Methods of treating cancer using a polynucleotide encoding GM-CSF and an additional agent

Abstract

Disclosed herein are compositions and methods for preventing ovarian cancer recurrence and treating BRCA1 / 2-wild-type ovarian cancer. In some embodiments, the compositions comprise an autologous tumor cell vaccine comprising cells genetically modified for furin knockdown and GM-CSF expression. In some embodiments, the methods comprise administering the autologous tumor cell vaccine followed by administering the autologous tumor cell vaccine in combination with atezolizumab. Also disclosed herein are methods for treating cancer in individuals identified as homologous recombination repair deficient (HRD)-negative, comprising a wild-type BRCA1 gene, a wild-type BRCA2 gene, or a combination thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and benefit of U.S. Provisional Application No. 62 / 960,583, filed January 13, 2020; U.S. Provisional Application No. 63 / 034,868, filed June 4, 2020; and U.S. Provisional Application No. 63 / 061,634, filed August 5, 2020. All of the foregoing provisional applications are incorporated herein by reference for all purposes. [Background technology]

[0002] Ovarian cancer has a high mortality rate among malignant tumors in women. This high mortality rate results from ovarian cancer's resistance to chemotherapy and frequent recurrence. Although drugs have been developed to treat ovarian cancer, the mortality and recurrence rates of ovarian cancer remain high. Typically, treatment for advanced ovarian cancer is based on a combination of surgery and chemotherapy. Surgery is followed by adjuvant platinum-based chemotherapy. The two most important prognostic factors for patients with advanced ovarian cancer are the amount of disease remaining after surgery and the response to platinum-based chemotherapy. Summary of the Invention

[0003] Disclosed herein is a method of treating cancer in an individual in need thereof, the method comprising administering to the individual an expression vector comprising: a. a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; and b. a second insert comprising two stem-loop structures, each having a miR-30a loop, wherein the first stem-loop structure has perfectly complementary guide and passenger strands, while the second stem-loop structure has a three base pair (bp) mismatch at positions 9-11 of the passenger strand, and wherein the individual is homologous recombination repair deficient (HRD) negative and / or the individual has a wild-type BRCA1 gene, a wild-type BRCA2 gene, or a combination thereof.

[0004] In some embodiments, the guide strand in the first stem-loop structure comprises the sequence of SEQ ID NO:6 and the passenger strand in the first stem-loop structure has the sequence of SEQ ID NO:5.

[0005] In some embodiments, the guide strand in the second stem-loop structure comprises the sequence of SEQ ID NO:6 and the passenger strand in the second stem-loop structure has the sequence of SEQ ID NO:7.

[0006] In some embodiments, the miR-30a loop comprises the sequence of SEQ ID NO:8.

[0007] Disclosed herein in certain embodiments are methods of treating cancer in an individual in need thereof, the method comprising administering to the individual an expression vector comprising: a. a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; and b. a second insert comprising a sequence according to SEQ ID NO: 2 or 4 (SEQ ID NO: 4). In some embodiments, the individual is homologous recombination repair deficient (HRD) negative. In some embodiments, the individual has substantially eradicated ovarian cancer, and the method prevents or delays relapse or recurrence of the substantially eradicated ovarian cancer. The term "relapse" is used interchangeably with "recurrence."

[0008] Disclosed herein in certain embodiments are methods for preventing or treating the recurrence of substantially eradicated ovarian cancer in an individual in need thereof, the method comprising administering to the individual autologous tumor cells transfected with an expression vector comprising: (a) a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; and (b) a second insert comprising a sequence according to SEQ ID NO: 2 or 4 (SEQ ID NO: 4). In some embodiments, the substantially eradicated ovarian cancer is stage III or stage IV ovarian cancer. In some embodiments, the individual comprises a wild-type BRCA1 gene, a wild-type BRCA2 gene, or a combination thereof. In some embodiments, the individual's recurrence-free survival (RFS) is extended compared to an individual whose ovarian cancer has been substantially eradicated but who has not received the transfected tumor cells.

[0009] In some embodiments, the individual has received a first therapy. In some embodiments, the first therapy comprises cytoreductive surgery, chemotherapy, or a combination thereof. In some embodiments, the chemotherapy comprises administering a platinum agent and a taxane. In some embodiments, the platinum agent comprises carboplatin. In some embodiments, the taxane comprises paclitaxel.

[0010] In some embodiments, the GM-CSF is a human GM-CSF sequence. In some embodiments, the expression vector further comprises a promoter. In some embodiments, the promoter is a cytomegalovirus (CMV) mammalian promoter. In some embodiments, the expression vector further comprises a CMV enhancer sequence and a CMV intron sequence. In some embodiments, the first insert and the second insert are operably linked to the promoter. In some embodiments, the expression vector further comprises a nucleic acid sequence encoding a picornavirus 2A ribosomal skipping peptide between the first and second nucleic acid inserts.

[0011] In some embodiments, the autologous tumor cells are administered in an amount of about 1×10 6 cells ~ approx. 5 x 10 7 In some embodiments, the autologous tumor cells are administered to the individual as a single dose of cells. In some embodiments, the autologous tumor cells are administered to the individual once per month. In some embodiments, the autologous tumor cells are administered to the individual for 1-12 months (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months). In some embodiments, the autologous tumor cells are administered to the individual by intradermal injection.

[0012] Disclosed herein in certain embodiments are methods of treating BRCA1 / 2 wild-type ovarian cancer in an individual in need thereof, the method comprising administering to the individual autologous tumor cells transfected with an expression vector comprising (a) a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence and (b) a second insert comprising a sequence according to SEQ ID NO: 2 or 4 (SEQ ID NO: 4). In some embodiments, the ovarian cancer is stage III or stage IV ovarian cancer. In some embodiments, the ovarian cancer is recurrent ovarian cancer. In some embodiments, the ovarian cancer is refractory ovarian cancer. In some embodiments, the refractory ovarian cancer is refractory to chemotherapy. In some embodiments, the chemotherapy comprises a platinum agent or a taxane. In some embodiments, the platinum agent comprises carboplatin. In some embodiments, the taxane comprises paclitaxel. In some embodiments, the ovarian cancer is recurrent / refractory (r / r) ovarian cancer. In some embodiments, recurrent or recurrent / refractory ovarian cancer is referred to as relapsed ovarian cancer.

[0013] In some embodiments, the method further comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent is selected from the group consisting of an angiogenesis inhibitor, a PARP inhibitor, and a checkpoint inhibitor to the individual. In some embodiments, the angiogenesis inhibitor is a vascular endothelial growth factor (VEGF) inhibitor. In some embodiments, the VEGF inhibitor is selected from the group consisting of sorafenib, sunitinib, bevacizumab, pazopanib, axitinib, cabozantinib, and levatinib. In some embodiments, the VEGF is bevacizumab. In some embodiments, the PARP inhibitor is selected from the group consisting of niraparib, olaparib, rucaparib, niraparib, talazoparib, veliparib, and pamiparib. In some embodiments, the PARP inhibitor is niraparib. In some embodiments, the checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor. In some embodiments, the checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, and ipilimumab.

[0014] In some embodiments, the GM-CSF is a human GM-CSF sequence. In some embodiments, the expression vector further comprises a promoter. In some embodiments, the promoter is a cytomegalovirus (CMV) mammalian promoter. In some embodiments, the expression vector further comprises a CMV enhancer sequence and a CMV intron sequence. In some embodiments, the first insert and the second insert are operably linked to the promoter. In some embodiments, the expression vector further comprises a nucleic acid sequence encoding a picornavirus 2A ribosomal skipping peptide between the first and second nucleic acid inserts.

[0015] In some embodiments, the autologous tumor cells are administered in an amount of about 1×10 6 cells ~ approx. 5 x 10 7In some embodiments, the autologous tumor cells are administered to the individual as a single dose of cells. In some embodiments, the autologous tumor cells are administered to the individual once per month. In some embodiments, the autologous tumor cells are administered to the individual for 1-12 months. In some embodiments, the autologous tumor cells are administered to the individual by intradermal injection.

[0016] Disclosed herein in certain embodiments are methods for preventing or treating the recurrence of substantially eradicated ovarian cancer in an individual in need thereof, the method comprising administering to the individual: a. at least one first dose of an autologous tumor cell vaccine comprising autologous tumor cells transfected with an expression vector comprising: a. a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; ii. a second insert comprising a sequence according to SEQ ID NO: 2 or 4 (SEQ ID NO: 4); and b. at least one second dose of the autologous tumor cell vaccine in combination with at least one dose of an additional therapeutic agent. In some embodiments, the substantially eradicated ovarian cancer is stage III or stage IV ovarian cancer. In some embodiments, the individual has a wild-type BRCA1 gene, a wild-type BRCA2 gene, or a combination thereof. In some embodiments, the individual's recurrence-free survival (RFS) is extended compared to an individual whose ovarian cancer has been substantially eradicated but who has not received the transfected tumor cells.

[0017] In some embodiments, the individual has received a first therapy. In some embodiments, the first therapy comprises cytoreductive surgery, chemotherapy, or a combination thereof. In some embodiments, the chemotherapy comprises administering a platinum agent and a taxane. In some embodiments, the platinum agent comprises carboplatin. In some embodiments, the taxane comprises paclitaxel. In some embodiments, the GM-CSF is a human GM-CSF sequence.

[0018] In some embodiments, the expression vector further comprises a promoter. In some embodiments, the promoter is a cytomegalovirus (CMV) mammalian promoter. In some embodiments, the expression vector further comprises a CMV enhancer sequence and a CMV intron sequence. In some embodiments, the first insert and the second insert are operably linked to the promoter. In some embodiments, the expression vector further comprises a nucleic acid sequence encoding a picornavirus 2A ribosomal skipping peptide between the first and second nucleic acid inserts.

[0019] In some embodiments, the additional therapeutic agent is selected from the group consisting of an angiogenesis inhibitor, a PARP inhibitor, and a checkpoint inhibitor for the individual. In some embodiments, the angiogenesis inhibitor is a vascular endothelial growth factor (VEGF) inhibitor. In some embodiments, the VEGF is selected from the group consisting of sorafenib, sunitinib, bevacizumab, pazopanib, axitinib, cabozantinib, and levatinib. In some embodiments, the VEGF is bevacizumab. In some embodiments, the PARP inhibitor is selected from the group consisting of niraparib, olaparib, rucaparib, niraparib, talazoparib, veliparib, and pamiparib. In some embodiments, the PARP inhibitor is niraparib. In some embodiments, the checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor. In some embodiments, the checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, and ipilimumab. In some embodiments, the checkpoint inhibitor is atezolizumab.

[0020] In some embodiments, the at least one dose of the additional therapeutic agent is between 1100 mg and 1300 mg. In some embodiments, the at least one first dose of the autologous tumor cell vaccine is between about 1 x 106 cells ~ approx. 5 x 10 7 In some embodiments, the at least one second dose of the autologous tumor cell vaccine comprises about 1 x 10 cells. 6 cells ~ approx. 5 x 10 7 In some embodiments, the at least one first dose of the autologous tumor cells is administered to the individual by intradermal injection. In some embodiments, the at least one second dose of the autologous tumor cell vaccine is administered to the individual by intradermal injection. In some embodiments, the at least one dose of the additional therapeutic agent is administered to the individual by intradermal injection.

[0021] In some embodiments, the at least one first dose of the autologous tumor cell vaccine comprises two doses (2, 3, 4, or 5 doses, or more). In some embodiments, each of the at least one first dose of the autologous tumor cell vaccine is administered to the individual once per month. In some embodiments, each of the at least one second dose of the autologous tumor cell vaccine is administered to the individual once per month. In some embodiments, each of the at least one dose of the additional therapeutic agent is administered to the individual at least once per month. In some embodiments, the at least one first dose of the autologous tumor cell vaccine and the at least one second dose of the autologous tumor cell vaccine comprise a total of at least 12 doses.

[0022] Disclosed herein in certain embodiments is a method of treating BRCA1 / 2 wild-type ovarian cancer in an individual in need thereof, the method comprising administering to the individual: a. at least one first dose of an autologous tumor cell vaccine comprising autologous tumor cells transfected with an expression vector comprising: a. a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; ii. a second insert comprising a sequence according to SEQ ID NO: 2 or 4 (SEQ ID NO: 4); and b. at least one second dose of the autologous tumor cell vaccine in combination with at least one dose of an additional therapeutic agent. In some embodiments, the ovarian cancer is stage III or stage IV ovarian cancer. In some embodiments, the ovarian cancer is refractory ovarian cancer. In some embodiments, the refractory ovarian cancer is refractory to chemotherapy. In some embodiments, the chemotherapy comprises a platinum agent or a taxane. In some embodiments, the platinum agent comprises carboplatin. In some embodiments, the taxane comprises paclitaxel. In some embodiments, the GM-CSF is a human GM-CSF sequence.

[0023] In some embodiments, the expression vector further comprises a promoter. In some embodiments, the promoter is a cytomegalovirus (CMV) mammalian promoter. In some embodiments, the expression vector further comprises a CMV enhancer sequence and a CMV intron sequence. In some embodiments, the first insert and the second insert are operably linked to the promoter. In some embodiments, the expression vector further comprises a nucleic acid sequence encoding a picornavirus 2A ribosomal skipping peptide between the first and second nucleic acid inserts.

[0024] In some embodiments, the additional therapeutic agent is selected from the group consisting of an angiogenesis inhibitor, a PARP inhibitor, and a checkpoint inhibitor for the individual. In some embodiments, the angiogenesis inhibitor is a vascular endothelial growth factor (VEGF) inhibitor. In some embodiments, the VEGF is selected from the group consisting of sorafenib, sunitinib, bevacizumab, pazopanib, axitinib, cabozantinib, and levatinib. In some embodiments, the VEGF is bevacizumab. In some embodiments, the PARP inhibitor is selected from the group consisting of niraparib, olaparib, rucaparib, niraparib, talazoparib, veliparib, and pamiparib. In some embodiments, the PARP inhibitor is niraparib. In some embodiments, the checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor. In some embodiments, the checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, and ipilimumab. In some embodiments, the checkpoint inhibitor is atezolizumab.

[0025] In some embodiments, the at least one dose of the additional therapeutic agent is between 1100 mg and 1300 mg. In some embodiments, the at least one first dose of the autologous tumor cell vaccine is between about 1 x 10 6 cells ~ approx. 5 x 10 7 In some embodiments, the at least one second dose of the autologous tumor cell vaccine comprises about 1 x 10 cells. 6 cells ~ approx. 5 x 10 7 In some embodiments, the at least one first dose of the autologous tumor cells is administered to the individual by intradermal injection. In some embodiments, the at least one second dose of the autologous tumor cell vaccine is administered to the individual by intradermal injection. In some embodiments, the at least one dose of the additional therapeutic agent is administered to the individual by intravenous infusion.

[0026] In some embodiments, the at least one first dose of the autologous tumor cell vaccine comprises two doses. In some embodiments, each of the at least one first dose of the autologous tumor cell vaccine is administered to the individual once per month. In some embodiments, each of the at least one second dose of the autologous tumor cell vaccine is administered to the individual once per month. In some embodiments, each of the at least one dose of the additional therapeutic agent is administered to the individual at least once per month. In some embodiments, the at least one first dose of the autologous tumor cell vaccine and the at least second dose of the autologous tumor cell vaccine comprise a total of at least 12 doses.

[0027] In some embodiments, the disclosure features a method of treating cancer in an individual in need thereof, the method including administering to the individual an expression vector including: a) a first insert including a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; and b) a second insert including a sequence according to SEQ ID NO: 2 or 4 (SEQ ID NO: 4), wherein the individual has a wild-type BRCA1 gene, a wild-type BRCA2 gene, or a combination thereof, and has been identified as homologous recombination repair deficient (HRD) negative.

[0028] In some embodiments, the GM-CSF is a human GM-CSF sequence.

[0029] In some embodiments, the expression vector further comprises a promoter. In some embodiments, the promoter is a cytomegalovirus (CMV) mammalian promoter. In some embodiments, the expression vector further comprises a CMV enhancer sequence and a CMV intron sequence. In some embodiments, the first insert and the second insert are operably linked to the promoter. In some embodiments, the expression vector further comprises a nucleic acid sequence encoding a picornavirus 2A ribosomal skipping peptide between the first and second nucleic acid inserts.

[0030] In some embodiments, the cancer is an HRD-negative wild-type BRCA1 / 2 cancer. In some embodiments, the cancer is selected from the group consisting of solid tumor cancer, ovarian cancer, adrenocortical carcinoma, bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer, glioblastoma, glioma, hepatocellular carcinoma, head and neck cancer, kidney cancer, leukemia, lymphoma, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, prostate cancer, sarcoma, gastric cancer, uterine cancer, thyroid cancer, and hematological cancer. In particular embodiments, the solid tumor cancer is selected from the group consisting of endometrial cancer, cholangiocarcinoma, bladder cancer, hepatocellular carcinoma, gastric / esophageal cancer, ovarian cancer, melanoma, breast cancer, pancreatic cancer, colorectal cancer, glioma, non-small cell lung cancer, prostate cancer, cervical cancer, renal cancer, thyroid cancer, neuroendocrine cancer, small cell lung cancer, sarcoma, head and neck cancer, brain cancer, clear cell renal carcinoma, skin cancer, endocrine tumors, thyroid cancer, tumors of unknown primary, and gastrointestinal stromal tumors. In one embodiment, the cancer is ovarian cancer.

[0031] In some embodiments, the method prevents or delays the relapse of substantially eradicated ovarian cancer. In particular embodiments, the substantially eradicated ovarian cancer is stage III or stage IV ovarian cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is lung cancer.

[0032] In some embodiments, the expression vector is in autologous cancer cells that are transfected with the expression vector. In particular embodiments, the autologous tumor cells are about 1 x 10 6 cells ~ approx. 5 x 10 7 In some embodiments, the autologous tumor cells are administered to the individual as a single dose of cells. In some embodiments, the autologous tumor cells are administered to the individual once per month. In some embodiments, the autologous tumor cells are administered to the individual for 1-12 months. In some embodiments, the autologous tumor cells are administered to the individual by intradermal injection.

[0033] In some embodiments, the individual has received a first therapy. In particular embodiments, the first therapy comprises cytoreductive surgery, chemotherapy, or a combination thereof. In some embodiments, the chemotherapy comprises administering a platinum compound and a taxane. In some embodiments, the platinum compound comprises carboplatin. In some embodiments, the taxane comprises paclitaxel.

[0034] In some embodiments, the method further comprises administering an additional therapeutic agent, hi one embodiment, the additional therapeutic agent is a member selected from the group consisting of an angiogenesis inhibitor, a PARP inhibitor, and a checkpoint inhibitor to the individual. [Brief explanation of the drawings]

[0035] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0036] [Figure 1] Figure 1 is a schematic diagram of bi-shRNAfurin (SEQ ID NO: 2), which contains two stem-loop structures, each carrying miR-30a; the first stem-loop structure has perfectly complementary guide and passenger strands, whereas the second stem-loop structure has a three base pair (bp) mismatch at positions 9-11 of the passenger strand.

[0037] [Figure 2A]Figures 2A-2D show patient recurrence-free survival (RFS) from surgery / harvesting or randomization to Vigil® versus placebo. Figure 2A shows RFS from surgery / harvesting in the BRCA1 / 2 wild-type (wt) population. Figure 2B shows RFS from randomization of the BRCA1 / 2 wild-type (wt) population to Vigil® versus placebo. Figure 2C shows RFS from surgery / harvesting regardless of BRCA1 / 2 status. Figure 2D shows RFS from randomization to Vigil® versus placebo regardless of BRCA1 / 2 status. Number at risk refers to the number of patients who are still recurrence-free and / or alive on the survival curve and whose follow-up has continued at least that far along the curve.

[0038] [Figure 2B] Figures 2A-2D show patient recurrence-free survival (RFS) from surgery / harvesting or randomization to Vigil® versus placebo. Figure 2A shows RFS from surgery / harvesting in the BRCA1 / 2 wild-type (wt) population. Figure 2B shows RFS from randomization of the BRCA1 / 2 wild-type (wt) population to Vigil® versus placebo. Figure 2C shows RFS from surgery / harvesting regardless of BRCA1 / 2 status. Figure 2D shows RFS from randomization to Vigil® versus placebo regardless of BRCA1 / 2 status. Number at risk refers to the number of patients who are still recurrence-free and / or alive on the survival curve and whose follow-up has continued at least that far along the curve.

