) as ligands.

[0062] The term "P2X7 receptor" as a whole refers to a purine receptor formed from three protein subunits or monomers, in which case at least one, preferably all three monomers substantially have the amino acid sequence shown in Sequence ID No. 1. Insofar as the P2X7 receptor is formed from three monomers, it is "trimer" or "trimeric." The "P2X7 receptor" may be a functional or non-functional receptor, as described below. The "P2X7 receptor" encompasses naturally occurring variants of the P2X7 receptor, for example, the P2X7 monomer is a splice variant, an allele variant, and an isoform, which include naturally occurring truncated or secreted forms of the monomers forming the P2X7 receptor (e.g., forms consisting of its extracellular domain sequence or truncated form), naturally occurring variant forms (e.g., alternative splicing forms), and naturally occurring allele variants. In certain embodiments of the present invention, the natural sequence P2X7 monomer polypeptide disclosed herein is a mature or full-length natural sequence polypeptide comprising the full-length amino acid sequence shown in SEQ ID NO: 1. In certain embodiments, the P2X7 receptor may have a modified amino acid sequence, for example, various amino acids in the sequence shown in SEQ ID NO: 1 may be substituted, deleted, or have residues inserted.

[0063] A "functional P2X7 receptor" as a whole refers to a form of P2X7 receptor that has a binding site or cavity for binding to ATP. When bound to ATP, the receptor forms a non-selective cation channel that converts into a pore-like structure that allows for increased influx of calcium ions and molecules up to 1000 Da into the cytosol, one consequence of which can be programmed cell death. In normal homeostasis, the expression of functional P2X7 receptors is generally limited to cells that induce programmed cell death, such as thymocytes, dendritic cells, lymphocytes, macrophages, and monocytes. Some expression of functional P2X7 receptors may also be present on erythrocytes and other cell types.

[0064] "Non-functional P2X7 receptors" or "nfP2XT" refer to a form of the P2X7 receptor in which the receptor as a whole has a conformation that prevents it from forming an apoptotic pore, but in which the apoptotic pore can still function as a non-selective channel. Isomerization can arise from any molecular event that leads to monomer misfolding, including, for example, mutations in the monomer primary sequence or abnormal post-translational processing. One consequence of isomerization is that the receptor is unable to expand the channel opening. Under these circumstances, the receptor cannot form a pore, which limits the extent to which calcium ions and molecules up to 1000 Da can enter the cytosol. Non-functional P2X7 receptors are expressed on a wide range of epithelial and hematopoietic cancers.

[0065] "Cancer-related P2X7 receptors" are P2X7 receptors found on cancer cells (including proneoplastic cells, neoplastic cells, malignant cells, benign cells, or metastatic cells) but not on non-cancer cells or normal cells.

[0066] The term "E200 epitope" refers to the epitope exposed on non-functional P2X7 receptors as a whole. In humans, the sequence is GHNYTTRNILPGLNITC (SEQ ID NO: 5).

[0067] The term "E300 epitope" refers to the epitope exposed on non-functional P2X7 receptors as a whole. In humans, the sequence is KYYKENNVEKRTLIKVF (Sequence ID 8).

[0068] A "composite epitope" refers to an epitope formed as a whole from the juxtaposition of E200 epitopes and E300 epitopes, or parts of these epitopes. As used herein, “targeted therapy of the P2X7 receptor” is any therapy that directly or indirectly reduces the viability of cancer cells expressing the P2X7 receptor, which is unable to form apoptotic pores under normal physiological conditions due to impaired response to ATP. Typically, such therapy involves the administration of a molecule that binds to, induces an immune response to, or reduces the level of the P2X7 receptor, which is unable to form apoptotic pores under normal physiological conditions due to impaired response to ATP. Preferably, the molecule that binds to the P2X7 receptor is an antibody or cell therapy. Preferably, the molecule that induces an immune response to the P2X7 receptor is an immunogen in an organism in the form of the P2X7 receptor, or a fragment of the P2X7 receptor that can induce an immune response to the P2X7 receptor in an organism, where the P2X7 receptor is unable to form apoptotic pores under normal physiological conditions due to impaired response to ATP. Preferably, the molecule that reduces the level of the P2X7 receptor is interfering RNA. Inhibition of P2X7 receptors that have impaired response to ATP may also include a reduction in the level or amount of P2X7 receptor protein, RNA, or DNA within cells, preferably cancer cells. These molecules may be specific to the P2X7 receptor and may exhibit only slightly lower levels of inhibitory activity against other P2X receptors.

[0069] "Antibodies," or "immunoglobulins," or "Ig," are gamma globulin proteins found in the blood or other bodily fluids of vertebrates that function in the immune system to bind to antigens and thus identify and / or neutralize foreign substances.

[0070] Antibodies are generally heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Each L chain is linked to an H chain by a single covalent disulfide bond. The two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges.

[0071] The H and L chains define specific Ig domains. More specifically, each H chain has at its N-terminus a variable domain (V H ), followed by three constant domains (C H ) for each of the α and γ chains and four C H domains for each of the μ isotype and ε isotype. Each L chain has at its N-terminus a variable domain (V L ), followed by a constant domain (C L ) at its other terminus. V L aligns with V H , and C L aligns with the first constant domain (C H 1) of the heavy chain.

[0072] Antibodies can be assigned to various classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each having a heavy chain named α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in the sequence and function of C H . For example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. L chains from all vertebrate species can be assigned to one of two distinct types called kappa and lambda based on the amino acid sequence of their constant domains.

[0073] The constant domain includes the Fc portion that contains the carboxy-terminal portions of both H chains linked by disulfide bonds. The effector functions of antibodies such as ADCC are determined by the sequences in the Fc region, which is also the part recognized by Fc receptors (FcR) found on certain types of cells.

[0074] V H and V LPair formation involves the joint formation of a "variable region" or "variable domain" containing the amino-terminal domain of the antibody's heavy or light chain. The variable domain of the heavy chain is "V H It can be called "V". The variable domain of the light chain is "V L The V domain may be called the "antigen-binding domain." The V domain contains an "antigen-binding site" that influences antigen binding and defines the specificity of a particular antibody to a particular antigen. The V region spans approximately 110 amino acid residues and consists of relatively invariant extensions called framework regions (FRs) (generally about 4) of 15 to 30 amino acids, separated by shorter, highly variable regions called "hypervariable regions" (generally about 3), each generally 9 to 12 amino acids long. The FRs mainly adopt a β-sheet configuration, and the hypervariable regions form loops connecting the β-sheet structures, although in some cases they form parts of the β-sheet structure.

[0075] A "hypervariable region" refers to a region of the antibody's variable domain that is hypervariable in sequence and / or forms a structurally defined loop. Generally, antibodies contain six hypervariable regions; V H Inside are three (H1, H2, H3) and V L There are three inside (L1, L2, L3).

[0076] A "framework" or "FR" residue is a variable domain residue other than a hypervariable region residue as defined herein. The term "antigen-binding site" refers to a molecule that, as a whole, includes at least a hypervariable region and a framework region necessary to confer antigen-binding function to the V domain. In the methods described herein, the antigen-binding site may be in the form of an antibody or antibody fragment (e.g., dAb, Fab, Fd, Fv, F(ab')2, or scFv).

[0077] "Intact" or "whole" antibodies are those that have an antigen-binding site along with the C2 antibody. L Furthermore, at least the heavy chain constant domain C H 1, C H 2, and C HIt includes 3. The constant domain may be the natural sequence constant domain (e.g., the human natural sequence constant domain) or an amino acid sequence variant thereof.

[0078] "All antibody fragments containing variable domains" include the Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0079] The "Fab fragment" is the variable region domain (V) of the H chain. H ) along with the entire L chain and the first constant domain (C) of one of the heavy chains H 1) consists of the following. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.

[0080] "Fab' fragment" contains one or more cysteines from the antibody hinge region, C H It differs from the Fab fragment by having a few additional residues at the carboxyl terminus of one domain. Fab'-SH is the name used herein for Fab' when the cysteine ​​residue of the constant domain possesses a free thiol group.

[0081] The "F(ab')2 fragment" roughly corresponds to two disulfide-linked Fab fragments that possess bivalent antigen-binding activity and can still cross-link to the antigen. "Fv" is the smallest antibody fragment containing a complete antigen recognition site and an antigen binding site. This fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain, tightly associated by non-covalent bonds.

[0082] In single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate in a "dimer" structure similar to that in double-chain Fv species. The folding of these two domains gives rise to six hypervariable loops (three from the H chain and three from the L chain), which contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody.

[0083] "Single-chain Fv," also abbreviated as "sFv" or "scFv," refers to V linked to form a single polypeptide chain. H antibody domain and V L It is an antibody fragment containing an antibody domain. Preferably, the scFv polypeptide allows the scFv to form a desired structure for antigen binding. H Domain and V L It further includes polypeptide linkers between domains.

[0084] A "single variable domain" is half of the Fv (containing only three antigen-specific CDRs), which has lower affinity than the entire binding site but still possesses the ability to recognize and bind to the antigen.

[0085] A "diabody" refers to an antibody fragment that has two antigen-binding sites, and this fragment is made up of the same polypeptide chain (V H -V L ) contains a light chain variable domain (V L ) connected to the heavy chain variable domain (V H ) contains. The small antibody fragment is such that interchain pairing rather than intrachain pairing occurs with respect to the V domain. H and V L It is prepared by constructing an sFv fragment (see previous section) which has a short linker (approximately 5-10 residues) between the domains, resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites.