[0039] [Figure 2C]Figures 2A-2D show patient recurrence-free survival (RFS) from surgery / harvesting or randomization to Vigil® versus placebo. Figure 2A shows RFS from surgery / harvesting in the BRCA1 / 2 wild-type (wt) population. Figure 2B shows RFS from randomization of the BRCA1 / 2 wild-type (wt) population to Vigil® versus placebo. Figure 2C shows RFS from surgery / harvesting regardless of BRCA1 / 2 status. Figure 2D shows RFS from randomization to Vigil® versus placebo regardless of BRCA1 / 2 status. Number at risk refers to the number of patients who are still recurrence-free and / or alive on the survival curve and whose follow-up has continued at least that far along the curve.

[0040] [Figure 2D] Figures 2A-2D show patient recurrence-free survival (RFS) from surgery / harvesting or randomization to Vigil® versus placebo. Figure 2A shows RFS from surgery / harvesting in the BRCA1 / 2 wild-type (wt) population. Figure 2B shows RFS from randomization of the BRCA1 / 2 wild-type (wt) population to Vigil® versus placebo. Figure 2C shows RFS from surgery / harvesting regardless of BRCA1 / 2 status. Figure 2D shows RFS from randomization to Vigil® versus placebo regardless of BRCA1 / 2 status. Number at risk refers to the number of patients who are still recurrence-free and / or alive on the survival curve and whose follow-up has continued at least that far along the curve.

[0041] [Figure 3A-B] Figures 3A-3B show the rate of any recurrent disease for Vigil® and placebo. Figure 3A shows the rate of any recurrent disease for BRCA1 / 2-wt patients for Vigil® (n=24) and placebo (n=24). Figure 3B shows the rate of any recurrent disease for Vigil® (n=46) and placebo (n=45) for all n=91 protocol-compliant patients.

[0042] [Figure 4]FIG. 4 shows forest plot key demographics for Vigil® / placebo patients.

[0043] [Figure 5] Figure 5 is a schematic diagram of bi-shRNAfurin (SEQ ID NO: 2), which contains two stem-loop structures, each carrying miR-30a; the first stem-loop structure has perfectly complementary guide and passenger strands, whereas the second stem-loop structure has a three base pair (bp) mismatch at positions 9-11 of the passenger strand.

[0044] [Figure 6A] Figures 6A-6F show recurrence-free survival (RFS) or overall survival (OS) for patients from surgery / harvesting or randomization with Vigil® versus placebo. Figure 6A shows RFS for all patients from randomization. Figure 6B shows RFS for all patients from surgery / harvesting. Figure 6C shows RFS for the BRCA1 / 2 wild-type (wt) population from randomization. Figure 6D shows RFS for the BRCA1 / 2 wild-type (wt) population from surgery / harvesting. Figure 6E shows OS for the BRCA1 / 2 wild-type (wt) population from randomization. Figure 6F shows OS for the BRCA1 / 2 wild-type (wt) population from surgery / harvesting. Number at risk refers to the number of patients who are still recurrence-free and / or alive on the survival curve and whose follow-up has continued at least that far on the curve.

[0045] [Figure 6B]Figures 6A-6F show recurrence-free survival (RFS) or overall survival (OS) for patients from surgery / harvesting or randomization with Vigil® versus placebo. Figure 6A shows RFS for all patients from randomization. Figure 6B shows RFS for all patients from surgery / harvesting. Figure 6C shows RFS for the BRCA1 / 2 wild-type (wt) population from randomization. Figure 6D shows RFS for the BRCA1 / 2 wild-type (wt) population from surgery / harvesting. Figure 6E shows OS for the BRCA1 / 2 wild-type (wt) population from randomization. Figure 6F shows OS for the BRCA1 / 2 wild-type (wt) population from surgery / harvesting. Number at risk refers to the number of patients who are still recurrence-free and / or alive on the survival curve and whose follow-up has continued at least that far on the curve.

[0046] [Figure 6C] Figures 6A-6F show recurrence-free survival (RFS) or overall survival (OS) for patients from surgery / harvesting or randomization with Vigil® versus placebo. Figure 6A shows RFS for all patients from randomization. Figure 6B shows RFS for all patients from surgery / harvesting. Figure 6C shows RFS for the BRCA1 / 2 wild-type (wt) population from randomization. Figure 6D shows RFS for the BRCA1 / 2 wild-type (wt) population from surgery / harvesting. Figure 6E shows OS for the BRCA1 / 2 wild-type (wt) population from randomization. Figure 6F shows OS for the BRCA1 / 2 wild-type (wt) population from surgery / harvesting. Number at risk refers to the number of patients who are still recurrence-free and / or alive on the survival curve and whose follow-up has continued at least that far on the curve.

[0047] [Figure 6D]Figures 6A-6F show recurrence-free survival (RFS) or overall survival (OS) for patients from surgery / harvesting or randomization with Vigil® versus placebo. Figure 6A shows RFS for all patients from randomization. Figure 6B shows RFS for all patients from surgery / harvesting. Figure 6C shows RFS for the BRCA1 / 2 wild-type (wt) population from randomization. Figure 6D shows RFS for the BRCA1 / 2 wild-type (wt) population from surgery / harvesting. Figure 6E shows OS for the BRCA1 / 2 wild-type (wt) population from randomization. Figure 6F shows OS for the BRCA1 / 2 wild-type (wt) population from surgery / harvesting. Number at risk refers to the number of patients who are still recurrence-free and / or alive on the survival curve and whose follow-up has continued at least that far on the curve.

[0048] [Figure 6E] Figures 6A-6F show recurrence-free survival (RFS) or overall survival (OS) for patients from surgery / harvesting or randomization with Vigil® versus placebo. Figure 6A shows RFS for all patients from randomization. Figure 6B shows RFS for all patients from surgery / harvesting. Figure 6C shows RFS for the BRCA1 / 2 wild-type (wt) population from randomization. Figure 6D shows RFS for the BRCA1 / 2 wild-type (wt) population from surgery / harvesting. Figure 6E shows OS for the BRCA1 / 2 wild-type (wt) population from randomization. Figure 6F shows OS for the BRCA1 / 2 wild-type (wt) population from surgery / harvesting. Number at risk refers to the number of patients who are still recurrence-free and / or alive on the survival curve and whose follow-up has continued at least that far on the curve.

[0049] [Figure 6F]Figures 6A-6F show recurrence-free survival (RFS) or overall survival (OS) for patients from surgery / harvesting or randomization with Vigil® versus placebo. Figure 6A shows RFS for all patients from randomization. Figure 6B shows RFS for all patients from surgery / harvesting. Figure 6C shows RFS for the BRCA1 / 2 wild-type (wt) population from randomization. Figure 6D shows RFS for the BRCA1 / 2 wild-type (wt) population from surgery / harvesting. Figure 6E shows OS for the BRCA1 / 2 wild-type (wt) population from randomization. Figure 6F shows OS for the BRCA1 / 2 wild-type (wt) population from surgery / harvesting. Number at risk refers to the number of patients who are still recurrence-free and / or alive on the survival curve and whose follow-up has continued at least that far on the curve.

[0050] [Figure 7A] Figures 7A-7B show forest plots. Figure 7A shows forest plots of major subgroups of PP with RFS calculated from the time of randomization to the date of first documented recurrence or death. Figure 7B shows forest plots of major subgroups of the BRCA-wt population with RFS calculated from the time of randomization to the date of first documented recurrence or death.

[0051] [Figure 7B] Figures 7A-7B show forest plots. Figure 7A shows forest plots of major subgroups of PP with RFS calculated from the time of randomization to the date of first documented recurrence or death. Figure 7B shows forest plots of major subgroups of the BRCA-wt population with RFS calculated from the time of randomization to the date of first documented recurrence or death.

[0052] [Figure 8]Figure 8 shows all atezolizumab-related adverse events (AEs) in Vigil®-1st and Atezo®-1st in Study Part 2. There were 24 grade 1 and 2 atezolizumab-related AEs in the Atezo-1st group and 50 in the Vigil®-1st group. There were 1 grade 1 and 2 Vigil®-related AE in the Atezo-1st group and 31 grade 3 and 4 atezolizumab-related AEs in the Atezo-1st group and 1 grade 3 and 4 Vigil®-related AE in the Vigil®-1st group. There were 0 grade 3 and 4 Vigil®-related AEs in the Atezo-1st group and 1 grade 3 and 4 Vigil®-related AE in the Vigil®-1st group.

[0053] [Figure 9A-B] Figures 9A-9D show efficacy analyses from the time of randomization in the study. Figure 9A shows overall survival (OS) for all study subjects in Vigil®-1st (n = 11) vs. Atezo-1st (n = 10). Median OS (mOS) was 10.8 months vs. non-treated patients, with a hazard ratio (HR) of 0.33 (95% CI [0.064-1.7], p = 0.097). Figure 9B shows OS in BRCA1 / 2-wt patients in Vigil®-1st (n = 6) vs. Atezo-1st (n = 7). Median OS was 5.2 months vs. non-treated patients, with a HR of 0.16 (95% CI [0.026-1.03], p = 0.027). Figure 9C shows investigator-assessed progression-free survival (PFS) for all study patients in Vigil®-1st (n=11) vs. Atezo-1st (n=10) by RECIST 1.1: median PFS (mPFS) 3.4 vs. 2.8 months, HR 0.76 (95% CI [0.28-2.0], p=0.29). Figure 9D shows PFS for BRCA1 / 2-wt patients in Vigil®-1st (n=6) vs. Atezo-1st (n=7): median PFS 3.5 vs. 2.8 months, HR 0.60 (95% CI [0.16-2.2], p=0.22).

[0054] [Figure 9C-D] Figures 9A-9D show efficacy analyses from the time of randomization in the study. Figure 9A shows overall survival (OS) for all study subjects in Vigil®-1st (n = 11) vs. Atezo-1st (n = 10). Median OS (mOS) was 10.8 months vs. non-treated patients, with a hazard ratio (HR) of 0.33 (95% CI [0.064-1.7], p = 0.097). Figure 9B shows OS in BRCA1 / 2-wt patients in Vigil®-1st (n = 6) vs. Atezo-1st (n = 7). Median OS was 5.2 months vs. non-treated patients, with a HR of 0.16 (95% CI [0.026-1.03], p = 0.027). Figure 9C shows investigator-assessed progression-free survival (PFS) for all study patients in Vigil®-1st (n=11) vs. Atezo-1st (n=10) by RECIST 1.1: median PFS (mPFS) 3.4 vs. 2.8 months, HR 0.76 (95% CI [0.28-2.0], p=0.29). Figure 9D shows PFS for BRCA1 / 2-wt patients in Vigil®-1st (n=6) vs. Atezo-1st (n=7): median PFS 3.5 vs. 2.8 months, HR 0.60 (95% CI [0.16-2.2], p=0.22).

[0055] [Figure 10A] Figures 10A-10D show patient recurrence-free survival (RFS) from surgery / harvesting or randomization to Vigil® versus placebo. Figure 10A shows RFS for the BRCA1 / 2 wild-type (wt) population from surgery / harvesting. Figure 10B shows RFS for the BRCA1 / 2 wild-type (wt) population from randomization to Vigil® versus placebo. Figure 10C shows RFS from surgery / harvesting regardless of BRCA1 / 2 status. Figure 10D shows RFS from randomization to Vigil® versus placebo regardless of BRCA1 / 2 status. Number at risk refers to the number of patients who are still recurrence-free and / or alive on the survival curve and whose follow-up has continued at least that far on the curve.

[0056] [Figure 10B] Figures 10A-10D show patient recurrence-free survival (RFS) from surgery / harvesting or randomization to Vigil® versus placebo. Figure 10A shows RFS for the BRCA1 / 2 wild-type (wt) population from surgery / harvesting. Figure 10B shows RFS for the BRCA1 / 2 wild-type (wt) population from randomization to Vigil® versus placebo. Figure 10C shows RFS from surgery / harvesting regardless of BRCA1 / 2 status. Figure 10D shows RFS from randomization to Vigil® versus placebo regardless of BRCA1 / 2 status. Number at risk refers to the number of patients who are still recurrence-free and / or alive on the survival curve and whose follow-up has continued at least that far on the curve.

[0057] [Figure 10C] Figures 10A-10D show patient recurrence-free survival (RFS) from surgery / harvesting or randomization to Vigil® versus placebo. Figure 10A shows RFS for the BRCA1 / 2 wild-type (wt) population from surgery / harvesting. Figure 10B shows RFS for the BRCA1 / 2 wild-type (wt) population from randomization to Vigil® versus placebo. Figure 10C shows RFS from surgery / harvesting regardless of BRCA1 / 2 status. Figure 10D shows RFS from randomization to Vigil® versus placebo regardless of BRCA1 / 2 status. Number at risk refers to the number of patients who are still recurrence-free and / or alive on the survival curve and whose follow-up has continued at least that far on the curve.

[0058] [Figure 10D]Figures 10A-10D show patient recurrence-free survival (RFS) from surgery / harvesting or randomization to Vigil® versus placebo. Figure 10A shows RFS for the BRCA1 / 2 wild-type (wt) population from surgery / harvesting. Figure 10B shows RFS for the BRCA1 / 2 wild-type (wt) population from randomization to Vigil® versus placebo. Figure 10C shows RFS from surgery / harvesting regardless of BRCA1 / 2 status. Figure 10D shows RFS from randomization to Vigil® versus placebo regardless of BRCA1 / 2 status. Number at risk refers to the number of patients who are still recurrence-free and / or alive on the survival curve and whose follow-up has continued at least that far on the curve.

[0059] [Figure 11A] Figures 11A and 11B show the rate of total recurrent disease for Vigil® and placebo. Figure 11A shows the rate of total recurrent disease for BRCA1 / 2-wt patients on Vigil® (n=24) and placebo (n=24). Figure 11B shows the rate of total recurrent disease for Vigil® (n=46) and placebo (n=45) for all n=91 protocol-compliant patients.

[0060] [Figure 11B] Figures 11A and 11B show the rate of total recurrent disease for Vigil® and placebo. Figure 11A shows the rate of total recurrent disease for BRCA1 / 2-wt patients on Vigil® (n=24) and placebo (n=24). Figure 11B shows the rate of total recurrent disease for Vigil® (n=46) and placebo (n=45) for all n=91 protocol-compliant patients.

[0061] [Figure 12] FIG. 12 shows forest plot key demographics for Vigil® / placebo patients.

[0062] [Figure 13]FIG. 13 shows the BRCA mutation status and HRD status of individuals receiving placebo and Vigil®.

[0063] [Figure 14] Figures 14 and 15 show forest plot key demographics for Vigil® / placebo patients.

[0064] [Figure 15] Figures 14 and 15 show forest plot key demographics for Vigil® / placebo patients.

[0065] [Figure 16] FIG. 16 shows the BRCA mutation status and HRD status of individuals receiving placebo and Vigil®.

[0066] [Figure 17] Figures 17-20 show forest plot key demographics for Vigil® / placebo patients.

[0067] [Figure 18] Figures 17-20 show forest plot key demographics for Vigil® / placebo patients.

[0068] [Figure 19] Figures 17-20 show forest plot key demographics for Vigil® / placebo patients.

[0069] [Figure 20] Figures 17-20 show forest plot key demographics for Vigil® / placebo patients.

[0070] [Figure 21] Figures 21 and 22 show the RFS of the patient population from surgery / harvesting (Figure 13) and randomization (Figure 14).

[0071] [Figure 22] Figures 21 and 22 show the RFS of the patient population from surgery / harvesting (Figure 13) and randomization (Figure 14).

[0072] [Figure 23] Figures 23 and 24 show overall survival (OS) for the patient population from surgery / harvesting (Figure 15) and randomization (Figure 16).

[0073] [Figure 24] Figures 23 and 24 show overall survival (OS) for the patient population from surgery / harvesting (Figure 15) and randomization (Figure 16).

[0074] [Figure 25] Figure 25 shows the probability of RFS for the patient population from randomization.

[0075] [Figure 26] Figure 26 shows the probability of OS for the patient population from randomization.

[0076] [Figure 27] FIG. 27 shows BRCA status, HRD status, and OS for patients receiving Vigil® versus other agents.

[0077] [Figure 28] Figure 28 shows HR-DDR mutation frequencies in various types of cancer.

[0078] [Figure 29] FIG. 29 shows the relationship between BRCA status, HRD status, and other protein expression.

[0079] [Figure 30] Figures 30 and 31 show forest plot key demographics for HRD-negative and HRD-positive patients.

[0080] [Figure 31]Figures 30 and 31 show forest plot key demographics for HRD-negative and HRD-positive patients.

[0081] [Figure 32] Figures 32 and 33 show forest plot key demographics of BRCA1 / 2-WT and HRD-positive patients.

[0082] [Figure 33] Figures 32 and 33 show forest plot key demographics of BRCA1 / 2-WT and HRD-positive patients.

[0083] [Figure 34] Figure 34 shows the profile of the study patients. BRCAWT = BRCA wild type. BRCAmut = BRCA mutated.

[0084] [Figure 35A-F] Figures 35A-35F show recurrence-free survival for all patients from randomization (35A) and tissue collection (35B). Recurrence-free survival for patients with BRCA wild-type disease from randomization (35C) and tissue collection (35D). Overall survival for all patients from randomization (35E) and tissue collection (35F). HR = Hazard Ratio.

[0085] [Figure 36A] Figures 36A and 36B show recurrence-free survival calculated from the time of randomization to the date of first documented recurrence or death for major subgroups of the per-protocol population (36A) and the BRCA wild-type population (36B). Data are number of events / number of patients unless otherwise stated. HR = hazard ratio. *>30% knockdown. †Above a release threshold of 30 pg per 106 cells.

[0086] [Figure 36B]Figures 36A and 36B show recurrence-free survival calculated from the time of randomization to the date of first documented recurrence or death for major subgroups of the per-protocol population (36A) and the BRCA wild-type population (36B). Data are number of events / number of patients unless otherwise stated. HR = hazard ratio. *>30% knockdown. †Above a release threshold of 30 pg per 106 cells.

[0087] [Figure 37A-B] Figures 37A-D show Kaplan-Meier analysis of the ITT population. Relapse-free survival for BRCA wild-type patients with intention-to-treat from tissue collection (A) and randomization (B). Relapse-free survival for all patients with intention-to-treat from tissue collection (C) and randomization (D).

[0088] [Fig. 37C-D] Figures 37A-D show Kaplan-Meier analysis of the ITT population. Relapse-free survival for BRCA wild-type patients with intention-to-treat from tissue collection (A) and randomization (B). Relapse-free survival for all patients with intention-to-treat from tissue collection (C) and randomization (D).

[0089] [Figure 38] Figure 38 shows a forest plot of overall survival of the patient population from the time of randomization.

[0090] [Figure 39] Figure 39 shows the overall survival forest plot for the adherent to protocol and BRCA wild-type populations.

[0091] [Figure 40A-B] Figures 40A-40D show overall survival of BRCA wild-type patients from the time of randomization (Figure 40A) and tissue collection (Figure 40B). Overall survival of BRCA mutant patients from the time of randomization (Figure 40C) and tissue collection (Figure 40D).

[0092] [Fig. 40C-D]Figures 40A-40D show overall survival of BRCA wild-type patients from the time of randomization (Figure 40A) and tissue collection (Figure 40B). Overall survival of BRCA mutant patients from the time of randomization (Figure 40C) and tissue collection (Figure 40D).

[0093] [Figure 41A-B] Figures 41A and 41B show recurrence-free survival of BRCA-mutated patients from the time of randomization (Figure 41A) and tissue collection (Figure 41B). DETAILED DESCRIPTION OF THE INVENTION

[0094] The majority of women diagnosed with ovarian cancer present at an advanced stage. Optimal standard treatment results in 5-year survival rates that vary by stage, ranging from 41% (stage IIa) to 20% (stage IV). Standard treatment for newly diagnosed ovarian cancer (stage III / IV) includes optimal cytoreductive surgery and frontline chemotherapy with paclitaxel and carboplatin. While most patients achieve a complete remission, approximately 75% relapse within approximately 12 months, including the 70% of patients who achieve a pathological complete response. Numerous studies have attempted to improve outcomes in initially treated ovarian cancer by administering maintenance therapy after patients achieve a complete response, followed by consolidation with paclitaxel and carboplatin; however, no studies have demonstrated a significant benefit in recurrence-free survival (RFS) or overall survival (OS).

[0095] Disclosed herein in certain embodiments is a method of treating cancer in an individual in need thereof, the method comprising administering to the individual an expression vector comprising: a. a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; and b. a second insert comprising two stem-loop structures, each having a miR-30a loop, wherein the first stem-loop structure has perfectly complementary guide and passenger strands, while the second stem-loop structure has a three base pair (bp) mismatch at positions 9-11 of the passenger strand, and the individual is homologous recombination repair deficient (HRD) negative and / or the individual has a wild-type BRCA1 gene, a wild-type BRCA2 gene, or a combination thereof. Descriptions of the miR-30a loop and its sequence are known in the art, see, e.g., Rao et al., Cancer Gene Ther. 17(11):780-91, 2010; Jay et al., Cancer Gene Ther. 20(12):683-9, 2013; Rao et al., Mol Ther. 24(8):1412-22, 2016; Phadke et al., DNA Cell Biol. 30(9):715-26, 2011; Barve et al., Mol Ther. 23(6):1123-1130, 2015; Rao et al., Methods Mol Biol. 942:259-78, 2013; and Senzer et al., Mol Ther. 20(3):679-86, 2012. In some embodiments, the miR-30a loop comprises the sequence GUGAAGCCACAGAUG (SEQ ID NO: 8). In some embodiments, the guide strand in the first stem-loop structure comprises SEQ ID NO: 6, and the passenger strand in the first stem-loop structure has SEQ ID NO: 5. In some embodiments, the guide strand in the second stem-loop structure comprises SEQ ID NO: 6, and the passenger strand in the second stem-loop structure has SEQ ID NO: 7.

[0096] Disclosed herein in certain embodiments is a method of preventing the relapse of substantially eradicated ovarian cancer in an individual in need thereof, the method comprising administering to the individual autologous tumor cells transfected with an expression vector comprising: a) a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; and b) a second insert comprising a sequence according to SEQ ID NO: 2 or 4 (SEQ ID NO: 4). Also disclosed herein in certain embodiments is a method of treating BRCA1 / 2 wild-type ovarian cancer in an individual in need thereof, the method comprising administering to the individual autologous tumor cells transfected with an expression vector comprising: a) a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; and b) a second insert comprising a sequence according to SEQ ID NO: 2 or 4 (SEQ ID NO: 4).

[0097] The majority of women diagnosed with ovarian cancer present at an advanced stage. Optimal standard treatments achieve 5-year survival rates that vary by stage, ranging from 41% (stage IIa) to 20% (stage IV). Standard treatment for newly diagnosed ovarian cancer (stage III / IV) involves primary cytoreductive surgery followed by adjuvant chemotherapy with paclitaxel and carboplatin, or neoadjuvant chemotherapy followed by cytoreductive surgery. While most patients achieve complete remission, nearly 75% relapse within two years. Numerous studies have attempted to improve outcomes in frontline-treated ovarian cancer by administering maintenance therapy after patients achieve a complete response. However, despite benefits in progression-free survival (PFS), no studies have demonstrated a significant benefit in RFS or OS, and toxicity is dose-limiting. Poly(ADP-ribose) polymerase (PARP) inhibitors have provided a new platform for frontline maintenance therapy in the clinic, but activity has been predominant in BRCA-m patients. PARP inhibitors are also approved for maintaining recurrent platinum sensitivity regardless of BRCA status, but the magnitude of benefit is greatest in patients who are BRCA-m or exhibit homologous recombination repair deficiency (HRD).

[0098] Disclosed herein in certain embodiments are methods for preventing or treating the recurrence of substantially eradicated ovarian cancer in an individual in need thereof, the method comprising administering to the individual autologous tumor cells transfected with an expression vector comprising: a) a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; and b) a second insert comprising a sequence according to SEQ ID NO: 2 or 4 (SEQ ID NO: 4). Also disclosed herein in certain embodiments are methods for treating BRCA1 / 2 wild-type ovarian cancer in an individual in need thereof, the method comprising administering to the individual autologous tumor cells transfected with an expression vector comprising: a) a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; and b) a second insert comprising a sequence according to SEQ ID NO: 2 or 4 (SEQ ID NO: 4). In some embodiments, the BRCA1 / 2 wild-type ovarian cancer does not have a germline mutation in a BRCA gene. In some embodiments, the BRCA1 / 2 wild-type ovarian cancer does not have a somatic mutation in a BRCA gene. In some embodiments, the BRCA genes are the BRCA1 gene, the BRCA2 gene, or the BRCA1 gene and the BRCA2 gene.

[0099] Disclosed in certain embodiments herein is a method of treating cancer in an individual in need thereof by administering to the individual an expression vector comprising: a) a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; and b) a second insert comprising a sequence according to SEQ ID NO: 2 or 4 (SEQ ID NO: 4), wherein the individual has a wild-type BRCA1 gene, a wild-type BRCA2 gene, or a combination thereof, and has been identified as homologous recombination repair deficient (HRD) negative.

[0100] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs.

[0101] Ranges and amounts are expressed herein by using "about" next to a specific value or range. About includes the exact amount. Thus, "about 5 μg" means "about 5 μg" as well as "5 μg." In general, the term "about" includes amounts that are expected to be within experimental error. In some embodiments, "about" refers to the stated number or value, plus or minus 20%, 10%, or 5% of that number or value.

[0102] The phrase "effective amount" or "therapeutically effective amount," as used herein, with respect to an administered drug or compound, refers to an amount sufficient to alleviate to some extent one or more symptoms of the disease or condition being treated, or to prevent the onset or recurrence of one or more symptoms of the disease or condition being treated. In some embodiments, the result is a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired change in a biological system. For example, an "effective amount" for therapeutic purposes is the amount of an autologous tumor cell vaccine required to provide a clinically significant alleviation of disease symptoms without undue adverse side effects. In another example, an "effective amount" for therapeutic purposes is the amount of an autologous tumor cell vaccine disclosed herein required to prevent the recurrence of disease symptoms without undue adverse side effects. The appropriate "effective amount" in any individual case can be determined using techniques such as a dose escalation study. The phrase "therapeutically effective amount" includes, for example, a prophylactically effective amount. An "effective amount" of a compound disclosed herein is an amount effective to achieve a desired effect or therapeutic improvement without undue adverse side effects. In some embodiments, an "effective amount" or "therapeutically effective amount" will vary from subject to subject due to variations in the subject's autologous tumor cell vaccine metabolism, age, weight, general condition, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician.

[0103] As used herein, the terms "subject," "individual," and "patient" are used interchangeably. Neither term should be construed as requiring the direction of a medical professional (e.g., a physician, nurse, physician's assistant, hospital attendant, or hospice worker). As used herein, a subject is an animal, which includes mammals (e.g., humans or non-human animals) and non-mammals. In one embodiment of the methods and autologous tumor cell vaccines presented herein, the mammal is a human.

[0104] As used herein, non-limiting examples of the terms "treat," "treating," or "treatment," and other grammatically equivalent terms, include alleviating, alleviating, or ameliorating one or more symptoms of a disease or condition; ameliorating, preventing, or alleviating the appearance, severity, or frequency of one or more additional symptoms of a disease or condition; ameliorating or preventing the metabolic causes underlying one or more symptoms of a disease or condition; inhibiting the disease or condition (e.g., halting the progression of the disease or condition, alleviating the disease or condition, causing regression of the disease or condition, alleviating conditions caused by the disease or condition, preventing recurrence of the disease or condition, or prophylactically treating, or prophylactically and / or therapeutically inhibiting symptoms of the disease or condition, etc.). In one non-limiting example, to obtain prophylactic benefit, the autologous tumor cell vaccines disclosed herein are administered to individuals at risk for developing a particular disease or condition, individuals prone to developing a particular disease or condition, or individuals who have previously suffered from or been treated for the disease or condition. In some embodiments, the disease or condition is ovarian cancer.

[0105] As used herein, the term "prevention" refers to prophylactic treatment administered before a subject suffers from a disease or before a previously diagnosed disease worsens, thereby avoiding, preventing, or reducing the likelihood of symptoms of the disease or associated illness in the subject. The subject may be at increased risk of developing a disease or at increased risk of worsening a previously diagnosed disease.

[0106] As used herein, the term "intradermal injection" refers to the superficial injection of a substance into the dermis, which is located between the epidermis and the subcutaneous tissue.

[0107] As used herein, the term "transfection" refers to the introduction of foreign DNA into a eukaryotic cell. In some embodiments, transfection involves any suitable technique (e.g., calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, or biolistics).

[0108] As used herein, the term "nucleic acid" or "nucleic acid molecule" refers to polynucleotides (e.g., deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)), oligonucleotides, fragments generated by polymerase chain reaction (PCR), and fragments generated by ligation, cleavage, endonuclease action, and exonuclease action. In some embodiments, nucleic acid molecules are composed of monomers that are natural nucleotides (e.g., DNA, RNA), or analogs of natural nucleotides (e.g., α-enantiomeric forms of natural nucleotides), or combinations thereof. In some embodiments, modified nucleotides have alterations in the sugar moiety and / or the pyrimidine or purine base moiety. Sugar modifications include replacement of one or more hydroxyl groups with, for example, halogens, alkyl groups, amines, or azide groups. Alternatively, sugars can be functionalized as ethers or esters. Additionally, in some embodiments, the entire sugar moiety is replaced with sterically or electronically similar structures (e.g., aza-sugars and carbocyclic sugar analogs). Examples of modifications of the base moiety include alkylated purine pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substituents. In some embodiments, nucleic acid monomers are linked by phosphodiester linkages or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroanilidates, phosphoramidates, and the like. In some embodiments, the terms "nucleic acid" or "nucleic acid molecule" also include so-called "peptide nucleic acids," which comprise natural or modified nucleobases attached to a polyamide backbone. In some embodiments, nucleic acids are single-stranded or double-stranded.

[0109] As used herein, the term "expression vector" refers to a nucleic acid molecule that encodes a gene to be expressed in a host cell. In some embodiments, an expression vector comprises a transcription promoter, a gene, and a transcription terminator. In some embodiments, gene expression is placed under the control of a promoter, and such a gene is said to be "operably linked" to that promoter. In some embodiments, a regulatory element and a core promoter are operably linked when the regulatory element modulates the activity of the core promoter. As used herein, the term "promoter" refers to any DNA sequence that, when associated with a structural gene in a host yeast cell, increases 1) transcription, 2) translation, or 3) mRNA stability (longer half-life of the mRNA) under appropriate growth conditions compared to transcription, translation, or mRNA stability in the absence of that promoter sequence.

[0110] As used herein, the term "bifunctional" refers to an shRNA that has two mechanistic pathways of action: the siRNA pathway and the miRNA pathway. The term "traditional" shRNA refers to an RNA-derived DNA transcription that acts via the siRNA mechanism. The term "dual" shRNA refers to two shRNAs that each disrupt the expression of two different genes but act in a "traditional" siRNA mode.

[0111] The additional therapeutic agent can be between 1100 mg and 1300 mg (e.g., 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, or 1300 mg).

[0112] Autologous tumor cell vaccine was administered at a dose of 1 × 10 6 cells ~ approx. 5 x 10 7 cells (1 × 10 6 cells, 2 x 10 6 cells, 3 x 10 6 cells, 4 x 10 6 cells, 5 x 10 6 cells, 6 x 10 6 cells, 7 x 10 6 cells, 8 x 106 9 x 10 cells 6 cells, 1 x 10 7 cells, 2 x 10 7 cells, 3 x 10 7 cells, 4 x 10 7 cells, or 5 x 10 7 cells, etc.

[0113] Stage III ovarian cancer means cancer found in one or both ovaries and has spread outside the pelvis to other parts of the abdomen and / or nearby lymph nodes. Spread to the surface of the liver is also considered stage III ovarian cancer. In stage IV ovarian cancer, the cancer has spread beyond the abdomen to other parts of the body, such as the lungs or tissue within the liver. Cancer cells in the fluid around the lungs are also considered stage IV ovarian cancer.

[0114] How to Treat Ovarian Cancer Disclosed in certain embodiments herein is a method for preventing or treating the recurrence of substantially eradicated ovarian cancer in an individual in need thereof, the method comprising administering to the individual an autologous tumor cell vaccine comprising autologous tumor cells transfected with an expression vector comprising a first nucleic acid encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) and a second nucleic acid encoding at least one short hairpin RNA (shRNA) capable of hybridizing to a region of an mRNA transcript encoding furin, thereby inhibiting the expression of furin through RNA interference. Further disclosed in certain embodiments herein are methods of treating BRCA1 / 2 wild-type ovarian cancer in an individual in need thereof, the method comprising administering to the individual an autologous tumor cell vaccine comprising autologous tumor cells transfected with an expression vector comprising a first nucleic acid encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) and a second nucleic acid encoding at least one short hairpin RNA (shRNA) capable of hybridizing to a region of an mRNA transcript encoding Furin, thereby inhibiting expression of Furin through RNA interference. In some embodiments, the second nucleic acid comprises a sequence according to SEQ ID NO: 2 or 4 (SEQ ID NO: 4). In some embodiments, the expression vector comprises a bishRNA. フーリン In some embodiments, inhibition of Furin expression inhibits expression of transforming growth factor beta (TGFβ). TGFβ includes the TGFβ isoforms TGFβ1 and TGFβ2.

[0115] Further disclosed in certain embodiments herein is a method for preventing or prophylactically treating recurrence of substantially eradicated ovarian cancer in an individual in need thereof, the method comprising: (a) administering to the individual at least one first dose of an autologous tumor cell vaccine comprising autologous tumor cells transfected with an expression vector comprising (i) a first nucleic acid encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) and (ii) a second nucleic acid encoding at least one short hairpin RNA (shRNA) capable of hybridizing to a region of an mRNA transcript encoding furin (thereby inhibiting expression of furin through RNA interference); and (b) administering to the individual at least one second dose of the autologous tumor cell vaccine in combination with at least one dose of an additional therapeutic agent. Further disclosed in certain embodiments herein is a method of treating BRCA1 / 2 wild-type ovarian cancer in an individual in need thereof, the method comprising: (a) administering to the individual at least one first dose of an autologous tumor cell vaccine comprising autologous tumor cells transfected with an expression vector comprising (i) a first nucleic acid encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) and (i) a second nucleic acid encoding at least one short hairpin RNA (shRNA) capable of hybridizing to a region of an mRNA transcript encoding furin (thereby inhibiting expression of furin through RNA interference); and (b) administering to the individual at least one second dose of the autologous tumor cell vaccine in combination with at least one dose of an additional therapeutic agent.

[0116] In some embodiments, the second nucleic acid comprises a sequence according to SEQ ID NO: 2 or 4 (SEQ ID NO: 4). In some embodiments, the expression vector comprises bishRNA フーリン In some embodiments, inhibition of Furin expression inhibits expression of transforming growth factor beta (TGFβ). In some embodiments, TGFβ includes the TGFβ isoforms TGFβ1 and TGFβ2.

[0117] Disclosed herein in certain embodiments is a method of treating cancer in an individual in need thereof by administering to the individual autologous tumor cells transfected with an expression vector comprising: a) a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; and b) a second insert comprising a sequence according to SEQ ID NO: 2 or 4 (SEQ ID NO: 4), wherein the individual has been identified as having a wild-type BRCA1 gene, a wild-type BRCA2 gene, or a combination thereof, and as homologous recombination repair deficient (HRD) negative.

[0118] Homologous recombination (HR) is a mechanism used by cells to repair double-stranded DNA breaks using homologous templates. HR repair defects can affect DNA repair. However, when HR alone is defective, activity of other DNA repair mechanisms can prevent the accumulation of excessive DNA damage and apoptosis. As used herein, the terms "homologous recombination repair defect-positive," "HRD-positive," and "HRD" are used interchangeably to refer to a state in which HR is defective. Conversely, the terms "homologous recombination negative," "HRD-negative," "homologous recombination proficient," and "HRP" are used interchangeably to refer to a state in which HR is not defective.

[0119] In some embodiments, HRD can be assessed by screening for germline or somatic mutations in genes involved in HR repair, for example, DNA from blood or other tissues can be analyzed by next-generation sequencing.

[0120] In some embodiments, an HRD score can be calculated to characterize an individual as HRD-positive or HRD-negative. In some embodiments, the HRD score can be calculated based on the scores of loss of heterozygosity (LOH), telomeric allele imbalance (TAI), and large-scale state transition (LST). In some embodiments, LOH is indicated by the presence of a single allele. In some embodiments, LOH is defined as the number of chromosome losses in heterozygous regions longer than 15 Mb. In some embodiments, TAI is indicated by a one-to-one allele ratio mismatch at the end of a chromosome. In some embodiments, LST is indicated by a transition point between a region of abnormal and normal DNA or between two different abnormal regions. In some embodiments, LST is defined as the number of breakpoints between regions longer than 10 Mb after excluding regions shorter than 3 Mb. In an embodiment, the HRD score is calculated as the sum of the LOH score, the TAI score, and the LST score. Methods for determining HRD scores are available in the art and are described, for example, in Takaya et al., Sci Rep. 10(1):2757, 2020, Telli et al., Clin Cancer Res 22(15):3764-73, 2016, and Marchetti and McNeish, Cancer Breaking News 5(1):15-20, 2017. Additionally, commercial services for determining HRD scores are available (e.g., services offered by Ambry Genetics, Caris Life Sciences, Counsylgenetic, Foundation Medicine, GeneDX, Integrated Genetics, Invitae, Myriad Genetics, and Neogenomics).

[0121] In the methods described herein, an individual has a wild-type BRCA1 gene, a wild-type BRCA2 gene, or a combination thereof. In some embodiments, an individual with a wild-type BRCA1 gene, a wild-type BRCA2 gene, or a combination thereof can be HRD-negative or HRD-positive. In another embodiment, a mutation in the BRCA1 / 2 gene can lead to HRD. In other words, a mutation in the BRCA1 / 2 gene can lead to an individual having an HRD-positive status. In particular embodiments, an individual identified as having an HRD-positive status has an HRD score of 42 or greater (e.g., 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or greater). Other mechanisms (e.g., germline and somatic mutations in other homologous recombination genes and epigenetic modifications) can also be involved in homologous recombination.

[0122] Expression vector In some embodiments, at least one shRNA is at least one bifunctional shRNA (bi-shRNA). In some embodiments, the bi-shRNA comprises a first stem-loop structure containing an siRNA component and a second stem-loop structure containing an miRNA component. In some embodiments, the bifunctional shRNA has two mechanistic pathways of action: the siRNA pathway and the miRNA pathway. Thus, in some embodiments, the bifunctional shRNAs described herein differ from traditional shRNAs, i.e., RNAs derived from DNA transcription that act by the action of the siRNA mechanism, or from "dual shRNAs," which refer to two shRNAs that each disrupt the expression of two different genes but act in a "traditional" siRNA mode. In some embodiments, the bi-shRNA comprises an siRNA (cleavage-dependent) motif and an miRNA (cleavage-independent) motif.

[0123] In some embodiments, the GM-CSF in the expression vector is a human GM-CSF sequence. In some embodiments, the expression vector further comprises a promoter, for example, the promoter is a cytomegalovirus (CMV) mammalian promoter. In some embodiments, the mammalian CMV promoter comprises a CMV immediate-early (IE) 5' UTR enhancer sequence and a CMV IE intron A. In further embodiments, the expression vector further comprises a CMV enhancer sequence and a CMV intron sequence.

[0124] The first and second inserts in the expression vector can be operably linked to a promoter. In particular embodiments, the expression vector further comprises a nucleic acid sequence encoding a picornavirus 2A ribosomal skipping peptide between the first and second nucleic acid inserts.