[0086] Diabodies can be bivalent or bispecific. A bispecific diabody is a heterodimer of two "crossover" sFv fragments, in this case, the V of two antibodies. H Domain and V L The domains are located on different polypeptide chains. Triabodies and tetrabodies are also commonly known in the art.

[0087] "Isolated antibodies" are those identified, isolated, and / or recovered from the components of their pre-existing environment. "Contaminants" are substances that are likely to interfere with the therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0088] A "human antibody" refers to an antibody that has an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but in which the endogenous gene locus has been deactivated.

[0089] The "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody containing a minimal sequence derived from the non-human antibody. For the most part, the humanized antibody is a human immunoglobulin (recipient antibody), in which case residues from the recipient's hypervariability region are replaced with residues from the hypervariability region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, that have the desired antibody specificity, affinity, and capabilities. In some cases, framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the recipient antibody or the donor antibody. These modifications are made to further refine the antibody performance. Generally, the humanized antibody will contain substantially all of at least one, typically two, variable domains, in which case all or substantially all of the hypervariability loops correspond to the hypervariability loops of the non-human immunoglobulin, and all or substantially all of the FRs are FRs of the human immunoglobulin sequence. Humanized antibodies will also, if necessary, include the immunoglobulin constant region (Fc), typically at least one portion of the immunoglobulin constant region of human immunoglobulin.

[0090] A "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are highly specific and directed to a single antigenic site or antigenic determinant on an antigen. In addition to their specificity, monoclonal antibodies have the advantage of being able to be synthesized without contamination by other antibodies. Monoclonal antibodies can be prepared by hybridoma. Monoclonal antibodies can also be isolated from phage antibody libraries using this technique.

[0091] The term “anti-P2X7 receptor antibody” or “antibody that binds to the P2X7 receptor” refers to an antibody that can bind to the P2X7 receptor with sufficient affinity to be useful as a diagnostic and / or therapeutic agent when targeting the P2X7 receptor, typically a non-functional P2X7 receptor. Preferably, the degree to which the P2X7 receptor antibody binds to unrelated proteins is less than approximately 10% of the antibody’s binding to the P2X7 receptor, as evaluated, for example, by radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), Biacore, or flow cytometry. In certain embodiments, the antibody that binds to the P2X7 receptor has a dissociation constant (Kd) of <1 μM, <100 nM, <10 nM, <1 nM, or <0.1 nM. An anti-non-functional P2X7 receptor antibody is an antibody that, as a whole, possesses some or all of these serological characteristics and binds to a non-functional receptor but not to a functional receptor.

[0092] A "affinity-mature" antibody is an antibody that, compared to a parent antibody that does not possess changes resulting in improved affinity for an antigen, has one or more changes in one or more hypervariable regions that result in improved affinity for the antigen. Preferred affinity-mature antibodies will have nanomolar or even picomolar affinity for the target antigen. Affinity-mature antibodies are produced by procedures known in the art.

[0093] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. A preferred blocking or antagonist antibody substantially or completely inhibits the biological activity of the antigen.

[0094] As used herein, "agonist antibody" refers to an antibody that mimics at least one of the functional activities of the target polypeptide. "Binding affinity" as a whole refers to the total strength of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by general methods known in the art, including those described herein. Low-affinity antibodies generally tend to bind to antigens slowly and dissociate easily, while high-affinity antibodies generally tend to bind to antigens more quickly and remain bound for longer. Various methods for measuring binding affinity are known in the art, and any of them can be used for the purposes of this invention.

[0095] An "epitope" as a whole refers to the portion of an antigen to which it is bound by the antigen-binding site of an antibody. An epitope can be described as "linear" in the sense that the hypervariable loop of the antibody CDR that forms the antigen-binding site binds to the amino acid sequence as it is in the primary protein structure. In certain embodiments, the epitope is a "concrete epitope," that is, an epitope in which the hypervariable loop of the CDR binds to the residues as they are presented in the tertiary or quaternary protein structure.

[0096] The term "treatment" as a whole refers to both therapeutic treatments and prophylactic or preventative measures. Patients requiring treatment include those who already have benign, precancerous, or non-metastatic tumors, as well as those for whom the development or recurrence of cancer should be prevented.

[0097] The objectives or outcomes of the treatment may be to reduce the number of cancer cells; reduce the size of the primary tumor; inhibit (i.e., slow down, preferably stop) the invasion of cancer cells into surrounding organs; inhibit (i.e., slow down, preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with the disorder.

[0098] The efficacy of a treatment can be measured by assessing survival time, time to disease progression, response rate (RR), duration of response, and / or quality of life. In one embodiment, the method of the present invention is particularly useful for delaying disease progression.

[0099] In one embodiment, the method of the present invention is particularly useful for extending human survival, including overall survival and progression-free survival. In one embodiment, the method of the present invention is particularly useful in achieving a complete response to therapy in which all signs of cancer disappear in response to the treatment. This does not necessarily mean that the cancer is cured.

[0100] In one embodiment, the method of the present invention is particularly useful for producing a partial response to therapy in which the size of one or more tumors or lesions or the extent of cancer within the body is reduced in response to the treatment.

[0101] "Precancerous" or "preneoplastic" generally refers to a condition or growth that usually precedes or develops into cancer. Precancerous growth may have cells characterized by abnormal cell cycle regulation, proliferation, or differentiation, which can be determined by cell cycle markers.

[0102] In one embodiment, the cancer is precancerous or preneoplastic. In one embodiment, the cancer is a secondary cancer or metastasis. Secondary cancers may be present in any organ or tissue, particularly those with relatively higher hemodynamic pressure, such as the lungs, liver, kidneys, pancreas, intestines, and brain.

[0103] Other examples of cancer include blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma and islet cell carcinoma), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancies, lung cancer including small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial carcinoma or uterine carcinoma, salivary gland carcinoma, kidney cancer or renal cancer This includes cancers such as prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, biliary tract tumors, and head and neck cancers.

[0104] "Conditions or symptoms associated with cancer" can be any medical condition that arises as a result of cancer, preceding cancer, or progressing from cancer. For example, if the cancer is skin cancer, the condition or associated symptoms may be a microbial infection. If the cancer is a secondary tumor, the condition or symptoms may be related to organ failure in the associated organs with tumor metastases. In one embodiment, the treatment methods described herein are for minimizing or treating conditions or symptoms associated with cancer in an individual.

[0105] A “non-self” molecule, such as a “non-self” antigen-binding site or “non-self” antibody, as a whole, refers to a molecule produced outside the body or that is exogenous to the body, in which case the molecule will be supplied, for example, for treatment. For example, synthetic or recombinant molecules are “non-self.” Furthermore, a molecule produced in one individual and administered to another individual for treatment is “non-self.” “Non-self” antigen-binding sites and antibodies can be used in accordance with the present invention for adoptive transfer of immunity, for example, in antibody injection. In contrast, a molecule produced within an individual being treated with a molecule is, as a whole, a “self” molecule or “endogenous” molecule. An example of a “self” molecule is an antigen-binding site or antibody that is produced or arises from an adaptive immune response to an immunogen.

[0106] In an individual, the "level of non-self antigen binding sites in circulation" refers to the overall concentration of antigen binding sites in body fluids, preferably in peripheral blood. "Substantially undetectable levels of non-self antigen-binding sites in circulation" refers to the overall concentration of exogenous antigen-binding sites (i.e., those administered by adoptive transfer), which is less than half the concentration of antigen-binding sites in circulation at the time of administration, preferably 25%, 10%, 5%, or 1%, or otherwise less than 0.001 mg / kg of the individual. This phrase may also refer to a situation where antigen-binding sites administered for the purpose of cancer immunotherapy are completely undetectable.

[0107] "Substantially undetectable" cancer, as a whole, refers to a situation where, due to the drastic reduction in the size, volume, or other physical measurements of the tumor, the cancer is clearly undetectable as a result of the therapy, using relevant standard detection techniques such as in vivo imaging. This phrase can also refer to a situation where the cancer is completely undetectable.

[0108] "Forming an immune response" as a whole refers to activating or inducing antigen-specific immunity through the adaptive immune system. As is commonly understood in the art, induction of antigen-specific immunity is distinguished from adoptive immune transfer, and standard cancer immunotherapy with the administration of exogenous or non-self antibodies is an example of the latter.

[0109] Individuals selected for treatment In one embodiment, individuals selected for treatment by the above method are individuals that have received or are currently receiving chemotherapy for the treatment of cancer. For example, individuals may be receiving one or more of the chemotherapy regimens described herein, including: oxazaphosphorine, topoisomerase I inhibitor, topoisomerase II inhibitor, proteasome inhibitor, folate antimetabolites, nitrogen mustard, ethyleneimine derivatives, alkyl sulfonates, nitrosourea, triazenes, folate analogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs, purine antagonists, antimetabolites, antibiotics, epipodophyllotoxin, platinum-based agents, ribonucleotide reductase inhibitors, vinca alkaloids, substituted ureas, hydrazine derivatives, corticosteroids, endostatins, camptothecin, oxaliplatin, doxorubicin and doxorubicin analogs, antibiotics, L-asparaginase, tyrosine kinase inhibitors, or derivatives or variants thereof.