[0125] In some embodiments, the expression vector comprises at least one bifunctional shRNA (bi-shRNA). In some embodiments, the bi-shRNA comprises a first stem-loop structure containing an siRNA component and a second stem-loop structure containing an miRNA component. In some embodiments, the bifunctional shRNA has two mechanistic pathways of action: the siRNA pathway and the miRNA pathway. Thus, in some embodiments, the bifunctional shRNAs described herein differ from traditional shRNAs, i.e., RNAs derived from DNA transcription that act via the siRNA mechanism, or from "dual shRNAs," which refer to two shRNAs that each disrupt the expression of two different genes but act in a "traditional" siRNA mode. In some embodiments, the bi-shRNA comprises an siRNA (cleavage-dependent) motif and an miRNA (cleavage-independent) motif.

[0126] In some embodiments, the at least one bi-shRNA can hybridize to one of more than one region of an mRNA transcript encoding Furin. In some embodiments, the mRNA transcript encoding Furin is the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the one or more regions of the mRNA transcript encoding Furin are selected from base sequences 300-318, 731-740, 1967-1991, 2425-2444, 2827-2851, and 2834-2852 of SEQ ID NO: 1. In some embodiments, the expression vector targets the coding region of a Furin mRNA transcript, the 3' UTR region of a Furin mRNA transcript, or simultaneously both the coding sequence and the 3' UTR sequence of a Furin mRNA transcript. In some embodiments, the bi-shRNA comprises SEQ ID NO: 2 or 4 (SEQ ID NO: 4). In some embodiments, a bi-shRNA capable of hybridizing to one or more regions of an mRNA transcript encoding Furin is referred to herein as a bi-shRNA. フーリン In some embodiments, the bi-shRNA フーリン comprises or consists of two stem-loop structures, each having a miR-30a loop. In some embodiments, the first of the two stem-loop structures comprises complementary guide and passenger strands (FIG. 1). In some embodiments, the second of the two stem-loop structures comprises three mismatches in the passenger strand. In some embodiments, the three mismatches are at positions 9-11 of the passenger strand. [Table 1-1] [Table 1-2]

[0127] An expression vector comprising a first nucleic acid encoding GM-CSF and a second nucleic acid encoding at least one bifunctional short hairpin RNA (bi-shRNA) capable of hybridizing to a region of an mRNA transcript encoding Furin is referred to as bi-shRNA. フーリン This is referred to as the / GMCSF expression vector.

[0128] In some embodiments, the first nucleic acid and the second nucleic acid are operably linked to a promoter. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the CMV promoter is a mammalian CMV promoter. In some embodiments, the mammalian CMV promoter comprises a CMV immediate-early (IE) 5' UTR enhancer sequence and a CMV IE intron A.

[0129] In some embodiments, the GM-CSF is human GM-CSF. In some embodiments, a nucleic acid sequence encoding a picornavirus 2A ribosomal skipping peptide sequence is intercalated between the first and second nucleic acid inserts.

[0130] In some embodiments, the expression vector plasmid can have a sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO: 3. The vector plasmid includes a first nucleic acid insert operably linked to a promoter, where the first nucleic acid insert encodes a GM-CSF cDNA, and a second nucleic acid insert operably linked to the promoter, where the second nucleic acid insert encodes one or more short hairpin RNAs (shRNAs) that can hybridize to a region of an mRNA transcript encoding Furin, thereby inhibiting expression of Furin through RNA interference. SEQ ID NO: 3

[0131] An expression vector comprising a first nucleic acid encoding GM-CSF and a second nucleic acid encoding at least one bifunctional short hairpin RNA (bi-shRNA) capable of hybridizing to a region of an mRNA transcript encoding Furin is referred to as bi-shRNA. フーリン This is referred to as the / GMCSF expression vector.

[0132] tumor cells In some embodiments, the cells are autologous tumor cells, xenograft-grown autologous tumor cells, allogeneic tumor cells, xenograft-grown allogeneic tumor cells, or a combination thereof. In some embodiments, the cells are autologous tumor cells. In some embodiments, the allogeneic tumor cells are established cell lines. In some embodiments, the autologous tumor cells are obtained from an individual in need thereof. In some embodiments, when the cells are autologous tumor cells, the composition is referred to as an autologous tumor cell vaccine.

[0133] In some embodiments, the cells are harvested from an individual. In some embodiments, the cells are harvested from a tissue of the individual. In some embodiments, the tissue is tumor tissue. In some embodiments, the tumor tissue is ovarian tumor tissue. In some embodiments, the tumor tissue is harvested during a biopsy or cytoreductive surgery on the individual. In some embodiments, the tumor tissue, or cells from the tumor tissue, are placed in an antibiotic solution in a sterile container. In some embodiments, the antibiotic solution comprises gentamicin, sodium chloride, or a combination thereof.

[0134] Usage In some embodiments, the ovarian cancer is stage III or stage IV ovarian cancer. In some embodiments, the stage III ovarian cancer is stage IIIb or worse. In some embodiments, the ovarian cancer is high-grade serous ovarian cancer, clear cell ovarian cancer, endometrioid ovarian cancer, mucinous ovarian cancer, or low-grade serous ovarian cancer.

[0135] In some embodiments, the individual has a wild-type BRCA1 gene, a wild-type BRCA2 gene, or both a wild-type BRCA1 gene and a wild-type BRCA2 gene. In some embodiments, the wild-type BRCA1 gene does not contain a mutation in the germline BRCA1 gene. In some embodiments, the wild-type BRCA2 gene does not contain a mutation in the germline BRCA2 gene. In some embodiments, ovarian cancer in an individual with wild-type BRCA1 and wild-type BRCA2 genes is referred to herein as BRCA1 / 2-wt ovarian cancer, BRCA-wt ovarian cancer, or BRCA1 / 2 wild-type ovarian cancer. Conversely, in some embodiments, ovarian cancer in an individual with a mutated BRCA1 gene, a mutated BRCA2 gene, or both a mutated BRCA1 gene and a mutated BRCA2 gene is referred to herein as BRCA1 / 2-m ovarian cancer or BRCA-m ovarian cancer. In some embodiments, the mutated BRCA1 gene or the mutated BRCA2 gene comprises a germline mutation. In some embodiments, the mutated BRCA1 gene or the mutated BRCA2 gene comprises a somatic mutation. In some embodiments, the recurrence-free survival (RFS) of the individual is increased compared to an individual who has not received the autologous tumor cell vaccine and whose ovarian cancer has been substantially eradicated.

[0136] In some embodiments, the cancer is an HRD-negative wild-type BRCA1 / 2 cancer. In some embodiments, the cancer is selected from the group consisting of solid tumor cancer, ovarian cancer, adrenocortical carcinoma, bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer, glioblastoma, glioma, hepatocellular carcinoma, head and neck cancer, kidney cancer, leukemia, lymphoma, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, prostate cancer, sarcoma, gastric cancer, uterine cancer, thyroid cancer, and hematological cancer. Non-limiting examples of solid tumor cancers include endometrial cancer, bile duct cancer, bladder cancer, hepatocellular carcinoma, gastric / esophageal cancer, ovarian cancer, melanoma, breast cancer, pancreatic cancer, colorectal cancer, glioma, non-small cell lung cancer, prostate cancer, cervical cancer, kidney cancer, thyroid cancer, neuroendocrine carcinoma, small cell lung cancer, sarcoma, head and neck cancer, brain tumor, clear cell renal carcinoma, skin cancer, endocrine tumors, thyroid cancer, tumors of unknown primary, and gastrointestinal stromal tumors.

[0137] As used herein, "recurrence-free survival" is used interchangeably with the term "relapse-free survival" and refers to the time that a cancer remains undetectable (i.e., until cancer recurrence) following administration of an initial therapy to treat the cancer. In some embodiments, the recurrence-free survival of an individual administered an autologous tumor cell vaccine is 5 to 11 months longer than the recurrence-free survival of an individual not administered an autologous tumor cell vaccine. In some embodiments, the recurrence-free survival of an individual administered an autologous tumor cell vaccine is at least 5, 6, 7, 8, 9, 10, or 11 months longer than the recurrence-free survival of an individual not administered an autologous tumor cell vaccine.

[0138] As used herein, the term "substantially eradicated" refers to ovarian cancer that is undetectable (e.g., below detectable levels or below the limit of detection (LOD)) in an individual following initial therapy for treating the ovarian cancer. In some embodiments, detection of ovarian cancer, or lack thereof, is by chest x-ray, computed tomography (CT) scan, magnetic resonance imaging (MRI), detection of cancer antigen 125 (CA-125) levels, physical examination, or the presence of symptoms suggestive of active cancer, or any combination thereof. In some embodiments, detection of cancer antigen 125 (CA-125) at levels of 35 units / ml or less indicates the absence of ovarian cancer in the individual. In some embodiments, ovarian cancer that is substantially eradicated is said to have achieved a clinical complete response (cCR). In some embodiments, ovarian cancer that is detected in a subject after the subject's ovarian cancer has previously been substantially eradicated is referred to as recurrent or relapsed ovarian cancer.

[0139] In some embodiments, the recurrence-free survival of individuals administered the autologous tumor cell vaccine is at least 5 months longer than the recurrence-free survival of individuals not administered the autologous tumor cell vaccine, regardless of BRCA1, BRCA2, or any combination thereof (i.e., mutant or non-mutant) status. In some embodiments, the recurrence-free survival of BRCA-wt individuals administered the autologous tumor cell vaccine is greater than 15 months from the time of cytoreductive surgery, and the recurrence-free survival of individuals not administered the autologous tumor cell vaccine is less than 15 months from the time of cytoreductive surgery. In some embodiments, the recurrence-free survival of BRCA-wt individuals administered the autologous tumor cell vaccine is at least 11 months longer than the recurrence-free survival of individuals not administered the autologous tumor cell vaccine.

[0140] In some embodiments, the individual has received initial therapy. In some embodiments, the cancer undergoes a clinically complete response to the therapy as a result of receiving the initial therapy. In some embodiments, the initial therapy comprises tumor debulking, chemotherapy, administration of a therapeutic agent, or a combination thereof. In some embodiments, the chemotherapy comprises a platinum agent, a taxane, or a combination thereof. In some embodiments, the platinum agent comprises cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin, tetranitrate, phenanthriplatin, picoplatin, satraplatin, or a combination thereof. In some embodiments, the platinum agent comprises carboplatin. In some embodiments, the taxane comprises paclitaxel, docetaxel, cabazitaxel, or a combination thereof. In some embodiments, the taxane comprises paclitaxel. In some embodiments, the therapeutic agent comprises an angiogenesis inhibitor, a PARP inhibitor, a checkpoint inhibitor, or a combination thereof. In some embodiments, the angiogenesis inhibitor comprises a vascular endothelial growth factor (VEGF) inhibitor. In some embodiments, the VEGF inhibitor comprises sorafenib, sunitinib, bevacizumab, pazopanib, axitinib, cabozantinib, levatinib, or a combination thereof. In some embodiments, the VEGF inhibitor is bevacizumab. In some embodiments, the PARP inhibitor comprises niraparib, olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib, or a combination thereof. In some embodiments, the PARP inhibitor is niraparib. In some embodiments, the checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or a combination thereof. In some embodiments, the checkpoint inhibitor comprises pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab, or a combination thereof. In some embodiments, the ovarian cancer is resistant or refractory to chemotherapy or a therapeutic agent.

[0141] In some embodiments, the method further comprises determining the status (i.e., wild type or mutant) of the BRCA1 gene, the BRCA2 gene, or a combination thereof in the individual. In some embodiments, determining comprises sequencing the BRCA1 gene, the BRCA2 gene, or a combination thereof. In some embodiments, sequencing comprises Sanger sequencing or next generation sequencing. In some embodiments, next generation sequencing comprises massively parallel sequencing. In some embodiments, determining comprises hybridizing nucleic acid extracted from the individual to an array. In some embodiments, the array is a microarray. In some embodiments, determining comprises array comparative genomic hybridization of nucleic acid extracted from the individual.

[0142] Administration, Formulation, and Dosage In some embodiments, the autologous tumor cell vaccine comprises approximately 1 x 10 cells transfected as described herein. 6 pieces or approximately 1 x 10 7 In some embodiments, the autologous tumor cell vaccine comprises at least 1 x 10 autologous tumor cells transfected as described herein. 6 or at least 1 x 10 7 In some embodiments, the autologous tumor cell vaccine comprises about 1 x 10 autologous tumor cells transfected as described herein. 6 ~Approx. 1×10 7 In some embodiments, the autologous tumor cell vaccine comprises about 1 x 10 autologous tumor cells transfected as described herein. 6 ~Approx. 2.5×10 7 In some embodiments, the autologous tumor cell vaccine comprises about 1 x 10 autologous tumor cells transfected as described herein. 6 ~Approx. 5×10 7 Contains autologous tumor cells.

[0143] In some embodiments, the autologous tumor cell vaccine further comprises one or more vaccine adjuvants.

[0144] In some embodiments, the autologous tumor cell vaccine is in a unit dosage form. The term "unit dosage form" is used herein to describe a physically discrete unit containing a predetermined amount of the autologous tumor cell vaccine described herein in combination with other components (e.g., a vaccine adjuvant). In some embodiments, the predetermined amount is a multiplicity of cells.

[0145] In some embodiments, an individual is administered one dose of the autologous tumor cell vaccine per month. In some embodiments, one dose of the autologous tumor cell vaccine is administered to an individual once per month for 1 month to 12 months. In some embodiments, an individual is administered at least one dose of the autologous tumor cell vaccine. In some embodiments, an individual is administered 12 or fewer doses of the autologous tumor cell vaccine. In some embodiments, an individual is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses of the autologous tumor cell vaccine. In some embodiments, the dose is a single unit dosage form of the autologous tumor cell vaccine. In some embodiments, one dose of the autologous tumor cell vaccine is administered to an individual every 3 months, every 2 months, once per month, twice per month, or three times per month. In some embodiments, the autologous tumor cell vaccine is administered to the individual within 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 18 months, 24 months, or 36 months. In some embodiments, the autologous tumor cell vaccine is administered to the individual by injection. In some embodiments, the injection is intradermal. In some embodiments, the first dose of the autologous tumor cell vaccine is administered to the individual after it has been confirmed that the individual has achieved a clinical complete response (cCR). In some embodiments, the first dose of the autologous tumor cell vaccine is not administered to the individual before the same day as the final treatment of the initial therapy. In some embodiments, the first dose of the autologous tumor cell vaccine is not administered to the individual more than 8 weeks after the final treatment of the initial therapy.

[0146] combination In some embodiments, the autologous tumor cell vaccine is administered to an individual together with an additional therapeutic agent. In some embodiments, at least one first dose of the autologous tumor cell vaccine is administered to an individual without an additional therapeutic agent, and at least one second dose of the autologous tumor cell vaccine is administered to the individual in combination with at least one dose of an additional therapeutic agent. In some embodiments, as used herein, "combined with" means that one dose of the additional therapeutic agent is administered within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks of one dose of the autologous tumor cell vaccine, or on the same day. The additional therapeutic agent can be administered before, at the same time as, or later than the autologous tumor cell vaccine.

[0147] In one representative example, an individual receives two doses of an autologous tumor cell vaccine by intradermal injection, spaced one month apart, and, beginning in the third month, receives, by intravenous infusion, both (i) 10 additional doses of the tumor cell vaccine, each spaced one month apart, and (ii) 12 doses of atezolizumab, each spaced three weeks apart.

[0148] In another representative example, an individual receives two doses of an autologous tumor cell vaccine by intradermal injection, one month apart, and, starting in month three, receives, by intravenous infusion, (i) 10 additional doses of the tumor cell vaccine, each spaced one month apart, and (ii) 10 doses of atezolizumab on the same days as the additional 10 doses of the tumor cell vaccine.

[0149] In some embodiments, administering an autologous tumor cell vaccine to an individual followed by a combination of the autologous tumor cell vaccine and an additional therapeutic agent first reduces toxicity compared to administering the additional therapeutic agent alone, in some embodiments, administering an autologous tumor cell vaccine to an individual followed by a combination of the autologous tumor cell vaccine and a checkpoint inhibitor first reduces toxicity compared to administering the checkpoint inhibitor alone.

[0150] In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses of the autologous tumor cell vaccine are administered to the individual before the autologous tumor cell vaccine is administered in combination with an additional therapeutic agent. In some embodiments, at least 2 doses of the autologous tumor cell vaccine are administered to the individual before the autologous tumor cell vaccine is administered in combination with an additional therapeutic agent.

[0151] In some embodiments, the additional therapeutic agent comprises an angiogenesis inhibitor, a PARP inhibitor, a checkpoint inhibitor, or a combination thereof. In some embodiments, the angiogenesis inhibitor comprises a vascular endothelial growth factor (VEGF) inhibitor. In some embodiments, the VEGF inhibitor comprises sorafenib, sunitinib, bevacizumab, pazopanib, axitinib, cabozantinib, levatinib, or a combination thereof. In some embodiments, the VEGF inhibitor is bevacizumab. In some embodiments, the PARP inhibitor comprises niraparib, olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib, or a combination thereof. In some embodiments, the PARP inhibitor is niraparib. In some embodiments, the checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or a combination thereof. In some embodiments, the checkpoint inhibitor comprises pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab, or a combination thereof. In some embodiments, the additional therapeutic agent is a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is atezolizumab. In some embodiments, the additional therapeutic agent is a VEGF inhibitor. In some embodiments, the VEGF inhibitor is bevacizumab. In some embodiments, the additional therapeutic agent is a PARP inhibitor. In some embodiments, the additional therapeutic agent is administered by intravenous infusion.

[0152] In some embodiments, the additional therapeutic agent comprises a therapeutically effective dose of atezolizumab. In some embodiments, the therapeutically effective dose of atezolizumab is about 900 mg to about 1500 mg, or about 1100 mg to about 1300 mg. In some embodiments, the therapeutically effective dose of atezolizumab is about 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, or 1500 mg. In some embodiments, the therapeutically effective dose of atezolizumab is about 1200 mg. In some embodiments, atezolizumab is administered by intravenous infusion.

[0153] In some embodiments, the additional therapeutic agent comprises a therapeutically effective dose of γIFN (gamma interferon). In some embodiments, the therapeutically effective dose of γIFN is about 50 μg / m 2 ~about 100 μg / m 2 In some embodiments, the therapeutically effective dose of γIFN is about 50 μg / m 2 , about 60 μg / m 2 , about 70 μg / m 2 , about 80 μg / m 2 , about 90 μg / m 2 , or approximately 100 μg / m 2 is.

[0154] In some embodiments, the expression vector or autologous tumor cell vaccine is administered with an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a therapeutically effective dose of γIFN (gamma interferon). In some embodiments, the therapeutically effective dose of γIFN is about 50 μg / m 2 ~about 100 μg / m 2 In some embodiments, the therapeutically effective dose of γIFN is about 50 μg / m 2 , about 60 μg / m 2 , about 70 μg / m 2 , about 80 μg / m 2 , about 90 μg / m 2 , or approximately 100 μg / m 2In some embodiments, the additional therapeutic agent comprises an angiogenesis inhibitor, a PARP inhibitor, a checkpoint inhibitor, or a combination thereof. In some embodiments, the angiogenesis inhibitor comprises a vascular endothelial growth factor (VEGF) inhibitor. In some embodiments, the VEGF inhibitor comprises sorafenib, sunitinib, bevacizumab, pazopanib, axitinib, cabozantinib, levatinib, or a combination thereof. In some embodiments, the VEGF inhibitor is bevacizumab. In some embodiments, the PARP inhibitor comprises niraparib, olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib, or a combination thereof. In some embodiments, the checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or a combination thereof. In some embodiments, checkpoint inhibitors comprise pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab, or combinations thereof.

[0155] manufacturing In some embodiments, the method of producing an autologous tumor cell vaccine described herein comprises the steps of: (i) aseptically harvesting one or more cancer cells from an individual; (ii) placing the harvested cells in an antibiotic solution in a sterile container; (iii) forming a cell suspension from the harvested solution, wherein the cells; (iv) forming the suspension is achieved by enzymatic comminution, mechanical division, or both; and (v) genetically modifying the cells by electroporation of the cell suspension to produce a bishRNA. フーリンThe method includes the steps of: (i) producing a vaccine having a GM-CSF / GMCSF expression vector plasmid, the vector plasmid comprising a first nucleic acid insert operably linked to a promoter, wherein the first insert encodes a GM-CSF cDNA, and a second nucleic acid insert operably linked to the promoter, wherein the second insert encodes one or more short hairpin RNAs (shRNAs) capable of hybridizing to a region of an mRNA transcript encoding furin, thereby inhibiting expression of furin through RNA interference; (vi) recovering the vaccine; (vii) irradiating the vaccine; and (viii) freezing the vaccine. In some embodiments, the antibiotic solution comprises gentamicin. In some embodiments, the one or more cancer cells are harvested from a patient suffering from ovarian cancer. In some embodiments, the genetically modified cells have been rendered proliferation-incompetent by irradiation. In some embodiments, the genetically modified cells are autologous, allogeneic, or xenograft-grown cells. In some embodiments, the method further comprises incubating the genetically modified cells with γIFN after transfection. In some embodiments, the dose of γIFN applied to the genetically modified cells after transfection is about 250 U / ml (500 U / ml at 24 hours to 100 U / ml at 48 hours). In some embodiments, the autologous tumor cell vaccine is frozen after being placed in culture medium. In some embodiments, the culture medium into which the autologous tumor cell vaccine is placed prior to freezing contains DMSO, human serum albumin (HSA), or a combination thereof. In some embodiments, the autologous tumor cell vaccine (optionally containing DMSO and HSA) is frozen to a final fill volume of 1.1 to 1.3 mL, or about 1.2 mL. In some embodiments, the autologous tumor cell vaccine is frozen at about -80°C.

[0156] Representative Embodiments The following are non-limiting embodiments of the present invention.

[0157] Embodiment 1. A method of preventing the relapse of substantially eradicated ovarian cancer in an individual in need thereof, comprising: a. a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; and b. A second insert comprising a sequence according to SEQ ID NO: 2 or 4 (SEQ ID NO: 4) administering to said individual autologous tumor cells transfected with an expression vector comprising:

[0158] Embodiment 2. The method of embodiment 1, wherein said substantially eradicated ovarian cancer is stage III or stage IV ovarian cancer.

[0159] Embodiment 3. The method of embodiment 1 or embodiment 2, wherein the individual comprises a wild-type BRCA1 gene, a wild-type BRCA2 gene, or a combination thereof.

[0160] Embodiment 4. The method of any one of embodiments 1-3, wherein the individual's recurrence-free survival (RFS) is extended compared to an individual whose ovarian cancer is substantially eradicated but who is not administered the transfected tumor cells.

[0161] Embodiment 5. The method of any one of embodiments 1-4, wherein said individual has received a first therapy.

[0162] Embodiment 6. The method of embodiment 5, wherein said initial therapy comprises cytoreductive surgery, chemotherapy, or a combination thereof.

[0163] Embodiment 7. The method of embodiment 6, wherein said chemotherapy comprises administering a platinum agent and a taxane.

[0164] Embodiment 8. The method of embodiment 7, wherein the platinum agent comprises carboplatin.

[0165] Embodiment 9. The method of embodiment 7, wherein the taxane comprises paclitaxel.

[0166] Embodiment 10. The method of any one of embodiments 1-9, wherein the GM-CSF is a human GM-CSF sequence.

[0167] Embodiment 11. The method of any one of embodiments 1 to 10, wherein the expression vector further comprises a promoter.

[0168] Embodiment 12. The method of embodiment 11, wherein said promoter is a cytomegalovirus (CMV) mammalian promoter.

[0169] Embodiment 13. The method of embodiment 12, wherein said expression vector further comprises a CMV enhancer sequence and a CMV intron sequence.

[0170] Embodiment 14. The method of any one of embodiments 11-13, wherein said first insert and said second insert are operably linked to said promoter.

[0171] Embodiment 15. The method of any one of embodiments 1 to 14, wherein the expression vector further comprises a nucleic acid sequence encoding a picornaviral 2A ribosomal skipping peptide between the first and second nucleic acid inserts.

[0172] Embodiment 16. About 1 x 10 the autologous tumor cells 6 cells ~ approx. 5 x 10 7 16. The method of any one of embodiments 1-15, wherein the cells are administered to said individual as a dose of cells.

[0173] Embodiment 17. The method of embodiment 16, wherein the autologous tumor cells are administered to the individual once a month.

[0174] Embodiment 18. The method of embodiment 17, wherein the autologous tumor cells are administered to the individual for 1 to 12 months.

[0175] Embodiment 19. The method of any one of embodiments 1-18, wherein the autologous tumor cells are administered to the individual by intradermal injection.

[0176] Embodiment 20. A method of treating BRCA1 / 2 wild-type ovarian cancer in an individual in need thereof, comprising: a. a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; and b. A second insert comprising a sequence according to SEQ ID NO: 2 or 4 (SEQ ID NO: 4) administering to said individual autologous tumor cells transfected with an expression vector comprising:

[0177] Embodiment 21. The method of embodiment 20, wherein said ovarian cancer is stage III or stage IV ovarian cancer.

[0178] Embodiment 22. The method of embodiment 20 or embodiment 21, wherein the ovarian cancer is refractory ovarian cancer.

[0179] Embodiment 23. The method of embodiment 22, wherein said refractory ovarian cancer is refractory to chemotherapy.

[0180] Embodiment 24. The method of embodiment 23, wherein said chemotherapy comprises a platinum agent or a taxane.

[0181] Embodiment 25. The method of embodiment 24, wherein the platinum agent comprises carboplatin.

[0182] Embodiment 26. The method of embodiment 24, wherein the taxane comprises paclitaxel.

[0183] Embodiment 27. The method of any one of embodiments 20-26, further comprising administering an additional therapeutic agent.

[0184] Embodiment 28. The method of embodiment 27, wherein said additional therapeutic agent is selected from the group consisting of an angiogenesis inhibitor, a PARP inhibitor, and a checkpoint inhibitor for said individual.

[0185] Embodiment 29. The method of embodiment 28, wherein the angiogenesis inhibitor is a vascular endothelial growth factor (VEGF) inhibitor.

[0186] Embodiment 30. The method of embodiment 29, wherein said VEGF is selected from the group consisting of sorafenib, sunitinib, bevacizumab, pazopanib, axitinib, cabozantinib, and levatinib.

[0187] Embodiment 31. The method of embodiment 29, wherein the VEGF is bevacizumab.

[0188] Embodiment 32. The method of embodiment 28, wherein said PARP inhibitor is selected from the group consisting of niraparib, olaparib, rucaparib, niraparib, talazoparib, veliparib, and pamiparib.

[0189] Embodiment 33. The method of embodiment 28, wherein the PARP inhibitor is niraparib.

[0190] Embodiment 34. The method of embodiment 28, wherein said checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor.

[0191] Embodiment 35. The method of embodiment 28, wherein the checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, and ipilimumab, and is administered by intravenous infusion.

[0192] Embodiment 36. The method of any one of embodiments 20-35, wherein the GM-CSF is a human GM-CSF sequence.

[0193] Embodiment 37. The method of any one of embodiments 20 to 36, wherein the expression vector further comprises a promoter.

[0194] Embodiment 38. The method of embodiment 37, wherein said promoter is a cytomegalovirus (CMV) mammalian promoter.

[0195] Embodiment 39. The method of embodiment 38, wherein said expression vector further comprises a CMV enhancer sequence and a CMV intron sequence.

[0196] Embodiment 40. The method of any one of embodiments 37-39, wherein said first insert and said second insert are operably linked to said promoter.

[0197]

[0032] Embodiment 41. The method of any one of embodiments 20 to 40, wherein the expression vector further comprises a nucleic acid sequence encoding a picornavirus 2A ribosomal skipping peptide between the first and second nucleic acid inserts.

[0198] Embodiment 42. About 1 x 10 the autologous tumor cells 6 cells ~ approx. 5 x 10 7 42. The method of any one of embodiments 20-41, wherein the cells are administered to said individual as a dose of cells.

[0199] Embodiment 43. The method of embodiment 42, wherein the autologous tumor cells are administered to the individual once a month.

[0200] Embodiment 44. The method of embodiment 43, wherein the autologous tumor cells are administered to the individual for 1 to 12 months.

[0201] Embodiment 45. The method of any one of embodiments 20-44, wherein the autologous tumor cells are administered to the individual by intradermal injection.

[0202] Embodiment 46. A method of preventing or treating the recurrence of substantially eradicated ovarian cancer in an individual in need thereof, comprising: ai a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; and ii. A second insert comprising a sequence according to SEQ ID NO: 2 or 4 (SEQ ID NO: 4) at least one first dose of an autologous tumor cell vaccine comprising autologous tumor cells transfected with an expression vector comprising b. A method comprising administering to said individual at least one second dose of said autologous tumor cell vaccine in combination with at least one dose of an additional therapeutic agent.

[0203] Embodiment 47. The method of embodiment 46, wherein said substantially eradicated ovarian cancer is stage III or stage IV ovarian cancer.

[0204] Embodiment 48. The method of embodiment 46 or embodiment 47, wherein the individual comprises a wild-type BRCA1 gene, a wild-type BRCA2 gene, or a combination thereof.

[0205] Embodiment 49. The method of any one of embodiments 46-48, wherein the individual's recurrence-free survival (RFS) is extended compared to an individual whose ovarian cancer is substantially eradicated but who has not been administered the transfected tumor cells.

[0206] Embodiment 50. The method of any one of embodiments 46-49, wherein said individual has received a first therapy.

[0207] Embodiment 51. The method of embodiment 50, wherein said initial therapy comprises cytoreductive surgery, chemotherapy, or a combination thereof.

[0208] Embodiment 52. The method of embodiment 51, wherein said chemotherapy comprises administering a platinum agent and a taxane.

[0209] Embodiment 53. The method of embodiment 52, wherein the platinum agent comprises carboplatin.

[0210] Embodiment 54. The method of embodiment 52, wherein the taxane comprises paclitaxel.

[0211] Embodiment 55. The method of any one of embodiments 46-54, wherein the GM-CSF is a human GM-CSF sequence.

[0212] Embodiment 56. The method of any one of embodiments 46 to 55, wherein the expression vector further comprises a promoter.

[0213] Embodiment 57. The method of embodiment 56, wherein said promoter is a cytomegalovirus (CMV) mammalian promoter.

[0214] Embodiment 58. The method of embodiment 57, wherein said expression vector further comprises a CMV enhancer sequence and a CMV intron sequence.

[0215] Embodiment 59. The method of any one of embodiments 56-58, wherein said first insert and said second insert are operably linked to said promoter.

[0216]

[0032] Embodiment 60. The method of any one of embodiments 46-59, wherein the expression vector further comprises a nucleic acid sequence encoding a picornavirus 2A ribosomal skipping peptide between the first and second nucleic acid inserts.

[0217] Embodiment 61. The method of any one of embodiments 46-60, wherein the additional therapeutic agent is selected from the group consisting of an angiogenesis inhibitor, a PARP inhibitor, and a checkpoint inhibitor for the individual.

[0218] Embodiment 62. The method of embodiment 61, wherein the angiogenesis inhibitor is a vascular endothelial growth factor (VEGF) inhibitor.

[0219] Embodiment 63. The method of embodiment 62, wherein said VEGF is selected from the group consisting of sorafenib, sunitinib, bevacizumab, pazopanib, axitinib, cabozantinib, and levatinib.

[0220] Embodiment 64. The method of embodiment 62, wherein the VEGF is bevacizumab.

[0221] Embodiment 65. The method of embodiment 61, wherein said PARP inhibitor is selected from the group consisting of niraparib, olaparib, rucaparib, niraparib, talazoparib, veliparib, and pamiparib.

[0222] Embodiment 66. The method of embodiment 61, wherein the PARP inhibitor is niraparib.

[0223] Embodiment 67. The method of embodiment 61, wherein said checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor.

[0224] Embodiment 68. The method of embodiment 61, wherein the checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, and ipilimumab.

[0225] Embodiment 69. The method of embodiment 61, wherein the checkpoint inhibitor is atezolizumab.

[0226] Embodiment 70. The method of any one of embodiments 46-69, wherein said at least one dose of said additional therapeutic agent is 1100 mg to 1300 mg.

[0227] Embodiment 71. The at least one first dose of the autologous tumor cell vaccine is about 1 x 10 6 cells ~ approx. 5 x 10 7 71. The method of any one of embodiments 46-70, comprising cells.

[0228] Embodiment 72. The at least one second dose of the autologous tumor cell vaccine is about 1 x 10 6 cells ~ approx. 5 x 10 7 72. The method of any one of embodiments 46-71, comprising cells.

[0229] Embodiment 73. The method of any one of embodiments 46-72, wherein said at least one first dose of said autologous tumor cells is administered to said individual by intradermal injection.

[0230] Embodiment 74. The method of any one of embodiments 46-73, wherein said at least one second dose of said autologous tumor cells is administered to said individual by intradermal injection.

[0231] Embodiment 75. The method of any one of embodiments 46-74, wherein said at least one dose of said additional therapeutic agent is administered to said individual by intravenous infusion.

[0232] Embodiment 76. The method of any one of embodiments 46-75, wherein said at least one first dose of said autologous tumor cell vaccine comprises two doses.

[0233] Embodiment 77. The method of any one of embodiments 46-76, wherein each dose of said at least one first dose of said autologous tumor cell vaccine is administered to said individual once a month.

[0234] Embodiment 78. The method of any one of embodiments 46-77, wherein each dose of said at least one second dose of said autologous tumor cell vaccine is administered to said individual once a month.

[0235] Embodiment 79. The method of any one of embodiments 46-78, wherein each dose of said at least one dose of said additional therapeutic agent is administered to said individual at least once per month.

[0236] Embodiment 80. The method of any one of embodiments 46-79, wherein the at least one first dose of the autologous tumor cell vaccine and the at least one second dose of the autologous tumor cell vaccine comprise a total of at least 12 doses.

[0237] Embodiment 81. A method of treating BRCA1 / 2 wild-type ovarian cancer in an individual in need thereof, comprising: ai a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; and ii. A second insert comprising a sequence according to SEQ ID NO: 2 or 4 (SEQ ID NO: 4) at least one first dose of an autologous tumor cell vaccine comprising autologous tumor cells transfected with an expression vector comprising b. A method comprising administering to said individual at least one second dose of said autologous tumor cell vaccine in combination with at least one dose of an additional therapeutic agent.

[0238] Embodiment 82. The method of embodiment 81, wherein said ovarian cancer is stage III or stage IV ovarian cancer.

[0239] Embodiment 83. The method of embodiment 81 or embodiment 82, wherein the ovarian cancer is refractory ovarian cancer.

[0240] Embodiment 84. The method of embodiment 83, wherein said refractory ovarian cancer is refractory to chemotherapy.

[0241] Embodiment 85. The method of embodiment 84, wherein said chemotherapy comprises a platinum agent or a taxane.

[0242] Embodiment 86. The method of embodiment 85, wherein the platinum agent comprises carboplatin.

[0243] Embodiment 87. The method of embodiment 85, wherein the taxane comprises paclitaxel.

[0244] Embodiment 88. The method of any one of embodiments 81-87, wherein the GM-CSF is a human GM-CSF sequence.

[0245] Embodiment 89. The method of any one of embodiments 81-88, wherein said expression vector further comprises a promoter.

[0246] Embodiment 90. The method of embodiment 89, wherein said promoter is a cytomegalovirus (CMV) mammalian promoter.

[0247] Embodiment 91. The method of embodiment 90, wherein said expression vector further comprises a CMV enhancer sequence and a CMV intron sequence.

[0248] Embodiment 92. The method of any one of embodiments 89-91, wherein said first insert and said second insert are operably linked to said promoter.

[0249]

[0033] Embodiment 93. The method of any one of embodiments 81-92, wherein the expression vector further comprises a nucleic acid sequence encoding a picornaviral 2A ribosomal skipping peptide between the first and second nucleic acid inserts.

[0250] Embodiment 94. The method of any one of embodiments 81-93, wherein the additional therapeutic agent is selected from the group consisting of an angiogenesis inhibitor, a PARP inhibitor, and a checkpoint inhibitor for the individual.

[0251] Embodiment 95. The method of embodiment 94, wherein said angiogenesis inhibitor is a vascular endothelial growth factor (VEGF) inhibitor.

[0252] Embodiment 96. The method of embodiment 95, wherein said VEGF is selected from the group consisting of sorafenib, sunitinib, bevacizumab, pazopanib, axitinib, cabozantinib, and levatinib.

[0253] Embodiment 97. The method of embodiment 95, wherein the VEGF is bevacizumab.

[0254] Embodiment 98. The method of embodiment 94, wherein said PARP inhibitor is selected from the group consisting of niraparib, olaparib, rucaparib, niraparib, talazoparib, veliparib, and pamiparib.

[0255] Embodiment 99. The method of embodiment 94, wherein the PARP inhibitor is niraparib.

[0256] Embodiment 100. The method of embodiment 94, wherein said checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor.

[0257] Embodiment 101. The method of embodiment 94, wherein said checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, and ipilimumab.

[0258] Embodiment 102. The method of embodiment 94, wherein said checkpoint inhibitor is atezolizumab.

[0259] Embodiment 103. The method of any one of embodiments 81-102, wherein said at least one dose of said additional therapeutic agent is 1100 mg to 1300 mg.

[0260] Embodiment 104. The at least one first dose of the autologous tumor cell vaccine is about 1 x 10 6 cells ~ approx. 5 x 10 7 104. The method of any one of embodiments 81-103, comprising cells.

[0261] Embodiment 105. The at least one second dose of the autologous tumor cell vaccine is about 1 x 10 6 cells ~ approx. 5 x 10 7 105. The method of any one of embodiments 81-104, comprising cells.

[0262] Embodiment 106. The method of any one of embodiments 81-105, wherein said at least one first dose of said autologous tumor cells is administered to said individual by intradermal injection.

[0263] Embodiment 107. The method of any one of embodiments 81-106, wherein said at least one second dose of said autologous tumor cells is administered to said individual by intradermal injection.

[0264] Embodiment 108. The method of any one of embodiments 81-107, wherein said at least one dose of said additional therapeutic agent is administered to said individual by intravenous infusion.

[0265] Embodiment 109. The method of any one of embodiments 81-108, wherein said at least one first dose of said autologous tumor cell vaccine comprises two doses.

[0266] Embodiment 110. The method of any one of embodiments 81-109, wherein each dose of said at least one first dose of said autologous tumor cell vaccine is administered to said individual once a month.

[0267] Embodiment 111. The method of any one of embodiments 81-110, wherein each dose of said at least one second dose of said autologous tumor cell vaccine is administered to said individual once a month.

[0268] Embodiment 112. The method of any one of embodiments 81-111, wherein each dose of said at least one dose of said additional therapeutic agent is administered to said individual at least once a month.

[0269] Embodiment 113. The method of any one of embodiments 81-112, wherein the at least one first dose of the autologous tumor cell vaccine and the at least one second dose of the autologous tumor cell vaccine comprise a total of at least 12 doses.

[0270] Embodiment 114. A method of treating cancer in an individual in need thereof, comprising: a. a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; and b. A second insert comprising a sequence according to SEQ ID NO: 2 or 4 (SEQ ID NO: 4) administering to the individual an expression vector comprising The method, wherein the individual contains a wild-type BRCA1 gene, a wild-type BRCA2 gene, or a combination thereof, and is identified as homologous recombination repair deficient (HRD) negative.

[0271] Embodiment 115. The method of embodiment 114, wherein said GM-CSF is a human GM-CSF sequence.

[0272] Embodiment 116. The method of embodiment 114, wherein said expression vector further comprises a promoter.

[0273] Embodiment 117. The method of embodiment 116, wherein said promoter is a cytomegalovirus (CMV) mammalian promoter.

[0274] Embodiment 118. The method of embodiment 114, wherein said expression vector further comprises a CMV enhancer sequence and a CMV intron sequence.

[0275] Embodiment 119. The method of embodiment 114, wherein said first insert and said second insert are operably linked to said promoter.

[0276] Embodiment 120. The method of embodiment 114, wherein said expression vector further comprises a nucleic acid sequence encoding a picornavirus 2A ribosomal skipping peptide between said first and said second nucleic acid inserts.