[0110] In one embodiment, the individual may receive chemotherapy that results in a reduced tumor volume, but still clinically or biochemically detectable tumor volume. For example, at the time P2X7 targeted treatment is applied, the cancer may have substantially decreased in size, volume, or other physical measurements as a result of the chemotherapy.

[0111] Furthermore, individuals selected for treatment using the above method may or may not have detectable cancer at the time of treatment. In another embodiment, the individuals selected for treatment are those that did not respond to chemotherapy, or no longer respond.

[0112] The objective of the treatment described above is to suppress cancer progression to at least a minimum extent. One approach to treatment involves inducing or forming an immune response in the individual against non-functional P2X7 receptors. Therefore, individuals selected for this form of treatment must be able to generate an immune response sufficient to satisfy this objective. Generally, the desired immune response includes the ability to produce either or both of circulating IgM and IgG when the individual is challenged by cancer, such as in cancer recurrence.

[0113] Antigen binding site and administration One approach to targeted therapy for the P2X7 receptor is the administration of an antigen-binding site or antibody that binds to the P2X7 receptor, which has a impaired response to ATP and therefore cannot form apoptotic pores under normal physiological conditions.

[0114] Typically, an antigen-binding site is a site that distinguishes between functional and non-functional P2X7 receptors, binding to the non-functional receptor but not to the functional receptor. Examples of such antigen-binding sites include those that bind to E200 epitopes, E300 epitopes, or complex epitopes, such as those described below: PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or corresponding U.S. Patents US No. 7,326,415, US No. 7,888,473, US No. 7,531,171, US No. 8,080,635, (to any one of US Patent No. 8,399,617, US Patent No. 8,709,425, US Patent No. 9,663,584, or US Patent No. 10,450,380), PCT / AU2007 / 001540 (or to the corresponding US Patent No. 8,067,550), PCT / AU2007 / 001541 (or to the corresponding US Publication No. 2010-0036101), PCT / AU2008 / 0 01364 (or to any one of the corresponding U.S. Patents US 8,440,186, US 9,181,320, US 9,944,701 or US 10,597,451), PCT / AU2008 / 001365 (or to any one of the corresponding U.S. Patents US 8,293,491 or US 8,658,385), PCT / AU2009 / 000869 (and (or to any one of the corresponding U.S. Patents U.S. 8,597,643, U.S. 9,328,155 or U.S. 10,238,716) and PCT / AU2010 / 001070 (or to any one of the corresponding U.S. Patents U.S. 9,127,059, U.S. 9,688,771 or U.S. 10,053,508), the entire contents of which are incorporated herein by reference.

[0115] In certain embodiments, the antigen-binding site includes a 2-2-1 CDR amino acid sequence described in PCT / AU2010 / 00170 (or any one of the corresponding U.S. Patent Nos. 9,127,059, 9,688,771, or 10,053,508), or BPM09 produced by hybridoma AB253 described in PCT / AU2007 / 001541 (or the corresponding U.S. Publication No. 2010-0036101) and deposited with the European Collection of Cultures (ECACC) under accession number 06080101.

[0116] Regardless of specificity (i.e., whether P2X7 receptor specific or not), the antigen-binding site can take the form of the entire antibody, or the entire antibody fragment, such as Fab, Fab', F(ab')2, and Fv, single-chain Fv, or a single variable domain.

[0117] The antigen-binding site may be of the same system, of the same but different system, or of different systems. Typically, the antigen-binding site is non-self or exogenous, meaning that the antigen-binding site was found or isolated outside the individual treated by the method of the present invention.

[0118] The antigen-binding site can undergo affinity maturation. Antigen-binding sites can have multiple specificities or binding valencies. The antigen-binding site can be adapted to suit the administration method of the selected type.

[0119] The antibody of the present invention may be a whole antibody of any isotype. The antibody of the present invention may be an antibody obtained from monoclonal or polyclonal antiserum. The antibody of the present invention may be produced by hybridoma or recombinant expression, or may be obtained from serum, as is available from mammals, particularly humans or mice. The antibody may also be obtained from birds.

[0120] The antibodies of the present invention may be chimeric, i.e., they may contain a human variable domain and a non-human constant domain. Alternatively, the antibodies of the present invention may be humanized, i.e., formed by grafting a non-human CDR onto a human antibody framework. Furthermore, the antibodies of the present invention may be fully human.

[0121] The antibodies of the present invention may be modified with respect to effector function, for example, to enhance the effectiveness of the antibody in treating cancer. If the antibody of the present invention is an antibody fragment, the antibody fragment is selected from the group consisting of dAb, Fab, Fd, Fv, F(ab')2, scFv, and CDR.

[0122] The dosage, frequency of administration, route of administration, etc., are described in detail below. Methods for preparing antibodies and administering them to subjects requiring them are well known or readily determined to those skilled in the art. Routes of administration can be, for example, oral, parenteral (e.g., intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, intradermal, rectal, or vaginal), by inhalation, or topically. One form of administration appears to be a solution for injection, particularly for intravenous or intra-arterial injection or infusion, which includes buffers (e.g., acetic acid, phosphoric acid, or citrate), surfactants (e.g., polysorbate), and optionally stabilizers (e.g., human albumin). Alternatively, antibodies may be delivered directly to the site of disease, thereby increasing exposure of diseased cells or tissues to the antibody.

[0123] Preparations for parenteral administration include sterile aqueous solutions (aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including physiological saline and buffered media) or non-aqueous solutions (non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate), suspensions, and emulsions. pharmaceutically acceptable carriers include 0.01-0.1 M, preferably 0.05 M phosphate buffer or 0.9% physiological saline. Other common parenteral vehicles include sodium phosphate solution, ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or non-volatile oils. Intravenous vehicles include replacement fluids and nutritional supplements, electrolyte replacement solutions such as those based on ringer's dextrose, etc. Preservatives and other additives may be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases.

[0124] More specifically, a pharmaceutical composition suitable for injection comprises a sterile aqueous solution (if water-soluble) or dispersant, and a sterile powder for the immediate preparation of a sterile injection solution or dispersant, in which case the composition must be sterile and should be fluid enough to allow for easy syringe passage. It must be stable under manufacturing and storage conditions and preferably protected against microbial contamination such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or suitable mixtures thereof. Adequate fluidity can be maintained, for example, by using a coating such as lecithin to maintain the particle size required in the case of a dispersion, and by using a surfactant. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., 16th edition (1980).

[0125] The action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyhydric alcohols such as mannitol and sorbitol, or sodium chloride. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents in the composition, such as aluminum monostearate and gelatin.

[0126] In any case, sterile injection solutions can be prepared by incorporating the required amount of an active compound (e.g., an antigen-binding site) in a suitable solvent with one or a combination of the components listed herein, followed by sterilization by filtration as necessary. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders for the preparation of sterile injection solutions, preferred methods of preparation are vacuum drying, freeze-drying, and spray-drying, which result in a powder having an active ingredient plus any additional desired components from a pre-sterilized filtered solution of the powder. The injection preparations are processed by methods known in the art and filled into containers such as ampoules, bags, bottles, syringes, or vials, and sealed under sterile conditions. Furthermore, the preparations may be packaged and sold in kit form. Such products would preferably be accompanied by a label or accompanying leaflet indicating that the accompanying composition is useful for treating subjects suffering from or susceptible to such disorders.

[0127] The effective dose of the composition of the present invention for the treatment of a disorder varies depending on a number of factors, including the means of administration, the target site, the patient's physiological state, whether the patient is human or animal, other pharmaceuticals administered, and whether the treatment is prophylactic or therapeutic, as described herein. Treatment dosing may be dose-adjusted using routine methods known to those skilled in the art to optimize safety and efficacy.

[0128] For the treatment of a specific disorder using antibodies, the dosage can range, for example, from about 0.0001 to 100 mg / kg of host body weight, more commonly from 0.01 to 5 mg / kg (e.g., 0.02 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 2 mg / kg, etc.). For example, the dosage can be in the range of 1 mg / kg body weight or 10 mg / kg body weight or 1 to 10 mg / kg, preferably at least 1 mg / kg. Doses in the intermediate ranges above are also intended to be within the scope of the present invention. Subjects may be administered such doses daily, every other day, weekly, or according to any other schedule determined by empirical analysis. Exemplary treatments require multiple doses over a long period, for example, at least 6 months. Further exemplary treatment regimens require administration every 2 weeks, monthly, or every 3 to 6 months. Exemplary dosing schedules include 1–10 mg / kg or 15 mg / kg daily, 30 mg / kg every other day, or 60 mg / kg weekly. In some methods, two or more antigen-binding sites with varying binding specificities are administered simultaneously, in which case the dosage of each administered antigen-binding site falls within the indicated range.