[0277] Embodiment 121. The method of embodiment 114, wherein said cancer is an HRD-negative wild-type BRCA1 / 2 cancer.

[0278] Embodiment 122. The method of embodiment 114, wherein the cancer is selected from the group consisting of solid tumor cancer, ovarian cancer, adrenocortical carcinoma, bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer, glioblastoma, glioma, hepatocellular carcinoma, head and neck cancer, kidney cancer, leukemia, lymphoma, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, prostate cancer, sarcoma, gastric cancer, uterine cancer, thyroid cancer, and blood cancer.

[0279] Embodiment 123. The method of embodiment 122, wherein said solid tumor cancer is selected from the group consisting of endometrial cancer, bile duct cancer, bladder cancer, hepatocellular carcinoma, gastric / esophageal cancer, ovarian cancer, melanoma, breast cancer, pancreatic cancer, colorectal cancer, glioma, non-small cell lung cancer, prostate cancer, cervical cancer, kidney cancer, thyroid cancer, neuroendocrine cancer, small cell lung cancer, sarcoma, head and neck cancer, brain cancer, renal clear cell carcinoma, skin cancer, endocrine tumors, thyroid cancer, tumors of unknown primary, and gastrointestinal stromal tumors.

[0280] Embodiment 124. The method of embodiment 122, wherein the cancer is ovarian cancer.

[0281] Embodiment 125. The method of embodiment 124, wherein the relapse of substantially eradicated ovarian cancer is prevented or delayed.

[0282] Embodiment 126. The method of embodiment 125, wherein said substantially eradicated ovarian cancer is stage III or stage IV ovarian cancer.

[0283] Embodiment 127. The method of embodiment 122, wherein the cancer is breast cancer.

[0284] Embodiment 128. The method of embodiment 122, wherein the cancer is melanoma.

[0285] Embodiment 129. The method of embodiment 122, wherein the cancer is lung cancer.

[0286] Embodiment 130. The method of embodiment 114, wherein the expression vector is in an autologous cancer cell that is transfected with the expression vector.

[0287] Embodiment 131. About 1 x 10 the autologous tumor cells 6 cells ~ approx. 5 x 10 7 131. The method of embodiment 130, wherein the cells are administered to the individual as a dose of cells.

[0288] Embodiment 132. The method of embodiment 131, wherein the autologous tumor cells are administered to the individual once a month.

[0289] Embodiment 133. The method of embodiment 131, wherein the autologous tumor cells are administered to the individual for 1 to 12 months.

[0290] Embodiment 134. The method of embodiment 114, wherein the autologous tumor cells are administered to the individual by intradermal injection.

[0291] Embodiment 135. The method of embodiment 114, wherein said individual has received a first therapy.

[0292] Embodiment 136. The method of embodiment 135, wherein said initial therapy comprises cytoreductive surgery, chemotherapy, or a combination thereof.

[0293] Embodiment 137. The method of embodiment 135, wherein said chemotherapy comprises administering a platinum agent and a taxane.

[0294] Embodiment 138. The method of embodiment 137, wherein the platinum agent comprises carboplatin.

[0295] Embodiment 139. The method of embodiment 137, wherein the taxane comprises paclitaxel.

[0296] Embodiment 140. The method of any one of embodiments 1-139, further comprising administering an additional therapeutic agent.

[0297] Embodiment 141. The method of embodiment 140, wherein said additional therapeutic agent is a member selected from the group consisting of an angiogenesis inhibitor, a PARP inhibitor, and a checkpoint inhibitor administered to said individual. [Example]

[0298] Example 1 - Recurrence-Free Survival and Overall Survival Benefit in BRCA1 / 2 Wild-Type Frontline Stage III / IV Ovarian Cancer with Vigil® Due to the limitations of frontline treatment for advanced ovarian cancer, the relationship between high TGF-β expression and immune suppression in ovarian cancer, and limited treatment options in BRCA1 / 2-wt patients, a study was initiated to investigate the use of Vigil® as frontline maintenance therapy in patients with ovarian cancer.

[0299] Materials and Methods Study design and treatment This phase 2b, double-blind study comparing Vigil® with placebo included 25 centers. Patients with stage III / IV high-grade serous ovarian cancer who achieved a clinical complete response (cCR) after surgery and a combination of chemotherapy including carboplatin and paclitaxel were included. Enrolled patients could have received either primary cytoreductive surgery followed by adjuvant chemotherapy or neoadjuvant chemotherapy followed by interval cytoreductive surgery and adjuvant chemotherapy. Tissue / blood germline and / or somatic BRCA1 / 2 molecular profiling was collected and analyzed (Ocean Ridge Biosciences, Deerfield Beach, FL). Patients received 1 × 10 7 Intradermal injections of cells / Vigil® or placebo were administered monthly (within 8 weeks of the end of the last chemotherapy treatment) for up to 12 doses. Treatment continued until disease recurrence or treatment supplies were exhausted. Toxicity was assessed using CTCAE v 4.03.

[0300] patient Women with histologically confirmed stage III or IV high-grade serous ovarian cancer (HGSC) who achieved a cCR after combined surgery and chemotherapy were included in the study.

[0301] Relationship between tumor collection and manufacturing Gradalis, Inc. (Carrollton, Texas) manufactured Vigil® from the harvested tumor tissue. A placebo (frozen medium) was manufactured at the same dose based on the number of vials of Vigil®. 10-30 grams of tissue was required for vaccine production. Lesions extending into the intestinal lumen were excluded (risk of bacterial infection).

[0302] Manufacturing of investigational drugs and placebos Surgically resected tumor tissue was harvested, cut into 1 / 4-inch pieces, placed in specimen containers supplemented with gentamicin (Fresenius Kabi), and packed on ice for overnight transport. On day 1, transport media and tumor specimens were tested for sterility (BacT / Alert 3D Microbial Identification System, BioMérieux). Tumor tissue was dissected with a scalpel, dissociated, enzymatically dissociated (type I collagenase solution), and incubated at 37°C to facilitate the formation of a single-cell suspension. The concentration of this suspension was adjusted to 40 million cells / mL and electroporated using a Gene Pulser XL (BioRad) to facilitate plasmid insertion.

[0303] On day 2, overnight cultures were harvested and resuspended in fresh X-VIVO medium. A QC sample and a minimum of four doses per patient were required before proceeding. Cells were washed with PlasmaLyte (Baxter) supplemented with 1% human serum albumin (HSA) (Octapharma), and a QC sample was removed. Cells were placed in freezing medium consisting of 10% DMSO (dimethyl sulfoxide; Cryoserv USP; Mylan), 1% HSA (Octapharma) in PlasmaLyte (Baxter) at pH 7.4, and aseptically placed at 1 × 10 cells per vial in a sterile 2.0 mL borosilicate glass vial (Algroup Wheaton Pharmaceutical and Cosmetics Packaging). 7The vials were dispensed at 1.2 cells / mL and sealed with butyl rubber stoppers coated with Flurotec® barrier files (West Pharmaceutical Services) to a final fill volume of 1.2 mL. The final vials were frozen at a controlled rate using CoolCell® freezing containers (Biocision) and placed in a -80°C freezer (Sanyo).

[0304] The placebo consisted of a freezing medium consisting of PlasmaLyte (Baxter) at pH 7.4 containing 10% DMSO (Cryoserv USP; Mylan) and 1% HSA (Octapharma). The medium was slowly cooled to -80°C and frozen, after which the vials were stored in vapor phase liquid nitrogen pending release testing for sterility and endotoxin. The placebo vial product matched the available product dose manufactured for the subjects.

[0305] Disease evaluation Subjects continued treatment until disease recurrence or the subject's supply of Vigil® or placebo was exhausted. Disease recurrence was assessed by WorldCare Clinical (WCC) (Boston, MA) using Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1).

[0306] Endpoints and statistical evaluation The primary endpoint was recurrence-free survival (RFS) from randomization comparing Vigil® with placebo. All planned statistical analyses were performed independently before unblinding (Stat Beyond Consulting, Irvine, CA). Secondary endpoints, in order of priority, included BRCA-wt RFS from time of harvest, BRCA-wt RFS from time of randomization, all-patient RFS from time of harvest, all-patient OS from time of randomization, and all-patient OS from time of harvest. BRCA-wt OS from time of randomization and harvest was a preplanned subanalysis. All patients (who received at least one dose of Vigil® or placebo) were included in the safety analysis. For protocol compliance, a one-sided p-value of 0.05 or less (stratified log-rank test) was considered to indicate statistical significance in the analysis. Based on sample size calculations, a total of 54 events were required for this analysis. The hazard ratio for RFS was calculated based on the randomization stratification factor. Cox proportional hazards models were used to assess RFS stratification by residual disease (macroscopic / microscopic) and frontline chemotherapy (neoadjuvant / adjuvant). The Kaplan-Meier method was used to assess RFS distribution. Planned subgroup analyses were performed using forest plots for all patients and BRCA-wt patients. Subgroups included number of injections, age, ECOG, BRCA-wt / m, race, stage, and manufacturing criteria (TGFβ1, GMCSF, and survival). P values ​​for baseline demographic variables were calculated using two-tailed Fisher's exact test for categorical data and two-tailed t-tests for continuous data without multiplicity adjustment. The Grambsch and Therneau test was used to assess the proportional hazards estimates for the stratified Cox models.

[0307] result patient Between February 2015 and March 2017, 310 patients were consented at 22 centers. 128 patients were deemed ineligible due to histology or screening dropout parameters. Of the remaining 181 patients who underwent randomization, 92 subjects (56%) were successfully consented and randomized, but one patient withdrew after randomization and prior to treatment for personal reasons of good health. 17 subjects did not sign consent for randomization, and 72 did not meet the product release criteria (65 had insufficient cells). 91 subjects received Vigil® (n=46) or placebo (n=45) and were analyzed for safety and efficacy. 67 subjects were BRCA-wt (Vigil®: 39 BRCA-wt, placebo: 28 BRCA-wt) and 24 were BRCA-m. Fifty-five recurrent events were observed in this study by an independent third-party radiology reviewer (WCC) through August 2019. Demographics are shown in Table 2. No significant differences were detected between cohorts, with the exception of 45.7% of Vigil® patients with an ECOG performance status of 1 compared with 20% for placebo. [Table 2-1] [Table 2-2]

[0308] Effectiveness The primary endpoint of median RFS, calculated from the time of randomization for all patients, was 12.7 vs. 8.4 months for Vigil® vs. placebo (HR 0.67, 90% CI [0.432-1.042], one-sided p=0.065). RFS from the time of surgery / harvesting was 18.3 vs. 15.9 months (Vigil® vs. placebo), HR 0.63 (90% CI [0.403-0.971], one-sided p=0.038) (Figures 6A-6B). Recurrence rates were 54% vs. 76% for Vigil® vs. placebo (Fisher's exact probability p=0.048). Furthermore, the 1-year RFS rate from the time of randomization was 51% for Vigil® versus 38% for placebo (p 0.13, one-sided Z-test), and the 2-year RFS rate was 33% for Vigil® versus 25% for placebo (p 0.22, one-sided Z-test). The median follow-up from the first dose of gemogenovatucel-T was 38.5 months versus 38.6 months for placebo. Both PP and ITT analyses revealed similar results. The median time from surgery to randomization was 7.0 versus 6.7 months, and the median time from the end of chemotherapy to the start of treatment was 1.6 versus 1.5 months (gemogenovatucel-T and placebo, respectively).

[0309] Stratification by BRCA status demonstrated improvement in Vigil® BRCA-wt patients. Median RFS from randomization in Vigil® BRCA-wt patients (Figure 6C) was 12.7 months compared with 8.0 months for placebo (HR 0.493, 90% CI [0.287-0.846], one-sided p=0.014), and from the time of surgery / harvesting was 19.4 months for Vigil® and 14.4 months for placebo (HR 0.459, 90% CI [0.268-0.787], one-sided p=0.007) (Figure 6D). Disease relapse occurred in 51% of Vigil® BRCA-wt1 / 2 patients compared with 79% of placebo BRCA1 / 2-wt patients (Fisher's exact probability p=0.039). An overall survival benefit was observed in planned subanalyses of BRCA-wt patients receiving Vigil® compared with placebo from the time of randomization and surgery / harvesting (Figures 6E-6F). Analyses of RFS and survival calculated from the time of randomization and surgery / harvesting revealed similar results. For BRCA-m patients, median RFS from randomization was 10.5 months with Vigil® and 13.3 months with placebo. Preliminary study demographics (planned subanalyses) related to disease effect, including prespecified stratification (residual disease, chemotherapy schedule) and product release criteria, are shown in Figures 7A-7B to explore the impact of BRCA-wt versus all patients on RFS from the time of randomization.

[0310] The secondary endpoint of median RFS from the time of surgery / harvesting in BRCA-m patients was compared between Vigil® and placebo: 19.4 months for gemogenovatucel-T and 14.4 months for placebo (HR 0.459, 90% CI [0.268-0.787], one-sided p 0.007) (Figure 6D). At the time of efficacy assessment, 51% of Vigil® BRCA1 / 2-wt patients had relapsed disease compared with 79% of placebo BRCA-wt patients (p 0.0195, one-sided Fisher's exact t-test). The 1-year RFS rate from the time of surgery / harvesting was 80% for Vigil® BRCA-wt patients compared with 64% for placebo BRCA-wt patients (p 0.075, one-sided Z-test), and the 2-year RFS rate was 43% for Vigil® compared with 23% for placebo (p 0.5, one-sided Z-test). From randomization, the median RFS for Vigil® BRCA-wt patients was 12.7 months compared with 8.0 months for placebo (HR 0.493, 90% CI [0.287-0.846], one-sided p 0.014) (Figure 6C). In BRCA-wt patients, the 1-year RFS rate from the time of randomization was 52% for Vigil® versus 27% for placebo (p 0.02, one-sided Z-test), and the 2-year RFS rate was 34% for Vigil® versus 13% for placebo (p 0.035, one-sided Z-test). Analysis of RFS calculated from the time of randomization versus surgery / harvesting revealed similar results (Figure 5A-D). For all patients, the median RFS from the time of surgery / harvesting was 18.3 versus 15.9 months (Vigil® vs. placebo), with a HR of 0.63 (90% CI [0.403-0.971], one-sided p 0.038) (Figure 6B).

[0311] A sensitivity analysis based on RMST was performed comparing Vigil® with placebo for BRCA-wt patients, and the results were consistent with those based on the stratified log-rank test. The RMST difference between Vigil® and placebo was 7.8 months (p 0.021) for RFS and 7.1 months (p 0.020) for OS from randomization for BRCA-wt patients. A cutoff point equal to the minimum of the longest follow-up time in either group was used in the RMST analysis.

[0312] safety A median of six Vigil® (range 1-12) or six placebo (range 3-12) injections was administered per patient. One treatment delay in a placebo patient was due to a documented pelvic infection, likely related to the study drug. Two placebo patients experienced grade 3 related toxicity; no grade 3 treatment-related adverse events were observed with Vigil®. Nine percent of reported adverse events were grade 2 or 3 adverse events with Vigil® compared with 2.9% with placebo. Eleven serious adverse events (SAEs) were reported in seven patients (four placebo; three Vigil®). All but one were likely unrelated or unrelated to study treatment.

[0313] Product shipping results Eighty-four (92%) subjects met all product release criteria. Median harvested tumor mass was 52 grams (range 8-137 grams). Median survival at product release was 88% (72-98%, n=91). Seven of the 91 patients (8%, 5 Vigil®, RFS randomized 7.4 months / 2 placebo) did not demonstrate a sufficient increase in GMCSF expression after plasmid transfer. However, six of these seven patients (6 / 7) demonstrated sufficient TGFβ1 knockdown. Additional product release results are shown in Table 3. Median evaluable GMCSF production (pg / 10 6The total number of cells (n = 84) was 860 (36-37669), with a median evaluable TGFβ1 knockdown of 100% (66-100%, n = 84). TGFβ1 knockdown results were equivocal in seven patients (six with adequate GMCSF expression, three with Vigil®, and a randomized RFS of 5.6 months / four with placebo), but 78 patients (86%) had TGFβ1 knockdown of 90% or greater, demonstrating robust activity associated with Furin bi-shRNAi knockdown. However, there was no significant difference in RFS. Baseline TGFβ1 production before plasmid transfection was detectable in all patients, suggesting a median TGFβ1 expression of 164 pg / 10 6 In both groups, BRCA1 / 2-wt TGFβ1 expression was found to be a median of 181 (mean 249, range 60–662) pg / 10 cells (mean 241, range 52–882). 6 The median BRCA1 / 2-m TGFβ1 level was 146 (mean 219, range 52-882) pg / 10 6 It was revealed that the number of cells was 100 (Table 4). [Table 3] [Table 4]

[0314] Effectiveness Of 91 evaluable patients, 62 patients had a BRCA1 / 2 molecular profile by local site analysis. The subgroup of BRCA1 / 2-wt patients demonstrated an RFS benefit in the Vigil® cohort compared with the placebo cohort. Results are shown in Figures 2A-2B. Median RFS, calculated from the time of surgery / harvesting, was not reached for BRCA-wt ovarian patients in the Vigil® cohort compared with 14.8 months in the placebo cohort (HR = 0.49, 90% CI, 0.25-0.97, one-sided p = 0.038). Similarly, when RFS was calculated from the time of randomization, median RFS in the BRCA-wt subgroup was 19.4 months in the Vigil® cohort compared with 8.0 months in the placebo group (HR = 0.51, 90% CI, 0.26-1.01, one-sided p = 0.050). Thirty-eight percent of BRCA1 / 2-wt patients treated with Vigil® exhibited relapsed disease compared to 71% of placebo BRCA1 / 2-wt patients (chi-squared = 0.021) (Figure 3A). 62% of Vigil® patients remained BRCA-wt and remained relapse-free to date (October 31, 2019).

[0315] RFS calculated from the time of cytoreductive surgery and the time of randomization, regardless of BRCA1 / 2 status, is shown in Figures 2C-2D. Median RFS calculated from the time of randomization was 13.67 months (416 days) in the Vigil® cohort compared with 8.38 months (255 days) in the placebo cohort (HR = 0.69, one-sided log-rank p = -.054). RFS calculated from the time of cytoreductive surgery improved, with a HR of 0.64 (one-sided p = 0.054). Relapse was reported in 48% of patients treated with Vigil® compared with 73% of placebo patients (chi-square p = 0.013) (Figure 3B). Other factors associated with a trend toward benefit for Vigil® response were younger age ≤65 years (p 0.057), late stage IIIb-IV disease (p 0.075), and microscopic residual disease (<10 mm) / no evidence of disease (p=0.066) (Figure 4).

[0316] Consideration BRCA1 / 2-wt patients demonstrated a significant RFS advantage (calculated from the time of cytoreductive surgery) with Vigil® over placebo (not reached vs. 14.8 months; HR=0.49, one-sided p=0.038) and had a lower incidence of post-treatment relapse (38% vs. 71%, p=0.021). These results supported Vigil® being considered maintenance in patients with BRCA1 / 2-wt ovarian cancer after complete cytoreductive surgery and adjuvant or neoadjuvant chemotherapy. Safety analyses demonstrated no evidence of toxic effects with Vigil® over placebo.

[0317] Increasing evidence suggests that GMCSF is involved in increasing tumor antigen presentation by dendritic cells (DCs). GMCSF has been shown to induce a subset of DCs that are superior at phagocytosis of apoptotic tumor cells. GMCSF induces higher levels of costimulatory molecules (which are characteristic of greater functional maturation and more efficient T cell stimulation), thereby expanding the arsenal of lymphocyte effector mechanisms induced by DCs. GMCSF also promotes lipid antigen presentation by DCs, which in turn leads to the activation of natural killer T cells (NKT cells), a population of lymphocytes that may be important in both innate and therapeutic responses to tumors. DCs initiate antigen-specific immune responses and express a variety of receptors that enable antigen recognition and capture in peripheral tissues such as the dermis. DCs efficiently process this material, directing it down the MHC class I and II presentation pathway, upregulating costimulatory molecules upon maturation, and trafficking to secondary lymphoid tissues, where they present antigens to T cells. Vigil® GMCSF protein was upregulated, 1806 / 826 pg / 10 6 This resulted in a robust mean / median of 100 cells. In 84 of the 91 (91%) patients in whom this was achieved, this was related to GMCSF plasmid transfer alone.