[0129] Antibodies for binding to non-functional P2X7 receptors expressed on cells may be administered in multiple doses. The interval between each dose may be weekly, monthly, or yearly. The interval may also be irregular, as indicated by measuring the blood levels of the target polypeptide or target molecule in the patient. In some methods, the dose is adjusted to obtain plasma polypeptide concentrations of 1–1000 μg / mL, and in other methods, 25–300 μg / mL. Alternatively, the antibody of the present invention may be administered as a sustained-release formulation, in which case a lower frequency of administration is required. The dosage and frequency will vary depending on the half-life of the antibody in the patient. The half-life of the antibody may also be extended via fusion to a stable polypeptide or moiety, such as albumin or PEG. Generally, humanized antibodies exhibit the longest half-lives, followed by chimeric antibodies and non-human antibodies. In one embodiment, the antibody of the present invention may be administered in a non-conjugate form. In another embodiment, the antibody of the present invention may be administered multiple times in a conjugate form. For certain therapeutic applications, relatively high doses at relatively short intervals (e.g., up to 400 mg / kg per dose of an anti-P2X7 conjugating molecule, e.g., an antibody) may be required until disease progression slows or terminates, preferably until the patient shows partial or complete improvement in the symptoms of the disease. The above amounts may be several logarithmic levels lower (i.e., 2-3 logarithmic levels lower) if the antibody is conjugated with a radioisotope or cytotoxic drug.

[0130] Therapeutic agents may be administered for prophylactic and / or therapeutic purposes by parenteral, topical, intravenous, oral, subcutaneous, intra-arterial, intracranial, intraperitoneal, intranasal, or intramuscular means, but in some methods the agonist is injected directly into specific tissues where non-functional P2X7 receptor cells accumulate, e.g., intracranial injection. Intramuscular injection or intravenous infusion is preferred for antibody administration.

[0131] Antibodies may be administered in combination with other agonists that are effective in treating disorders or conditions requiring treatment (e.g., prophylactic or therapeutic), as needed. Examples include agonists commonly used in oncology with chemotherapy or radiotherapy. Additionally or alternatively, the antibodies or agonists of the present invention may be administered before, during, or after surgical intervention for the excision or removal of tumors or tissues.

[0132] Formation of immunogens and immune responses Another type of P2X7 receptor targeted therapy involves inducing an immune response in the treated individual against P2X7 receptors, particularly non-functional P2X7 receptors. Generally, immunogens used for this purpose are those that induce an immune response against non-functional P2X7 receptors but not against functional P2X7 receptors.

[0133] The immunogen may contain or be derived from a peptide containing the sequence of the P2X7 receptor. The peptide may contain at least one sequence that can be presented on a major histocompatibility complex class II molecule or that can interact with a B cell receptor or B cell membrane-bound immunoglobulin. Typically, the peptide contains the sequence of the human P2X7 receptor or a fragment thereof.

[0134] The scope of peptide immunogens is known and described below: PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or any one of the corresponding U.S. Patents No. 7,326,415, No. 7,888,473, No. 7,531,171, No. 8,080,635, No. 8,399,617, No. 8,709,425, No. 9,663,584, or No. 10,450,380), PC T / AU2008 / 001364 (or to any one of the corresponding U.S. Patents U.S. 8,440,186, U.S. 9,181,320, U.S. 9,944,701 or U.S. 10,597,451) and PCT / AU2009 / 000869 (or to any one of the corresponding U.S. Patents U.S. 8,597,643, U.S. 9,328,155 or U.S. 10,238,716), the entire contents of which are incorporated herein by reference.

[0135] Exemplary peptide immunogens within their specifications, including epitopes for generating an immune response to non-functional P2X7 receptors, are described below.

[0136] [Table 1]

[0137] It will be understood that these are merely examples of immunogens that may be useful in forming an immune response by the methods of the present invention described herein. Furthermore, the present invention encompasses the use of other peptides described in those applications that are useful in forming an immune response to non-functional P2X7 receptors.

[0138] Typically, an immunotherapy regimen requires two or more immunizations. The first immunization may aim to develop an IgM response to the immune system. The second immunization may aim to develop an IgG response. Further immunizations may aim to boost the IgG response, as will be discussed further below.

[0139] If the immunogen is a peptide, the peptide may be supplied in an amount of about 0.1 to 2 mg, preferably about 0.25 to 1 mg, preferably about 0.5 mg per dose. An additional dose of approximately 0.25–1 mg of the peptide may be applied as a booster.

[0140] In one embodiment, the first immunization is performed when the circulating levels of the antigen-binding site administered for antibody immunotherapy are substantially undetectable. In other words, circulating antibodies against the relevant cancer biomarker are undetectable in the peripheral blood. Subsequently, the level of IgM production is monitored over the following weeks. Approximately 4-5 weeks after the first immunization, the level of IgM antibodies may have decreased to a very low circulating level. At this point, the second immunization is then performed, and the level of IgG production is monitored over the following weeks.

[0141] Following the boost, the level of antibodies produced can be 0.1–25 mg / kg, for example, 0.1–10 mg / kg, preferably 5 mg / kg, or 10–25 mg / kg, preferably 15 mg / kg and greater than 10 mg / kg. Whether this amount is detectable in circulation depends on the presence or absence of existing tumor volume. If there is existing tumor volume that can bind to antibodies formed by the humoral response, the level of antibodies detectable in circulation may be at the lower end of the above range, actually outside the lower end of the above range (i.e., less than 0.1 mg / kg), or otherwise substantially undetectable. If there is no detectable tumor volume, the level of antibodies formed from the humoral response may be at the upper end of the above range, although in certain embodiments, an amount of approximately 5 mg / kg of antibody may be sufficient in such situations. Further testing of the immunization may be carried out over the following months / years, but boost immunization may be supplied if necessary.

[0142] The degree or frequency of boosts may depend on the patient's condition and response. If scanning or absence of circulating free antibodies indicates an existing tumor burden, boosts may be performed monthly, ideally to ensure a full immune response. If serum antibody release levels increase, boosts may then be eased and possibly applied 6 to 12 times monthly, subject to clinical observation.

[0143] As discussed above, the immune response can target biomarkers different from those targeted by antibody immunotherapy. For example, while anti-CD20 antibodies may be used for antibody immunotherapy, non-functional P2X7 immunogens may be used to generate the immune response.

[0144] In another embodiment, a single biomarker is targeted by antibody immunotherapy and immunization. For example, a monoclonal antibody directed to one epitope on the P2X7 receptor (e.g., the E300 epitope) may be used for antibody immunotherapy, and an immunogen to form an immune response targeting a different epitope on P2X7 (e.g., the E200 epitope) may be used for immunization.

[0145] The peptide immunogens for use in the methods of the present invention as described herein may have a length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 residues.

[0146] In one embodiment, the immunogen for forming an immune response by the method of the present invention is a peptide having a P2X7 receptor sequence which may or may not have Pro210 in cis-conformation.

[0147] The immunogen may be in the form of one or more of the P2X7 extracellular domain or P2X7 isoforms. The immunogen may be supplied for administration in a soluble form or in combination with a solid phase such as a cell membrane, beads, or other surface.

[0148] A method for screening peptides that can be used as immunogens to form an immune response by the method of the present invention is disclosed herein. One example involves the use of erythrocytes in a rosette formation assay. In this assay, an antibody that binds to a functional receptor is used as a positive control in which a rosette is observed. A test antibody is determined not to bind to the functional receptor if it does not form a rosette. A test antibody is determined to bind to a non-functional receptor if it is observed to bind to a non-functional receptor-expressing cell line, including those discussed herein.

[0149] The peptides of the present invention can be prepared by several techniques known in the art, including solid-phase synthesis and recombinant DNA technology. As is known in this field, a carrier is a substance that can be conjugated to a peptide epitope, thereby enhancing its immunogenicity. Some carriers achieve this enhancement by binding to multiple peptides, supplying a larger molecular weight antigen to a host in which an immune response is to be developed.

[0150] Preferred carriers include bacterial toxins or toxoids. Other preferred carriers include meningococcal (N. meningitides) outer membrane protein, albumins such as bovine serum albumin, synthetic peptides, heat shock proteins, KLH, pertussis protein, Haemophilus influenzae (H. influenzae) protein D, and C. difficile toxins A, B, or C.

[0151] If the carrier is a bacterial toxin or toxoid, diphtheria toxoid or tetanus toxoid is preferred. Preferably, the support contains a functional group that can react with the peptide of the present invention, or can be modified to react with the peptide of the present invention. Immunogens can be administered subcutaneously, intradermally, and / or intramuscularly.

[0152] Adjuvant In a preferred embodiment, the composition for inducing an immune response to the P2X7 receptor for use in the method of the present invention as described herein comprises an adjuvant or compound for enhancing the immune response.

[0153] Numerous adjuvants are known; see also Allison (1998, Dev. Biol. Stand., 92:3~11; incorporated herein by reference), Unkeless et al. (1998, Annu. Rev. Immunol., 6:251~281), and Phillips et al. (1992, Vaccine, 10:151~158). Exemplary adjuvants that may be used in accordance with the present invention include, but are not limited to, cytokines, aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, etc.; Baylor et al., Vaccine, 20:S18, 2002), gel-type adjuvants (e.g., calcium phosphate, etc.); microbial adjuvants (e.g., immunomodulatory DNA sequences containing CpG motifs); endotoxins such as monophosphoryl lipid A (Ribi et al., 1986, Immunology and Immunopharmacology of bacterial endotoxins, Plenum). Publ. Corp., NY, p407, 1986); exotoxins such as cholera toxin, Escherichia coli (E. coli) heat-unstable toxin and pertussis toxin; muramyl dipeptides, etc.; oil emulsions and emulsifier-based adjuvants (e.g., Freund's adjuvant, MF59 [Novartis], SAF, etc.); particulate adjuvants (e.g., liposomes, biodegradable microspheres, etc.); synthetic adjuvants (e.g., nonionic block copolymers, muramyl peptide analogs, polyphosphazenes, synthetic polynucleotides, etc.); and / or combinations thereof.Other exemplary adjuvants include: certain polymers (e.g., polyphosphazenes; described in U.S. Patent No. 5,500,161), Q57, saponins (e.g., QS21, Ghochikyan et al., Vaccine, 24:2275, 2006), squalene, tetrachlorodecaoxide, and CPG. 7909 (Cooper et al., Vaccine, 22:3136, 2004), poly[di(carboxylatofenoxy)phosphazene] (PCCP; Payne et al., Vaccine, 16:92, 1998), interferon-γ (Cao et al., Vaccine, 10:238, 1992), block copolymer P1205 (CRL1005; Katz et al., Vaccine, 18:2177, 2000), interleukin-2 (IL-2; Mbwuike et al., Vaccine, 8:347, 1990), polymethyl methacrylate (PMMA; Kreuter et al., J. Pharm. ScL, 70:367, 1981), etc.

[0154] In one embodiment, a peptide immunogen containing a P2X7 receptor sequence is supplied onto the surface of a bacteriophage for individual immunization according to the method of the present invention as described herein.

[0155] cell therapy Other P2X7 receptor-targeted therapies include cell therapy. Specifically, the present invention relates to a method for treating cancer in individuals who have not responded to or no longer respond to chemotherapy, - A process of preparing individuals that did not respond to or no longer respond to chemotherapeutic agents; - A step of supplying a cell therapy targeting cancer cells expressing the P2X7 receptor in an individual, - Here, the P2X7 receptor has a impaired response to ATP and therefore cannot form an apoptotic pore under normal physiological conditions. This allows for the treatment of cancer in an individual. Provide a method.

[0156] In any embodiment, cell therapy targeting cancer cells expressing the P2X7 receptor may be CAR-T cells or other cytotoxic cells that have the ability to bind to cancer cells expressing the P2X7 receptor.

[0157] Chimeric antigen receptor T cells (CAR-T cells) are T cells that have been genetically engineered to produce artificial T cell receptors (chimeric antigen receptors). The chimeric antigen receptor useful in this invention comprises an antigen recognition domain and a signaling domain, where the antigen recognition domain recognizes the P2X7 receptor, which has a impaired response to ATP and therefore cannot form an apoptotic pore under normal physiological conditions.

[0158] Chimeric antigen receptors (CARs) consist of an extracellular domain and an intracellular domain. The extracellular domain contains a target-specific binding element, also known as the antigen-binding portion. The intracellular domain, or cytoplasmic domain, contains a costimulatory signaling region and a zeta chain portion. The costimulatory signaling region refers to the portion of the CAR that contains the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for the efficient response of lymphocytes to antigens.

[0159] A spacer domain may be incorporated between the extracellular domain and the transmembrane domain of a CAR, or between the cytoplasmic domain and the transmembrane domain of a CAR. As used herein, the term “spacer domain” means any oligo or polypeptide as a whole that functions to link a transmembrane domain to either the extracellular domain or the cytoplasmic domain of a polypeptide chain. A spacer domain may contain up to 600 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. A spacer domain may consist of the entire antibody Fc domain and, additionally, may possess a suitable linker that can separate one or more binding domains.

[0160] Antigen recognition domain The CAR useful in the present invention includes a target-specific binding element, also called an antigen-recognition domain or antigen-binding moiety, that binds to the P2X7 receptor. Preferably, the P2X7 receptor has a impaired response to ATP and therefore cannot form an apoptotic pore under normal physiological conditions.

[0161] Appropriate antigen-recognition domains or antigen-binding regions are described herein, including any antigen-binding sites or antigen-binding domains described herein in one or more formats appropriately isolated for the purpose of structural optimization. transmembrane domain With respect to the transmembrane domain, CARs may be designed to include a transmembrane domain that fuses to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that naturally associates with one of the extracellular domains of the CAR is used. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid such domain binding to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex.

[0162] In various embodiments, the transmembrane domain may originate from either a natural or synthetic source. If the source is natural, the domain may originate from any membrane-bound or transmembrane protein. Transmembrane regions particularly useful in the present invention may originate from (i.e., including at least the following transmembrane regions): the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. The transmembrane domain may also be synthetic, in which case it will mainly consist of hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine will be found at each terminus of the synthetic transmembrane domain.

[0163] If necessary, short, oligo or polypeptide linkers, preferably with a length of 2 to 10 amino acids, can form a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. Glycine-serine doublets provide particularly suitable linkers.

[0164] Preferably, the transmembrane domain in the CAR of the present invention is a CD8 transmembrane domain. In some cases, the transmembrane domain of the CAR of the present invention includes a CD8a hinge domain.

[0165] Cytoplasmic domain The cytoplasmic domain of the CAR of the present invention, or in other words, the intracellular signaling domain, is responsible for activating at least one of the normal effector functions of the immune cell to which the CAR is located. The term “effector function” refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity including cytokine secretion. Therefore, the term “intracellular signaling domain” refers to a portion of a protein that transmits effector function signals and induces the cell to perform its specialized function. While the entire intracellular signaling domain may usually be used, in many cases it is not necessary to use the entire chain. Since a shortened portion of the intracellular signaling domain is used, such a shortened portion can be used in place of the intact chain as long as it transmits effector function signals. The term intracellular signaling domain, therefore, shall include any shortened portion of the intracellular signaling domain that is sufficient to transmit effector function signals.

[0166] Preferred examples of intracellular signaling domains for use in the CAR of the present invention include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act cooperatively to induce signal transduction upon binding to antigen receptors, as well as any derivatives or variants of those sequences and any synthetic sequences having the same functional capacity.

[0167] It is known that signals generated through the TCR alone are insufficient for complete T cell activation, and that secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two distinct classes of cytoplasmic signaling sequences: those that induce antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to produce secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0168] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex either stimulatingly or inhibitorily. Primary cytoplasmic signaling sequences that act stimulatingly may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs).

[0169] Examples of ITAM-containing primary cytoplasmic signaling sequences particularly useful in the present invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d. The cytoplasmic signaling molecule in the CAR of the present invention is particularly preferably a cytoplasmic signaling sequence derived from CD3 zeta.

[0170] In preferred embodiments, the cytoplasmic domain of the CAR may be designed to include a CD3-zeta signaling domain, either by itself or in combination with any other desired cytoplasmic domain useful in the context of the CAR of the present invention. For example, the cytoplasmic domain of the CAR may include a CD3 zeta chain portion and a co-stimulatory signaling region. The co-stimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient lymphocyte response to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83. Thus, while the present invention is primarily illustrated using 4-1BB as a co-stimulatory signaling element, other co-stimulatory elements are also within the scope of the present invention.

[0171] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the present invention can be linked to each other in a random or specified order. If necessary, short, oligo, or polypeptide linkers, preferably with a length of 2 to 10 amino acids, can form the linkage. Glycine-serine doublets provide particularly suitable linkers.

[0172] In one embodiment, the cytoplasmic domain is designed to include a CD3-zeta signaling domain and a CD28 signaling domain. In another embodiment, the cytoplasmic domain is designed to include a CD3-zeta signaling domain and a 4-1BB signaling domain. In yet another embodiment, the cytoplasmic domain is designed to include a CD3-zeta signaling domain as well as CD28 and 4-1BB signaling domains. In one embodiment, the cytoplasmic domain of the CAR of the present invention is designed to include a 4-1BB signaling domain and a CD3-zeta signaling domain.

[0173] The CAR-T cells useful in this invention include those described in PCT / AU2016 / 050851 (or the corresponding U.S. Publication No. 2019-0365805), the full contents of which are incorporated by reference.

[0174] Interfering RNA and administration One approach to treatment is the administration of interfering RNA to reduce the level of P2X7 receptors on the surface of cancer cells. The interfering RNA itself does not need to be specific to P2X7 receptors that are unable to form apoptotic pores under normal physiological conditions because they have a impaired response to ATP. Instead, the interfering RNA can be directed to target cells that express P2X7 receptors that are unable to form apoptotic pores under normal physiological conditions because they have a impaired response to ATP, and can reduce the total P2X7 receptor level within the cell. In other words, the interfering RNA can be directed to target cancer cells using any method known in the art.

[0175] Exemplary interfering RNA molecules are described in Gilbert et al., Oncogene. 2019 / 1 / 38(2):194-208, and their full content is incorporated by reference. Examples of siRNA target sequences include: A:5'-CCCGCAGAGCAAAGGAATTCAGACC-3'(Sequence ID 13) B:5'-GAGATATTGTGAGGACAAATTGAGA-3'(Sequence No. 12) It includes.

[0176] Cancer and related conditions Preneoplastic, neoplastic, and metastatic diseases are specific examples to which the methods of the present invention may be applied. A wide range of examples include breast tumors, colorectal tumors, adenocarcinomas, mesotheliomas, bladder tumors, prostate tumors, germ cell tumors, liver / bile duct tumors, carcinomas, neuroendocrine tumors, pituitary neoplasms, small round cell tumors, squamous cell carcinomas, melanomas, atypical fibroxanthomas, seminomas, non-seminomas, stromal Lydic cell tumors, Sertoli cell tumors, skin tumors, kidney tumors, testicular tumors, brain tumors, ovarian tumors, gastric tumors, oral tumors, bladder tumors, bone tumors, cervical tumors, esophageal tumors, laryngeal tumors, liver tumors, lung tumors, vaginal tumors, and Wilms' tumors.