[0318] It has also been previously shown that expression of TGF-β in ovarian malignant cells is significantly higher in malignant ovarian cancer tissues than in non-malignant ovarian cancer tissues. A gene expression meta-analysis of over 1,500 ovarian cancer patients identified high- and low-risk groups based on the expression of several genes, and the results were validated by IHC or qRT-PCR. Pathway analysis of gene signatures demonstrated enrichment of TGF-β in patients with poor prognosis. TGF-β signals are transmitted through binding to the serine / threonine kinase receptors TGF-βRI and II, leading to the phosphorylation and activation of the intracellular effectors Smad2 and Smad3. Smad2 / 3 form a transcriptional complex with Smad4, translocate to the nucleus, and regulate the expression of TGF-β-regulated genes. TGF-β is involved in the progression of non-invasive serous ovarian tumors to invasive serous ovarian tumors. TGF-β can be activated by both Smad-dependent and Smad-independent pathways and is thought to promote tumor metastasis in ovarian cancer. A correlation between overexpression of TGFβ and tumor cell proliferation and metastasis has also been described in prostate, colon, and renal cell carcinomas.

[0319] TGF-β secreted by ovarian cancer cells induces the proliferation of immunosuppressive regulatory T cells (Tregs) (CD4+CD25+) in the tumor microenvironment. This has been shown to be associated with poor prognosis in patients with frontline-treated high-grade serous ovarian cancer. TGF-β inhibits the maturation of bone marrow-derived dendritic cells (DCs) and the induction of GMCSF-induced expression of MHC class II molecules and costimulatory molecules. TGF-β inhibits activated macrophages, including their antigen-presenting function, and inhibits the expression of PD-L1 ligands by ovarian cancer and tumor-associated myeloid cells, which is further associated with poor overall survival in ovarian cancer. Evidence reveals that modulation of associated antitumor immunity can restore TGF-β-related immune surveillance.

[0320] TGF-β1 has also been shown to downregulate the miR181 / BRCA1 axis, thereby modulating DNA repair dysregulation and inducing "BRCA-ness" in breast cancers with wild-type BRCA genes. The term "BRCA-ness" has been used to identify sporadic tumors with clinicopathological and molecular features similar to those associated with BRCA1 / 2 germline mutations (DNA repair inhibition). In addition, increased TGF-β signaling has been demonstrated in low-HRD tumors, which can induce NF-κB activation and elicit antitumor immune responses in lymphocytes. Therefore, in addition to BRCA molecular signaling, homologous recombination repair dysregulation (HRD) scores may also be relevant in predicting outcome and response to TGF-β-modulating immunotherapy.

[0321] The improved RFS results in the BRCA-wt population in this study further support the relationship between TGFβ inhibition and immune responsiveness, thereby supporting the use of Vigil® in BRCA-wt patients and other cancer subpopulations, possibly with other histological types, that have high TGFβ expression (e.g., prostate cancer, renal cell carcinoma, and colorectal cancer). High levels of furin mRNA and protein are also widely expressed in ovarian cancer and many other human tumors where gene expression differs between malignant (high) and non-malignant (low) cell populations. Expression of furin, a converting enzyme of TGFβ1 and TGFβ2, appears to be inversely correlated with survival and likely contributes significantly to the maintenance of TGFβ-mediated peripheral immune tolerance toward tumors. Downregulation of furin using bi-shRNAi induced a significant reduction in TGFβ1 expression, as validated in this study. Notably, 73% of the constructed Vigil® vaccines had 90% or greater TGFβ1 knockdown compared to untransfected tumor cells from the same patients.

[0322] BRCA-wt, HRD-low ovarian cancers also exhibit elevated tumor-infiltrating lymphocyte (TIL) rates, high PD-L1 expression, high type 1 IFN-gamma signaling activity in mononuclear cells, and high perforin-1 expression in the tumor microenvironment, which may suggest a generally improved immune response. However, several attempts to enhance both frontline and relapsed / refractory ovarian responses to checkpoint inhibitor therapy have failed. Hypothetically, this may also be related to the high TGF-β suppressive effect within the local tumor microenvironment, as previously described.

[0323] PARP inhibitors not only block the catalytic activity of PARP proteins on the surface of DNA, but also bind to and capture the protein, leading to numerous double-stranded DNA breaks and ultimately cell death. Lynparza® (olaparib) was one of the first PARP inhibitors approved for patients with high-grade serous ovarian cancer who harbor a germline BRCA mutation. Previous studies have shown improved progression-free survival in BRCA1 / 2-mu ovarian cancer (HR 0.3, p<0.0001). A recent long-term follow-up study (Study 19) of olaparib in patients with platinum-sensitive recurrent ovarian cancer showed favorable OS results, with an HR of 0.73 (p = 0.0138) for all patients enrolled. However, activity appeared to be limited to the BRCA-mu population, with an HR of 0.62 (p = 0.02140), in contrast to the BRCA-wt population, which showed no benefit, with an HR of 0.84 (p = 0.34) (82). However, some BRCA-wt patients with relapsed or refractory disease and homologous recombination repair deficiency (HRD) may benefit from PARP inhibitors. Recent reports have shown that some HRD-high tumors are associated with a better response to niraparib. Furthermore, recent studies have demonstrated that BRCA-wt, HRD-low tumors have elevated immunogenic signals, including increased TIL abundance, thereby demonstrating increased IFN-gamma signaling. This is consistent with the observed benefit of Vigil® on RFS, which was most significantly demonstrated in the BRCA-wt population. Further clinical trials of PARP-inhibiting Vigil® in BRCA-wt ovarian cancer are warranted.

[0324] Bevacizumab is the recommended maintenance therapy for frontline-treated ovarian cancer, but no evidence of a recurrence-free or overall survival benefit has been observed with bevacizumab alone as maintenance therapy. Vascular endothelial growth factor (VEGF) inhibitors (i.e., bevacizumab) target the polymorphic growth factor VEGF, which is not only a key regulator of tumor angiogenesis but also suppresses the immune system. The hypoxic tumor microenvironment promotes the secretion of the proangiogenic factor VEGF-A (also known as VEGF), which binds to the receptor tyrosine kinases VEGF receptor (VEGFR)-1 (FLT1) and -2 (FLK1 / KDR) and the VEGFR co-receptors neuropilin (NRP) 1 and 2. This proangiogenic switch in the tumor microenvironment not only promotes tumor angiogenesis, tumor maturation, and metastatic dissemination, but also exerts immunosuppressive effects, such as inhibiting dendritic cell (DC) maturation, promoting regulatory T cell function and enhancing tumor-associated macrophage development, and accumulating myeloid-derived suppressor cells. PD-1 expression on the surface of CD8+ T cells is also increased. VEGF-A and its receptors VEGFR1, VEGFR2, and NRP1 are commonly upregulated in ovarian cancer.

[0325] However, the clinical efficacy of bevacizumab in ovarian cancer remains challenging due to its association with limited clinical responses and a moderate toxicity profile, as well as the relatively rapid activation of anticancer resistance after the initiation of antiangiogenic therapy. However, a potential direction for the use of antiangiogenic agents is being explored: enhancing immunotherapeutic activity. There is evidence that combining bevacizumab with therapeutic vaccines may induce an infiltrating T lymphocyte response, shifting the balance of immunosuppression from T regulatory suppression to CD8 T cell activation, creating a T cell-inflamed tumor microenvironment ("hot tumor") that may be more immunologically responsive to immunotherapy.

[0326] Results demonstrating an advantage in the BRCA1 / 2-wt population may be related to clonal neoantigen exposure, which may be diluted compared to subclonal neoantigen exposure in BRCA1 / 2-m patients in the context of increased repair abnormalities. Compared with BRCA-deficient ovarian cancers, BRCA-wt ovarian cancers exhibit an elevated proportion of tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment, elevated PD-L1 expression, elevated type 1 IFN-gamma signaling activity in mononuclear cells, and elevated perforin 1 expression, which are also consistent with a greater proportion of clonal neoantigens. BRCA1-wt expression also significantly impacts autophagy and its association with MHC expression of tumor antigens. BRCA1-m thus disrupts this process, potentially limiting neoantigen visibility and Vigil® activity.

[0327] GMCSF is involved in increasing tumor antigen presentation by dendritic cells (DCs) and induces higher levels of costimulatory molecules, which then lead to more efficient T cell stimulation. GMCSF also promotes the presentation of lipid antigens by dendritic cells, which in turn leads to the activation of natural killer T cells (NKT cells).

[0328] Additionally, TGFβ is expressed at higher levels in malignant ovarian tissues than in nonmalignant ovarian tissues. A gene expression meta-analysis of over 1,500 ovarian cancer patients identified high- and low-risk groups based on the expression of several genes, and the results were validated by IHC or qRT-PCR. Pathway analysis of gene signatures showed that TGFβ signaling was enriched in patients with poor prognosis. TGFβ is involved in the progression of noninvasive serous ovarian tumors to invasive serous ovarian tumors. Furthermore, overexpression of TGFβ correlates with tumor cell proliferation and metastasis.

[0329] TGF-β secreted by ovarian cancer cells induces the proliferation of immunosuppressive regulatory T cells (Tregs) (CD4+CD25+) in the tumor microenvironment. This has been shown to be associated with poor prognosis in patients with frontline-treated high-grade serous ovarian cancer. TGF-β inhibits the maturation of bone marrow-derived DCs and the induction of GMCSF-induced expression of MHC class II molecules and costimulatory molecules. TGF-β inhibits activated macrophages, including their antigen-presenting function, and inhibits the expression of PD-L1 ligands by ovarian cancer and tumor-associated myeloid cells, which is further associated with poor overall survival in ovarian cancer. Knockdown of TGF-β1, as demonstrated in this study, may contribute to the suppression of TGF-β effects, which may be more important in BRCA-wt patients.

[0330] Given that Vigil® is well tolerated, easy to administer, and demonstrates promising efficacy, it represents an ideal maintenance therapy for patients with ovarian cancer. Strengths of the presented results include safety in BRCA-wt patients and clinically validated RFS and OS benefits. However, the BRCA-wt subset is a secondary endpoint, and the use of autologous tumor harvest as a component of product manufacturing poses limitations to the product's application. Further evaluation of combinations with bevacizumab and / or niraparib is reasonable.

[0331] In conclusion, Vigil® demonstrated compelling RFS benefit and low toxicity as monotherapy maintenance therapy in frontline ovarian (stage III / IV) cancer patients with a BRCA1 / 2-wt molecular profile. Further investigation as a single agent and in combination with antiangiogenic agents, PARP inhibitors, and checkpoint inhibitors is also warranted.

[0332] Example 2 – Proof-of-principle study of the sequential combination of atezolizumab and Vigil® in relapsed ovarian cancer While there is impressive evidence favoring immunomodulation over checkpoint inhibitor (CI) therapy in many cancer types, little benefit has been demonstrated in OC. Theories suggest this inadequacy likely stems from the lack of specificity of CIs combined with the significant heterogeneity and genetic instability of OC. Recent reports have detailed how ovarian cancer (OC) cells acquire potential escape mechanisms, allowing them to evade host immunity through several immunosuppressive factors, including loss of MHC expression and upregulation of immunosuppressants, including transforming growth factor beta (TGFβ), indoleamine 2,3-dioxygenase (IDO), and cyclooxygenases (COX-1 and COX-2).

[0333] Vigil® is an autologous tumor cell vaccine in which tumor cells are harvested from patients at the time of cytoreductive surgery and transfected via electroporation with a plasmid encoding the GMCSF gene, an immune-stimulating cytokine, and a bifunctional short hairpin RNA (bi-shRNA) that specifically knocks down the expression of furin, a crucial converting enzyme responsible for the activation of two TGFβ isoforms, TGFβ-1 and TGFβ-2 (a cancer immune effector suppressor). By downregulating TGFβ expression, cancer cells are less able to evade the host immune response. Vigil® is a personalized "neoantigen-educating" immunotherapy, delivering 2.5×10 7It has been safely administered at doses up to 100 cells / injection and has shown evidence of benefit in phase 2 trials in cancer patients, including OC. It is hypothesized that the improved expression of clonal tumor neoantigens and the reduced tumor-suppressive effect of TGFβ synergistically increase the activity of checkpoint inhibitor therapy. Furthermore, preclinical evidence supports that pre-administration of neoantigen-education therapy before cycle 1 (C1) enhances the immunotherapy anticancer activity of C1. To evaluate safety and preliminary evidence of benefit, the first clinical combination using Vigil® and atezolizumab as single agents sequentially at previously clinically validated safe dose levels is being evaluated. Vigil® is being evaluated first (Vigil®-1). st ) and when using atezolizumab first (Atezo-1 st ) was compared and investigated.

[0334] method Study design and treatment Parts 1 and 2 of this phase, an open-label trial, were conducted at six centers across the United States. Part 1 examined the safety of Vigil® and atezolizumab. Part 2 was a randomized study comparing Vigil® first with atezolizumab first, followed by the Vigil® + atezolizumab combination. Patients entering the Vigil® vaccine configuration protocol for ovarian cancer were eligible for this study. Tissue and peripheral blood mononuclear cell samples were collected and analyzed for BRCA1 / 2 molecular profiling using a call quality of 40 and a minimum allele depth of 5 (Ocean Ridge Biosciences, Deerfield Beach, FL). Patients received Vigil® (1 × 10 intradermally). 6 ~10 7 Patients were administered 12 doses of 1200 mg / dose (1000 cells / dose) and atezolizumab (1200 mg / dose by intravenous infusion) every 3 weeks for up to 12 doses. Informed consent was obtained before collection and before primary study enrollment / randomization. Written IRB approval of the full protocol and consent documents were required before patients could be enrolled at any site.

[0335] patient Women with relapsed ovarian cancer, in stable medical condition, and who had failed at least one prior systemic therapy in the setting of recurrence or platinum-resistant disease were eligible for the study. Subjects were required to receive at least four manufactured vials of Vigil®, have an ECOG performance status (PS) of ≤1, and have normal organ and bone marrow function, which was defined as protocol compliance (absolute neutrophil count ≥1,500 / mm3). 3 ;platelets ≧100,000 / mm 3 (Hemoglobin ≥ 5.59 mmol / L; serum bilirubin ≤ 1.5 × institutional upper limit of normal; AST / ALT ≤ 2.5 × institutional upper limit of normal; creatinine > 50 mL / min; TSH within institutional limits). Patients with previous immunotherapy and active autoimmune disease were excluded.

[0336] Tumor harvesting and manufacturing Gradalis, Inc. (Carrollton, Texas) manufactured Vigil® from harvested tumor tissue. Manufacturing was a two-day process. The equivalent of a "golf ball-sized" mass (10-30 g of tissue, cumulative) was required for vaccine production (3 cm by radiological scan). Lesions extending into the intestinal lumen were excluded due to the risk of bacterial infection.

[0337] Manufacturing of investigational drugs and placebos Surgically removed tumor tissue during tumor debulking procedures was harvested, cut into ¼- to ½-inch sections, and then placed into up to four specimen containers containing sterile 0.9% sodium chloride (Baxter) supplemented with gentamicin (Fresenius Kabi). The samples were then packaged on ice for overnight transport to the manufacturing facility. On day 1, the transport medium and tumor samples in each container were checked for sterility (BacT / Alert 3D Microbial Identification System, BioMérieux). Tumor tissue was trimmed to remove fat, connective / necrotic tissue, and sutures / staples, mechanically dissociated with a scalpel, and then enzymatically dissociated (type I collagenase solution) and incubated at 37°C for up to 45 minutes to form a single-cell suspension. The cell suspension was filtered through a sterile 100 μm filter (Corning) to separate the cells from cellular debris. The free cells were washed with PlasmaLyte (Baxter) supplemented with 1% human serum albumin (Octapharma) and manually counted using a hemocytometer (InCyto). Quality control (QC) samples were retained and removed for immune monitoring. Pre-transfection cultures were initiated and baseline cytokine levels were obtained by ELISA for GMCSF (R&D Systems) and TGFβ1 (R&D Systems). The cell suspension concentration was adjusted to 40 million cells / mL and electroporated using a Gene Pulser XL (BioRad) to insert the plasmid DNA into the cells. The transfected cells were then transferred to sterile T-225cm tubes. 2 1 × 10 cells in X-VIVO 10 medium (Lonza) supplemented with gentamicin (Fresenius Kabi) in a flask (Corning). 6 The cells were plated at 100x the cell mass per mL and incubated overnight (14-22 hours) at 37°C in a 5% CO2 overlay (Sanyo) to allow the bi-shRNA Furin and GMCSF mRNA to be incorporated into the tumor cells.

[0338] On day 2, overnight cultures were harvested by detaching cells from each flask. The medium containing free-floating cells was collected and resuspended in fresh X-VIVO medium. Cells were manually counted to ensure a minimum of 80 million cells were available for a QC sample and a minimum of four doses before proceeding. Two 1-mL mycoplasma samples containing cells were collected and frozen at -80°C. Cells were irradiated with 4 × 25 Gy cycles using a gamma irradiator to arrest replication / proliferation. Cells were washed with PlasmaLyte (Baxter) supplemented with 1% human serum albumin (Octapharma), and a QC sample was retained and removed for immunomonitoring. Post-transfection cultures were initiated to monitor transfected cytokine levels by ELISA (or ELLA machine for one patient) for GMCSF and TGFβ1. Cells were placed in freezing medium consisting of PlasmaLyte (Baxter) at pH 7.4 containing 10% DMSO (dimethyl sulfoxide; Cryoserv USP; Mylan), 1% human serum albumin (Octapharma), and 1 × 10 6 cells / mL or 1 x 10 7 Cells were aseptically dispensed at 1.2 cells / mL into sterile 2.0 mL borosilicate glass vials (Algroup Wheaton Pharmaceutical and Cosmetic Packaging); the final fill volume was adjusted to 1.2 mL and sealed with butyl rubber stoppers coated with Flurotec® barrier files (West Pharmaceutical Services). Final product vials were frozen at a controlled rate using CoolCell® freezing containers (Biocision) placed in a -80°C freezer (Sanyo). After freezing, cells were stored in a vapor-phase liquid nitrogen tank during release testing. Frozen product vials were certified for sterility (USP 1000) by gelation (Limulus Amoebocyte Lysate, Lonza). <71> ) and endotoxin tests.

[0339] Atezolizumab was provided by Roche / Genentech and distributed by Gradalis, Inc. Atezolizumab was formulated as 60 mg / mL atezolizumab in 20 mM histidine acetate, 120 mM sucrose, and 0.04% polysorbate 20 (pH 5.8) (Phase III formulation). Atezolizumab contains no preservatives and is provided for single use only. Atezolizumab in formulation F03 (1200 mg per vial) was administered in a 250 mL 0.9% NaCl IV infusion bag, prepared, and diluted under sterile conditions.

[0340] Disease evaluation Subjects remained on treatment until disease progression, death, or product toxicity. Disease progression was determined radiographically by local investigators using Response Evaluation Criteria in Solid Tumors Version 1.1 (RECIST 1.1). In Part 1, disease progression was assessed at baseline and every third cycle thereafter. In Part 2, disease was assessed at baseline, at the end of cycle 2 of monotherapy, and every third cycle thereafter.

[0341] Endpoints and statistical evaluation The data cutoff date for the primary analysis was arbitrary, December 20, 2019. The primary endpoint was safety. Efficacy assessments and endpoints included Vigil®-1 st Group and Atezo-1 st Progression-free survival (PFS) and overall survival (OS) between groups were included. Time to progression was calculated from the date of randomization to the first documented date of progression or death. PFS and OS were analyzed sequentially in the BRCA1 / 2-wt subgroup and compared with Vigil®-1. st and Atezo-1 stComparisons were made between groups. Median follow-up time was calculated from the data cutoff date subtracted from the time of treatment initiation. A one-sided p value of 0.05 or less (log-rank) was considered to indicate statistical significance in the analysis. Hazard ratios (HRs) for PFS and OS were estimated using the Mantel-Haenszel hazard model. PFS and OS distributions were estimated using the Kaplan-Meier method. Toxicity was assessed using NCI CTCAE v 4.03.