[0177] Examples of specific cancers include, but are not limited to, adenocarcinoma, adenoma, adenofibrilloma, adenolymphoma, odontoma, AIDS-related cancer, acoustic neuroma, acute lymphoblastic leukemia, acute myeloid leukemia, adenocyscal carcinoma, adrenocortical carcinoma, idiopathic myelometaplasia, alopecia, hydatidiform soft tissue sarcoma, myeloblastoma, angiokeratomas, angiolymphoid hyperplasia with eosinophilia, sclerosing hemangioma, hemangiomatosis, apdoma, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, telangiectasia ataxia, basal cell carcinoma (skin), bladder cancer, bone cancer, intestinal cancer, brainstem glioma, tumors of the brain and CNS, breast cancer, branchiomas, CNS tumors Cervical tumors, carcinoid tumors, cervical cancer, pediatric brain tumors, pediatric cancers, pediatric leukemia, pediatric soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, cutaneous T-cell lymphoma, carcinomas (e.g., Walker's tumor, basal cell tumor, basal squamous cell tumor, Brown-Pierce tumor, glandular tumor, Ehrlich tumor, Krebs II tumor, Merkel cell tumor, mucinous, non-small cell lung tumor, oat cell tumor, papillary, hard, bronchiolar, bronchogenic, squamous epithelial cell tumor, and transitional cell tumors), carcinosarcoma, cervical malformations, phyllodes cyssarcoma, cementoma, chordoma, spondylolysis, chondrosarcoma, chondroblastoma, craniopharyngioma, bile duct Tumors, cholesteatoma, cystic adenoma, cystadenocarcinoma, cysdenoma, dermatofibrosarcoma protuberans, fibroplastic small round cell tumors, tubal carcinoma, undifferentiated germ cell tumor, endocrine cancer, endometrial cancer, ependymal cell tumor, esophageal cancer, Ewing's sarcoma, extrahepatic cholangiocarcinoma, eye cancer, eye: melanoma, retinoblastoma, fallopian tube cancer, Fanconi anemia, fibroma, fibrosarcoma, gallbladder cancer, stomach cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, genitourinary cancer, germ cell tumor, gestational trophoblastic disease, glioma, gynecological cancers, giant cell tumor, gangliomas, gliomas, glomus hemangioma, granulosa cell tumor, male and female germ cell tumors, hematological malignancies, hairy Cellular leukemia, head and neck cancer, hepatocellular carcinoma, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole, hypercalcemia, hypopharyngeal cancer, hamartoma, hemangioendothelioma, hemangioma, periangiocarcinoma, angiosarcoma, angiosarcoma, histiocytic disorders, malignant tumors of histiocytosis, histiocytoma, liver cancer, sweat adenoma, chondrosarcoma, immunoproliferative microcarcinoma, opoma, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Lie-Fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma,Hodgkin lymphoma, non-Hodgkin lymphoma, leiomyosarcoma, leukemia (e.g., B-cell, mixed-cell, null-cell, T-cell, chronic T-cell, HTLV-II-associated, lymphangiosarcoma, acute lymphoblastic, chronic lymphoblastic, mast cell, and myeloid), leukemosarcoma, Leydig cell tumor, liposarcoma, leiomyoma, leiomyosarcoma, lymphangiomas, lymphangiocytomas, lymphangiomyoma, lymphangiomyoma, lymphangiomyoma, lymphangiosarcoma, male breast cancer, malignant rhabdoid tumor of the kidney, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer Oral cancer, multiple endocrine neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloma, myeloproliferative disorders, malignant carcinoid syndrome, carcinoid heart disease, medulloblastoma, meningioma, melanoma, mesenchymal cell tumor, mesonephroma, mesothelioma, rhabdomyoblastoma, myoma, sarcoma, myxoma, myxosarcoma, nasal cavity cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmihen chromosomal instability syndrome, non-melanoma skin cancer, non-small cell lung cancer (NSCLC), schwannoma, neuroblastoma, neuroepithelioma, neurofibromatosis, neurofibroma, neuromatosis, neoplasms (e.g., bone, breast, digestive system, colorectal, liver) Organ cancer, eye cancer, esophageal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ostomy ovarian cancer, pancreatic cancer, paranasal sinus cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral neuroectodermal tumor, pituitary cancer, polycythemia vera, prostate cancer, osteoma, osteosarcoma, ovarian cancer, papilloma, paraganglioma, nonchromophilic paraganglioma, pineal gland tumor, plasmacytoma, proto-oncogene, rare cancers and related disorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rohtmunnd-Thomson syndrome, retinoendotheliosis, rhabdomyomas, salivary gland cancer, sarcoma, Schwann cell tumor, Sézary syndrome, Skin cancer, small cell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma, spinal cord tumor, squamous cell carcinoma (skin), gastric cancer, synovial sarcoma, sarcoma (e.g., Ewing's experimental sarcoma, Kaposi's sarcoma, and mast cell sarcoma), Sertoli cell tumor, synovial sarcoma, testicular cancer, thymic cancer, thyroid cancer, transitional cell carcinoma (bladder), transitional cell carcinoma (kidney-pelvis- / -ureter), trophoblastic carcinoma, teratoma, follicular cell tumor, thymoma, trophoblastic tumor, urethral cancer, urinary tract cancer, uroplakin, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Valdenström macroglobulinemia, and Wilms tumor.

[0178] Cancer may be 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.

[0179] Cancer may be selected from lung cancer, esophageal cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer, cervical cancer, vaginal cancer, epithelial cell carcinoma, skin cancer, hematological cancer, breast cancer, endometrial cancer, uterine cancer, cervical cancer, and testicular cancer.

[0180] Cancer can be metastatic. The cancer can be classified as stage III cancer. The cancer can be classified as stage IV cancer.

[0181] kit In another embodiment, a kit or product is provided that includes the following: - Reactive with P2X7 receptors, preferably non-functional P2X7 receptors, with an immunoglobulin variable domain, antibody, dAb, Fab, Fd, Fv, F(ab')2, scFv or CDR, and an antigen-binding site; - Immunogens for generating an immune response to non-functional P2X7 receptors; - A label or accompanying document containing instructions for use in the method described herein.

[0182] amino acid sequence The following are exemplary amino acid sequences described herein: 1 MPACCSCSDV FQYETNKVTR IQSMNYGTIK WFFHVIIFSY VCFALVSDKL YQRKEPVISS 61 VHTKVKGIAE VKEEIVENGV KKLVHSVFDT ADYTFPLQGN SFFVMTNFLK TEGQEQRLCP 121 EYPTRRTLCS SDRGCKKGWM DPQSKGIQTG RCVVHEGNQK TCEVSAWCPI EAVEEAPRPA 181 LLNSAENFTV LIKNNIDFPG HNYTTRNILP GLNITCTFHK TQNPQCPIFR LGDIFRETGD 241 NFSDVAIQGG IMGIEIYWDC NLDRWFHHCR PKYSFRRLDD KTTNVSLYPG YNFRYAKYYK 301 ENNVEKRTLI KVFGIRFDIL VFGTGGKFDI IQLVVYIGST LSYFGLAAVF IDFLIDTYSS 361 NCCRSHIYPW CKCCQPCVVN EYYYRKKCES IVEPKPTLKY VSFVDESHIR MVNQQLLGRS 421 LQDVKGQEVP RPAMDFTDLS RLPLALHDTP PIPGQPEEIQ LLRKEATPRS RDSPVWCQCG 481 SCLPSQLPES HRCLEELCCR KKPGACITTS ELFRKLVLSR HVLQFLLLYQ EPLLALDVDS 541 TNSRLRHCAY RCYATWRFGS QDMADFAILP SCCRWRIRKE FPKSEGQYSG FKSPY (Sequence ID 1) HNYTTRNIL (Sequence ID 2) GHNYTTRNIL (Sequence ID 3) DFPGHNYTTRNIL (Sequence ID 4) GHNYTTRNILPGLNITC (Sequence ID 5) 1 MPACCSCSDV FQYETNKVTR IQSMNYGTIK WFFHVIIFSY VCFALVSDKL YQRKEPVISS 61 VHTKVKGIAE VKEEIVENGV KKLVHSVFDT ADYTFPLQGN SFFVMTNFLK TEGQEQRLCP 121 EYPTRRTLCS SDRGCKKGWM DPQSKGIQTG RCVVHEGNQK TCEVSAWCPI EAVEEAPRPA 181 LLNSAENFTV LIKNNIDFPG HNYTTRNIL (Sequence ID 6) KTTNVSLYPGYNFRYAKYYKENNVEKRTLIKVFGIRFDILVFGTGGKFD(Sequence ID 7) KYYKENNVEKRTLIKVF (Sequence ID 8) GHNYTTRNILP (Sequence ID 9) AKYYKENNVEK (Sequence ID 10) GHNYTTRNILPGAGAKYYKENNVEK(Sequence ID 11)

[0183] It will be understood that the present invention disclosed and defined herein encompasses all of two or more selective combinations of the individual features mentioned or evident from the text or drawings. All of these various combinations constitute various selective embodiments of the present invention. [Examples]

[0184] The experimental data below identified that chemotherapy or radiotherapy increases the level of P2X7 receptors that are unable to form apoptotic pores on living cells. Furthermore, the data indicates that the level of P2X7 receptors that are unable to form apoptotic pores increases on cells that have developed complete or partial resistance to chemotherapy. These findings are independent of the type of chemotherapy, as they are different structural classes of chemotherapeutic agents with different mechanisms of action; all result in an increase in the level of P2X7 receptors that are unable to form apoptotic pores. Moreover, these findings apply to a wide variety of cancers, regardless of tissue origin (e.g., blood-derived or solid). Finally, the experimental data indicates that these chemotherapy-driven level increases occur in both patient-derived cell lines and primary cells. Cancer cells pretreated with chemotherapy, and therefore possessing elevated levels of P2X7 receptors that are unable to form apoptotic pores, are sensitive to various interventions targeting the P2X7 receptor.

[0185] Example 1 Chemotherapy treatment in myeloma RPMI-8226 cell line and neuroblastoma Kelly cell line. material and method 4,000 cells were seeded into 96-well plates and allowed to adhere overnight. The cells were then treated with either increased doses of chemotherapy or a vehicle control for 72 hours to induce variable levels of cell death. Cell viability was then measured using the Promega CellTiter-Blue Cell Viability Assay, according to the manufacturer's instructions.

[0186] Normalized ethidium influx in response to 0.5 mM BzATP stimulation in myeloma RPMI-8226 cell line and neuroblastoma Kelly cell line. The mean of three independent experiments is shown.

[0187] result Figure 1A shows that the myeloma cell line RPMI-8226 can open P2X7 pores in response to 0.5 mM BzATP, whereas the neuroblastoma cell line Kelly cannot. The RPMI-8226 and Kelly cell lines were selected as representative models for hematological cancers (myeloma) and solid tumors (neuroblastoma). Figures 1B, C, and D show the effects of dose-increasing chemotherapy (doxorubicin and 5Fu) on RPMI-8226 and Kelly cells, as measured using the CellTitle-Blue (CTB) assay. These data were used to select the chemotherapy concentrations used in Figure 2.

[0188] Doxorubicin, also known as Adriamycin, belongs to the anthracycline family. Its mechanism of action is mediated by the blocking of topoisomerase 2, which inhibits DNA replication and cell proliferation and ultimately leads to cell death, as observed in Figures 1B and 1C. 5Fu, also known as fluorouracil, is a chemotherapy drug whose mechanism of action is primarily mediated by the inhibition of thymidylate synthase, which subsequently blocks the synthesis of pyrimidine thymidine, a nucleoside necessary for DNA replication. This results in the inhibition of cell proliferation and ultimately cell death, as observed in Figure 1D. Chemotherapy with doxorubicin and 5Fu was selected for these distinctly different modes of action.

[0189] Example 2 Chemotherapy treatment and induction of nfP2X7 detected by BPM09 in functional myeloma RPMI-8226 cell line and non-functional neuroblastoma Kelly cell line.

[0190] material and method 50,000 cells were seeded in 6-well plates and allowed to adhere overnight. The cells were then treated with either increased-volume chemotherapy or a vehicle control for 72 hours to induce variable levels of cell death. The remaining viable cells were dissociated with a PBS-based enzyme-free dissociation buffer, washed, and resuspended in a staining buffer (PBS, 2% FCS). The cells were then stained for 1 hour with a primary antibody produced against non-functional P2X7 (here, 2-2-1hFc), washed three times with the staining buffer, and incubated with a fluorescently conjugated secondary antibody and 7AAD for 1 hour. Fluorescence staining of viable cells was acquired using a BD Accuri flow cytometer and analyzed with FlowJo flow cytometry software. The median fluorescence intensity of the viable cell population was analyzed using 7AAD viability staining.

[0191] result Figures 2A-C show that increasing the dose of chemotherapy (doxorubicin and 5Fu) to RPMI-8226 cells and Kelly cells leads to a dose-dependent increase in nfPX7 antibody binding (here, 2-2-1hFc). This demonstrates that cancer cell lines containing either functional P2X7 (RPMI-8226) or non-functional P2X7 (Kelly) show increased nfP2X7 antibody binding in response to various chemotherapy treatments (doxorubicin, 5Fu, cisplatin, etc.). These data support the rationale for combination therapy with chemotherapy and nfP2X7 targeted therapy.

[0192] Example 3 Cell lines that exhibit acquired resistance to chemotherapy have increased expression of nfP2X7. material and method Cultured A2780 parent cells, A2780 cells with acquired resistance to doxorubicin, and A2780 cells with acquired resistance to cisplatin were obtained from the ECACC repository (References: Proc Amer Assoc Cancer Res 1984;25:336;Semin Oncol 1984;11:285;Cancer Res 1987;47:414;Cancer Res 1988;48:5713).

[0193] 50,000 A2780 cells, including doxorubicin-resistant and cisplatin-resistant A2780 cells, were seeded into 6-well plates and allowed to adhere overnight. The cells were dissociated with a PBS-based enzyme-free dissociation buffer, washed, and resuspended in a staining buffer (PBS, 2% FCS). The cells were then stained for 1 hour with a primary antibody produced against non-functional P2X7 (here, BPM09), washed three times with the staining buffer, and incubated with a fluorescently conjugated secondary antibody and 7AAD for 1 hour. Fluorescence staining of live cells was acquired using a BD Accuri flow cytometer and analyzed with FlowJo flow cytometry software. The median fluorescence intensity of the live cell population was analyzed using 7AAD viability staining.

[0194] result Experiments were conducted to determine the degree of binding of antibodies that specifically bind to nfP2X7 on living ovarian A2780 parental cells and A2780 cells that have acquired resistance to doxorubicin or cisplatin.

[0195] Figure 3 shows the changes in nfP2X7 antibody (here, BPM09) binding to living ovarian A2780 parental cells and A2780 cells with acquired resistance to doxorubicin and cisplatin. The data indicate that ovarian cancer cells with acquired chemotherapy resistance to doxorubicin and cisplatin have increased nfP2X7 antibody binding compared to parental A2780 cells.

[0196] These data demonstrate that chemotherapy-induced increases in nfP2X7 are durable and persistent. The data also show that cells that acquired chemotherapy resistance had increased nfP2X7 exposure, which can be targeted by nfP2X7-targeted therapy.

[0197] Example 4 Xenografts derived from patients previously treated with chemotherapy and / or radiotherapy exhibit increased expression of nfP2X7. material and method Immunohistochemistry was performed as previously described by Gilbert et al. (Br J Dermatol. 2017). 5-micrometer thick sections were excised from formalin-fixed, paraffin-embedded tissue from TMA slides containing duplicate core biopsies from patient-derived xenograft (PDX) models. Heat antigen retrieval was performed, followed by staining with primary mouse monoclonal anti-E200 antibody (BPM09) at final concentrations of 1 μg / ml to 25 μg / ml for 60 minutes, followed by staining with Mach 4 mouse probe (Biocare, USA) for 15 minutes and Mach 4 universal polymer HRP for 25 minutes. Each step was separated by rinsing the tissue with Tris-buffered saline for 5 minutes. Dako liquid DAB was used as chromatogen (5 minutes), and hematoxylin (5 seconds) was used as counterstain. Slides were examined using a 20× objective lens. The intensity of membrane staining was scored by a pathologist.

[0198] result BPM09 membrane staining was scored after immunohistochemistry in a series of patient-derived xenograft (PDX) models, some previously treated with chemotherapy and / or radiotherapy, and others not. The treated PDX models included in this study were: colorectal cancer (16 samples), testicular cancer (2 samples), sarcoma (4 samples), melanoma cancer (3 samples), bladder cancer (1 sample), pancreatic cancer (2 samples), small cell lung cancer (1 sample), non-small cell lung cancer (2 samples), ovarian cancer (1 sample), cervical cancer (2 samples), and breast cancer (6 samples). Various chemotherapy regimens were used as monotherapy or in combination, including the following: 5-FU, folinic acid, bleomycin, etoposide, cisplatin, capecitabine, oxaliplatin, dacarbazine, cyclophosphamide, vincristine, doxorubicin, irinotecan, gemcitabine, mitomycin C, gemcitabine, carboplatin, paclitaxel, pemetrexed, hydroxyethyl-chloroethylnitrosourea (HeCNU), tamoxifen, methotrexate, epirubicin, vindesine, erlotinib, bevacizumab, cetuximab, and radiotherapy.

[0199] Figure 4 shows that xenografts derived from patients previously treated with chemotherapy and / or radiotherapy (multiple chemotherapy regimens and other treatments such as radiotherapy were pooled in this analysis) exhibited elevated nfP2X7 antibody binding (here, BPM09). These data demonstrate that multiple non-targeted therapy regimens, including chemotherapy and radiotherapy, lead to increased nfP2X7 exposure on the surface of tumor cells and justify the use of nfP2X7-targeted therapy in patients previously treated with such non-targeted treatments.

[0200] Example 5 Cancer cells pretreated with chemotherapy are sensitive to nfP2X7 targeting. material and method Untreated or Kelly cells treated with 4 μM 5-FU for 72 hours were seeded at 50,000 cells per well in a black, clear-bottomed 96-well plate and allowed to adhere overnight. The cells were then loaded with 6.6 μg / ml of calcein in preheated medium for 30 minutes. After loading, the cells were washed three times with serum-free RPMI 1640, and then 5 μl of mouse serum from mice immunized with PBS or BIL06v was added to 50 μl of serum-free RPMI 1640 per well (serum from 4 PBS-immunized mice and 16 BIL06v-immunized mice in each well). The cells were incubated on ice for 30 minutes. 50 μl of 80% rabbit complement (Cedar Lane-cl3441-s50) from preheated serum-free RPMI 1640 was added to each well. The plate was incubated at 37°C for 2 hours. After 2 hours, the culture medium was transferred to a V-bottom plate and centrifuged at 2000 g for 5 minutes to pellet all cells. 80 μl of supernatant per well was transferred to a black 96-well plate with a clear bottom. Fluorescence was measured at 495 nm / 515 nm. The data shown represent calcein fluorescence from wells containing serum from BIL06v-immunized mice with untreated or 5-FU pretreated cells, normalized to the mean calcein fluorescence from wells containing serum from PBS-immunized mice with the same pretreatment.

[0201] result The effect of 5Fu on nfP2X7 antibody (here, polyclonal mouse antibody)-mediated complement-dependent cytotoxicity in Kelly cells.

[0202] The data demonstrate that combination therapy between chemotherapy and nfP2X7 targeted therapy can be implemented to enhance antitumor effects, as evidenced by the increase in complement-dependent cytotoxic nfP2X7-targeted antibodies against chemotherapy-treated Kelly cells (Figure 5). Overall, across various chemotherapy treatments (doxorubicin, 5Fu, cisplatin, etc.) and cancer models (myeloma, neuroblastoma, ovarian, colorectal), the data show that chemotherapy treatment leads to an increase in nfP2X7 levels on the surface of cancer cells, which can be used as a rationale for combination therapy with nfP2X7-targeted antibodies.

[0203] Example 6 The data shown in Figure 6a suggests that DAMPs such as HMGB1 are likely to mediate the effect. Both chemotherapy and radiotherapy drive the release of DAMPs such as HMGB1, which in turn drive the increase in nfP2X7.

[0204] Figure 6b shows data demonstrating that the P2X7 inhibitor (A 740003-N-[1-[[(cyanoamino)(5-quinolinylamino)methylene]amino]-2,2-dimethylpropyl]-3,4-dimethoxybenzeneacetamide, Cat. No. 3701, Tocris) does not block conditioned medium-induced nfP2X7 induction. Surprisingly, this indicates that ATP-mediated activation of the P2X7 receptor does not mediate the chemotherapeutic effect.

Claims

1. A pharmaceutical composition for use in treating cancer in individuals that have not responded to or are no longer responding to chemotherapy and / or radiotherapy, comprising a P2X7 receptor targeted therapy agent, The aforementioned P2X7 receptor target therapy agents are It is a molecule that binds to the P2X7 receptor, which has a impaired response to ATP and therefore cannot form an apoptotic pore under normal physiological conditions. Here, the molecule is an antibody or its antigen-binding fragment, or a cell therapy agent containing cytotoxic cells that target cancer cells expressing the P2X7 receptor. The aforementioned pharmaceutical composition.

2. The molecule is a whole antibody or its antigen-binding fragment containing a variable domain for binding to the P2X7 receptor expressed by the individual. The pharmaceutical composition according to claim 1.

3. The pharmaceutical composition according to claim 2, wherein the molecule is an antibody fragment selected from the group consisting of dAb, Fab, Fd, Fv, F(ab')2 and scFv.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the molecule is an antibody or antigen-binding fragment thereof that does not bind to a functional P2X7 receptor.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the molecule is an antibody or an antigen-binding fragment thereof that binds to a polypeptide containing the amino acid sequence described in any one of SEQ ID NOs: 1 to 11.

6. The pharmaceutical composition according to claim 5, wherein the molecule is an antibody or antigen-binding fragment thereof that binds to a polypeptide containing the amino acid sequence described in any one of SEQ ID NOs: 2 to 5.

7. The pharmaceutical composition according to claim 1, wherein the cytotoxic cells have the ability to bind to cancer cells expressing the P2X7 receptor, and the cytotoxic cells are selected from leukocytes, peripheral blood mononuclear cells (PBMCs), lymphocytes, T cells, CD4+ T cells, CD8+ T cells, natural killer cells, or natural killer T cells.

8. The pharmaceutical composition according to claim 1, wherein the cytotoxic cells are CAR-T cells that target cancer cells expressing the P2X7 receptor.

9. Cytotoxic cells express chimeric antigen receptors that include an antigen recognition domain and a signaling domain, where the antigen recognition domain recognizes a dysfunctional or non-functional P2X7 receptor (i.e., the P2X7 receptor has a impaired response to ATP and therefore cannot form apoptotic pores under normal physiological conditions). The pharmaceutical composition according to claim 1.

10. The pharmaceutical composition according to claim 9, wherein the antigen recognition domain binds to a dysfunctional or non-functional P2X7 receptor having reduced ATP-binding capacity compared to that of a wild-type (functional) P2X7 receptor.

11. The pharmaceutical composition according to claim 10, wherein the dysfunctional or non-functional P2X7 receptor has a stereostructural change that makes the receptor dysfunctional or non-functional.

12. The pharmaceutical composition according to claim 10, wherein the antigen recognition domain recognizes an epitope comprising one or more amino acid residues spanning from glycine at amino acid position 200 to cysteine ​​at amino acid position 216 of a dysfunctional P2X7 receptor.

13. The pharmaceutical composition according to claim 12, wherein the antigen recognition domain comprises an amino acid sequence of a fragment-antigen binding (Fab) portion, a single-chain variable fragment (scFv), or a single antibody domain (dAb) of an antibody that binds to a dysfunctional or non-functional P2X7 receptor.

14. The pharmaceutical composition according to claim 12, wherein the antigen recognition domain comprises an amino acid sequence of a multivalent single-chain variable fragment (scFv) that binds to a dysfunctional P2X7 receptor.

15. The pharmaceutical composition according to claim 14, wherein the polyvalent single-chain variable fragment (scFv) is divalent scFv or trivalent scFv.

16. The pharmaceutical composition according to any one of claims 9 to 15, wherein the signal transduction domain includes a portion derived from an activating receptor and / or a co-stimulatory receptor.

17. The activating receptor is either a member of the CD3 coreceptor complex, or Here, the portion derived from the CD3 coreceptor complex is either CD3-zeta or Here, the activating receptor is an Fc receptor, and preferably the portion derived from the Fc receptor is an Fc epsilon RI or an Fc gamma RI, or Here, the co-stimulatory receptor is selected from the group consisting of CD27, CD28, CD30, CD40, DAP10, OX40, 4-1 BB (CD137), and ICOS. The pharmaceutical composition according to claim 16.

18. Antibodies are produced by immunogens in the form of P2X7 receptors or fragments of P2X7 receptors that can induce an immune response to P2X7 receptors in an individual. Here, the P2X7 receptor has a impaired response to ATP and therefore cannot form an apoptotic pore under normal physiological conditions. The pharmaceutical composition according to claim 2.

19. The pharmaceutical composition according to claim 18, wherein the P2X7 receptor fragment has the amino acid sequence described in any one of SEQ ID NOs: 1 to 10.

20. The immunogen is supplied in the initial administration to the individual, thereby initiating a response in the individual that includes IgM production. In some cases, it is subsequently supplied in a further dose following the initial dose, thereby forming a response including IgG production. The pharmaceutical composition according to claim 18 or 19.

21. The chemotherapeutic agent in which the individual did not respond, or no longer responds, is one or more chemotherapeutic agents selected from the group consisting of oxazaphosphorine, topoisomerase I inhibitor, topoisomerase II inhibitor, proteasome inhibitor, folic acid antimetabolites, nitrogen mustard, ethyleneimine derivatives, alkyl sulfonates, nitrosourea, triazenes, folic acid analogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs, purine antagonists, antimetabolites, antibiotics, epipodophyllotoxin, platinum-based drugs, ribonucleotide reductase inhibitors, vinca alkaloids, substituted ureas, hydrazine derivatives, adrenal cortical depressants, endostatins, camptothecin, oxaliplatin, doxorubicin and doxorubicin analogs, antibiotics, L-asparaginase, tyrosine kinase inhibitors, and derivatives or variants thereof, according to any one of claims 1 to 20.

22. The chemotherapeutic agents include doxorubicin, cisplatin, vincristine, dacarbazine (DTIC), cyclophosphamide, CPT-11, oxaliplatin, gemcitabine, and 5-fluorouracil / leucovorin, and / or 5-FU, folinic acid, bleomycin, etoposide, cisplatin, capecitabine, oxaliplatin, dacarbazine, cyclophosphamide, vincristine, doxorubicin, irinotecan, gemcitabine, mitomycin C, gemcitabine, carboplatin, paclitaxel, pemetrexed, hydroxyethyl-chloroethylnitrosourea (HeCNU), tamoxifen, methotrexate, epirubicin, vindesine, erlotinib, bevacizumab, and cetuximab, Selected from, The pharmaceutical composition according to claim 21.

23. The pharmaceutical composition according to any one of claims 1 to 22, wherein the cancer is selected from brain cancer, esophageal cancer, oral cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, kidney cancer, colorectal 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.

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