[0342] result patient From June 2, 2017 to February 13, 2019, 3 patients entered Part 1 of the study and 21 were randomized for Part 2 of the study (n=11 Vigil®-1 st :n=10 Atezo-1 st All patients had failed at least one prior systemic therapy (Part 1 / Vigil®-1 st / Atezo-1 st (0 / 5 / 2 patients received one treatment, 1 / 4 / 3 patients received two treatments, 1 / 2 / 4 patients received three treatments, and 1 / 0 / 1 patient received four treatments). Summary demographics for individuals (Part 2 only) and all patients are shown in Table 5. Germline and somatic BRCA (g / sBRCA) status was determined for all 24 patients. Median follow-up time was 29.3 months for Part 1 and 21.3 months for Part 2. [Table 5]

[0343] safety No grade 3 or 4 treatment-related toxicities were observed in Part 1 and therefore could occur in Part 2. st There were 30 treatment-related adverse events in the Vigil®-1 group. st There were 83 events in the group: 30 Atezo-1 stTwenty-five of the events (83.3%) were grade 1 or 2, and the majority were attributable to atezolizumab treatment (24 / 25). st Eighty-one of the 83 events (97.6%) in the treatment group were grade 1 or 2 events, with 61.7% (50 / 81) attributable to atezolizumab treatment and 38.3% (31 / 81) related to Vigil® treatment.

[0344] Vigil®-1 st Group and Atezo-1 st Comparison of atezolizumab-related total events and grade 3 and 4 events between groups is shown in Figure 8. Grade 3 and 4 treatment-related events were significantly higher in Atezo-1 than in placebo. st The Vigil®-1 group (17.2%) st (5.1%). st Grade 3 atezolizumab-related events in the 100% group included blood and lymphatic system disorders (6.9%), general disorders (3.5%), injuries (3.5%), and respiratory, thoracic, and mediastinal disorders (3.5%). st No adverse events were observed in the Vigil®-1 group. st There were two grade 3 urinary events in the Atezolizumab group (one related to atezolizumab and one related to Vigil®). Additionally, atezolizumab-related adverse events (all grades) were more frequent in the Atezo-1 group than in the Vigil® group. st Vigil®-1 in the group (96.7%) st This was significantly higher than the group (61.3%).

[0345] Product shipping results Vigil® vaccine from 20 subjects (83.3%) met all product release criteria. The median tumor volume harvested was 51.65 g (range, 11.52 g to 114.23 g). Four subjects (16.7%) failed to demonstrate a sufficient increase in GMCSF expression after plasmid transfection, an exception to this study (n=1 in Part 1, n=3 in Part 2). Key product release results are shown in Table 6. Significant TGFβ1 knockdown was detectable in 23 / 24 (95.8%) subjects. Fifteen (62.5%) patients had 90% or greater TGFβ1 knockdown, demonstrating robust activity associated with Furin bi-shRNAi knockdown. From detectable baseline TGFβ1 production in all patients before plasmid transfection, a median TGFβ1 expression level of 158 pg / 10 was achieved. 6 TGFβ1 expression in BRCA1 / 2-wt cells (n=13, part 2) revealed that Atezo-1 st Group and Vigil®-1 st The median value for the group was 139 pg / 10 6 TGFβ1 expression in BRCA1 / 2-m patients (n=8, part 2) revealed a median of 223 pg / 10 6 cells (mean 215, range 111-307). [Table 6]

[0346] Effectiveness Median OS was 1.2% with Vigil®-1 st Not reached in cohort, Atezo-1 st The median follow-up period for Part 2 was 10.8 months in the BRCA1 / 2-wt cohort (HR 0.33; 95% confidence interval (CI) [0.064-1.7]; p=0.097) (Figure 9A). The median follow-up period was 21.3 months for Part 2. Subset analysis by BRCA status demonstrated that ovarian cancer in BRCA1 / 2-wt patients was significantly associated with Vigil®-1. stSo, Atezo-1 st In the BRCA-m cohort, Vigil®-1 demonstrated an improvement over Vigil®-1 (HR 0.16, 95% CI [0.026-1.03]; p=0.027, Figure 9B). st Cohort and Atezo-1 st There was no OS benefit among the cohorts, and no significant benefit was observed in PFS (Figures 9C-9D).

[0347] Consideration Although most ovarian cancer patients achieve a complete clinical response after primary surgery and combination chemotherapy, the vast majority (75%) of these patients unfortunately relapse within 16–24 weeks and ultimately succumb to their disease. Treatment for these relapsed patients is typically based on the relapse-free interval. Women who relapse more than 6 months after completing platinum-based chemotherapy are traditionally considered platinum-sensitive and are typically treated with maintenance therapy, often with a platinum doublet and / or a PARP inhibitor. Patients who relapse within 6 months of completing platinum-based chemotherapy (platinum-resistant) are typically treated with non-platinum-based therapy, with or without bevacizumab. PARP inhibitors have demonstrated improved progression-free survival (PFS) after initial chemotherapy and as maintenance therapy after platinum-sensitive recurrent OC therapy. Bevacizumab has been found to improve PFS when combined with initial chemotherapy or chemotherapy after relapse and is commonly used in combination therapy for relapsed OC.

[0348] The results demonstrated here justify the continuation of the Vigil® and atezolizumab combination study in recurrent ovarian cancer, and suggest that Vigil® followed by Vigil® / atezolizumab combination therapy may be beneficial for patients with Atezo-1 st This more favorable toxicity profile with promising clinical benefit supports the use of Vigil®-1, especially in BRCA-wildtype patients. stThis suggests a good therapeutic index for delivery of ovarian cancer, which is particularly encouraging given the limited benefit of single-agent checkpoint inhibitor therapy.

[0349] Because TGFβ expression is elevated in ovarian cancer tissue compared with many other cancers (especially recurrent disease), and Vigil®'s direct activity—knockdown of TGFβ1 and TGFβ2 through inhibition of furin—may directly affect the resistance mechanism mediated through TGFβ1 expression in ovarian cancer. Based on these results, Vigil® may be a practical mechanism for improving neoantigen exposure and T cell activation, thereby enabling checkpoint inhibitor therapy to function optimally despite TGFβ. TGFβ secreted by ovarian cancer cells generates immunosuppressive Treg cells (CD4+CD25+) from peripheral CD4+CD25- cells within the tumor microenvironment. Tumor infiltration of Tregs is associated with a poor prognosis in patients with high-grade serous ovarian cancer treated with neoadjuvant chemotherapy. Therefore, the CD8 / Treg ratio is also a prognostic indicator. At the start of this study, Vigil®-1 before the combination of Vigil® + atezolizumab st We hypothesized that therapeutic focus on visualization of immune effector cells targeting tumor neoantigens prior to functional checkpoint activation would limit off-target toxicity and potentially maximize immune effector cell availability and response. These observations support this hypothesis and the work of others in preclinical models, including evidence of selective TGFβ knockdown in overcoming resistance to checkpoint inhibitors.

[0350] To generate an effective antitumor immune response, T cells must be educated against the tumor's neoantigen repertoire. These T cells then differentiate into memory T cells, providing long-term immunological memory and potentially sustainable disease control. Tumors with low neoantigen heterogeneity have been reported to respond better to checkpoint blockade using pembrolizumab. The same study also demonstrated that T cells may be unable to recognize subclonal neoantigens (indicating the importance of abundant visible neoantigens for recognition) and may dilute the visibility of clonal neoantigens. The likelihood of T cells recognizing tumor-specific clonal neoantigens for the same patient's disease is greater when using autologous (individualized) tumor tissue rather than allogeneic tumor tissue.

[0351] More than 1,400 doses of Vigil® have been administered in clinical trials. The most frequently reported adverse reactions attributed to the administration of Vigil®-engineered cells were injection site reactions of mild to moderate intensity, consisting primarily of redness and swelling at the injection site, which are thought to be related to immune activation at the injection site. No serious or life-threatening (CTC grade 3 or 4) adverse events have been attributed to Vigil® treatment. No new or unexpected toxic effects were observed with Vigil® in combination with atezolizumab.

[0352] In conclusion, a personalized autologous ovarian cancer vaccine followed by a checkpoint inhibitor (atezolizumab) demonstrated encouraging efficacy with minimal toxicity.

[0353] Example 3 - BRCA1 / 2 Wild-Type Frontline Stage III / IV Ovarian Cancer Recurrence-Free Survival Benefit with Vigil® Use Because frontline treatment options for advanced ovarian cancer, particularly high TGFβ-expressing ovarian cancer, are limited, and because of the involvement of the BRCA1 / 2-wt population (85% of ovarian cancer patients), we conducted a double-blind, placebo-controlled trial of Vigil® as frontline maintenance therapy after cytoreductive surgery and the combination of paclitaxel and carboplatin in ovarian cancer patients with stage III / IV disease.

[0354] 1. Materials and Methods Study design and treatment This phase IIb, double-blind study was conducted at 25 centers. It was a placebo-controlled trial of Vigil® in patients with stage IIIa-c or IV high-grade serous, clear cell, or endometrioid ovarian cancer, classified as Level 1 according to the NCCN Guidelines (Version 3.2015), who had achieved a clinical complete response (cCR) after surgery and five to eight cycles of consolidation chemotherapy containing carboplatin and paclitaxel. Tumor tissue was obtained at the time of cytoreductive surgery. Tissue was used for vaccine construction and histological confirmation of disease by the local pathology department. Germline and / or somatic BRCA1 / 2 molecular profiling was collected when available. Peripheral blood mononuclear cell samples were analyzed for germline BRCA1 / 2 molecular profiling (Ocean Ridge Biosciences, Deerfield Beach, FL). Patients who achieved a cCR after primary cytoreductive surgery and adjuvant platinum / taxane chemotherapy were randomized 1:1 to either the Vigil® (Vigil® group, VG) or placebo (control group, CG) cohorts. Randomization was stratified by i) extent of cytoreductive surgery (complete / microscopic NED vs. macroscopic residual disease) and ii) neoadjuvant vs. adjuvant chemotherapy. Patients were randomized to receive 1x10 7Intradermal injections of cells / Vigil® or placebo were administered monthly (within 8 weeks of the last chemotherapy treatment) for up to 12 doses. Treatment continued until disease recurrence or the patient's supply of vaccine or placebo was exhausted. A Drug Safety Monitoring Board was established before the start of the study to maintain the safety of all study patients. No unacceptable toxic effects were determined. Informed consent was obtained before sampling. Written IRB approval of the full protocol and consent forms were required before patients could be enrolled at any site.

[0355] patient Women with histologically confirmed stage IIIa-c or IV high-grade papillary serous, clear cell, or endometrioid ovarian cancer were included in the study. After completion of cytoreductive surgery and chemotherapy, a chest x-ray (CT scan was acceptable) and a CT scan or MRI of the abdomen and pelvis showing no evidence of malignancy, a CA-125 antigen level of 35 units / mL or less, and no findings on physical examination or symptoms suggestive of active cancer were required at the time of randomization. Acceptable chemotherapy regimens included five to eight cycles of standard platinum / taxane, divided into neoadjuvant and adjuvant therapy. Subjects had to have initiated adjuvant chemotherapy within 8 weeks of primary cytoreductive surgery. An ECOG performance status (PS) of 0–1 and normal organ and bone marrow function were required for protocol compliance (absolute granulocyte count ≥ 1,500 / mm3; absolute lymphocyte count ≥ 500 / mm3; platelets ≥ 75,000 / mm3; total bilirubin ≤ 2 mg / dL; AST (SGOT) / ALT (SGPT) ≤ 2 × institutional upper limit of normal; creatinine < 1.5 mg / dL). All patients were required to be able to understand and willing to sign written, protocol-specific informed consent.

[0356] Tumor harvesting and manufacturing Gradalis, Inc. (Carrollton, Texas) manufactured Vigil® from the harvested tumor tissue. A placebo (freezing medium) was manufactured at the same dose based on the number of vials of Vigil®. Manufacturing was a two-day process. An equivalent "golf ball-sized" mass (10-30 g of tissue, cumulative) was required for vaccine production (3 cm by radiological scan). Lesions extending into the intestinal lumen were excluded due to the risk of bacterial infection.

[0357] Manufacturing of investigational drugs and placebos During tumor debulking, surgically resected tumor tissue (mean value = 55 g) was harvested, cut into 1 / 4-inch sections, and then placed into up to four sample containers containing 0.9% sodium chloride (Baxter) supplemented with gentamicin (Fresenius Kabi) and packaged on ice for overnight transport to the manufacturing facility. On day 1, the transport medium and tumor samples in each container were checked for sterility (BacT / Alert 3D Microbial Identification System, BioMérieux). Tumor tissue was trimmed to remove fat, connective / necrotic tissue, and sutures / staples, mechanically dissociated with a scalpel, and then enzymatically dissociated (type I collagenase solution) and incubated at 37°C for up to 45 minutes to form a single-cell suspension. The cell suspension was filtered through a sterile 100 μm filter (Corning) to separate the cells from cellular debris. The free cells were washed with PlasmaLyte (Baxter) supplemented with 1% human serum albumin (Octapharma) and manually counted using a hemocytometer (InCyto). Quality control (QC) samples were retained and removed for immune monitoring. Pre-transfection cultures were initiated and baseline cytokine levels were obtained by ELISA for GMCSF (R&D Systems), TGFβ1 (R&D Systems), and TGFβ2 (R&D Systems). The cell suspension concentration was adjusted to 40 million cells / mL and electroporated using a Gene Pulser XL (BioRad) to insert the plasmid DNA into the cells. The transfected cells were then transferred to sterile T-225cm tubes. 2 1 × 10 cells in X-VIVO 10 medium (Lonza) supplemented with gentamicin (Fresenius Kabi) in a flask (Corning). 6 The cells were plated at 100x the cell mass per mL and incubated overnight (14-22 hours) at 37°C in a 5% CO2 overlay (Sanyo) to allow the bi-shRNA Furin and GMCSF mRNA to be incorporated into the tumor cells.

[0358] On day 2, overnight cultures were harvested by detaching cells from each flask. The medium containing free-floating cells was collected and resuspended in fresh X-VIVO medium. Cells were manually counted to ensure a minimum of 80 million cells were available for a QC sample and a minimum of four doses before proceeding. Two 1-mL mycoplasma samples containing cells were collected and frozen at -80°C. Cells were irradiated with 4 x 25 Gy cycles using an RS-3400 X-ray (RadSource) to arrest replication / proliferation. Cells were washed with PlasmaLyte (Baxter) supplemented with 1% human serum albumin (Octapharma), and a QC sample was retained and removed for immunomonitoring. Post-transfection cultures were initiated to obtain levels of transfected cytokines, including GMCSF, TGFβ1, and TGFβ2. Cells were placed in freezing medium consisting of PlasmaLyte (Baxter) at pH 7.4 containing 10% DMSO (dimethyl sulfoxide; Cryoserv USP; Mylan), 1% human serum albumin (Octapharma), and 1 × 10 7 Cells were aseptically dispensed at 1.2 cells / mL into sterile 2.0 mL borosilicate glass vials (Algroup Wheaton Pharmaceutical and Cosmetic Packaging); the final fill volume was adjusted to 1.2 mL and sealed with butyl rubber stoppers coated with Flurotec® barrier files (West Pharmaceutical Services). Final product vials were slowly frozen using CoolCell® freezing containers (Biocision) placed in a -80°C freezer (Sanyo). After freezing, cells were stored in a vapor-phase liquid nitrogen tank during release testing. Frozen product vials were certified for sterility (USP 1000) by gelation (Limulus Amoebocyte Lysate, Lonza). <71> ) and endotoxin testing. After the study, assay validation for (GMCSF, TGFβ1, TGFβ2) was completed. Data shown are calculated using the appropriate validated parameters.

[0359] The placebo consisted of a freezing medium consisting of PlasmaLyte (Baxter) at pH 7.4 containing 10% DMSO (dimethyl sulfoxide; Cryoserv USP; Mylan) and 1% HSA (Octapharma). After freezing, the medium was slowly cooled to -80°C, and the vials were stored in vapor phase liquid nitrogen pending release testing for sterility and endotoxin alone. The placebo vial product matched the available product dose manufactured for the subjects. Study drug (Vigil® or placebo) was distributed monthly to the sites via portable liquid nitrogen containers. After product collection, site pharmacy staff were instructed to apply blinding tape to the syringe barrels to conceal any visible differences between Vigil® and placebo to maintain blinding.

[0360] Disease evaluation Subjects continued treatment until disease recurrence or the subject's supply of Vigil® or placebo was exhausted. Disease relapse was assessed by WorldCare Clinical (WCC) (Boston, MA) using Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1). Two reviewers were assigned to each case, with an arbitrator assigned when necessary. Data were provided to a third-party statistician for analysis. Treatment group assignment was discussed between the QA department and the statistician, who were blinded to other departments. Disease recurrence was defined by RECIST 1.1 as the appearance of any measurable or evaluable disease or asymptomatic CA-125 levels >35 U / ml on two consecutive measurements at least 1 month apart.

[0361] Endpoints and statistical evaluation The primary endpoint was recurrence-free survival (RFS) in the Vigil® group compared with the placebo group. Tumor assessments were performed at randomization (baseline) and at protocol-specified intervals until disease recurrence or death by WorldCare Clinical (WCC) (Boston, MA) according to Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1). Time to recurrence was calculated from 1) the date of randomization and 2) the date of cytoreduction surgery / harvesting to the first documented date of relapse or death. RFS was analyzed sequentially in the BRCA-wt subgroup and compared between Vigil® and placebo. All patients who received at least one dose of Vigil® or placebo were included in the safety analysis. A one-sided p value of 0.05 or less (log-rank) was considered to indicate statistical significance in the analysis. Hazard ratios for recurrence-free survival were assessed using a Cox proportional hazards model without covariates. The distribution of RFS was estimated using the Kaplan-Meier method. A chi-square test for goodness of fit was used to assess whether the number of patients who relapsed in the placebo group differed from the number of patients who relapsed in the Vigil® group. Patients in the BRCA-wt subgroup were assessed for whether the number of patients who relapsed in the placebo group was significantly different from the number of patients who relapsed in the Vigil® group.

[0362] 2.Results patient Between February 2015 and March 2017, 310 ovarian cancer patients at 22 centers consented and had tissue harvested for Vigil® manufacturing. 128 patients were deemed ineligible due to pathology or screening dropout parameters (i.e., staging). Of the remaining 181 patients who completed manufacturing, 92 subjects (56%) successfully underwent manufacturing and were randomized; 17 chose other treatment options, and 72 did not meet the product release criteria (65 for insufficient cells). One patient withdrew after randomization but before treatment for personal reasons of good health; this patient was not included in the analysis. 91 subjects were analyzed for safety and response; 46 patients were randomized to Vigil® and 45 patients were randomized to placebo. Fifty-five relapse events were observed by an independent third-party radiology review (WCC) through August 2019. Demographics are shown in Table 7. No significant differences were detected between cohorts, with the exception of 21 / 46, or 45.7%, Vigil® patients having a lower performance status score (ECOG 1) compared to placebo (9 / 45, 20.0%) (p=0.0093). [Table 7-1] [Table 7-2]

[0363] safety A median of six Vigil® (range 1-12) injections or six placebo (range 3-12) injections was administered per patient. No treatment delays or withdrawals due to treatment-related toxic effects were reported. One patient in the Vigil® group experienced a grade 3 drug-related toxic effect (nausea and vomiting), and two patients in the placebo group experienced grade 3-related toxicities (one with bone pain, global muscle weakness, dyspnea, and syncope; the other with arthralgia). More grade 2 and 3 adverse events were observed in the placebo group compared with the Vigil® group (18% vs. 8%). Fourteen serious adverse events (SAEs) were reported in seven patients (four placebo, three Vigil®). In two, the SAEs were probably related (one placebo / one Vigil®). Otherwise, the SAE events were likely unrelated to study treatment or unrelated.

[0364] Product shipping results Eighty-four subjects (92%) met all product release criteria. The median tumor mass harvested was 52 grams (range 8-137 grams). Seven of 91 patients (8%) did not demonstrate a sufficient increase in GMCSF expression after plasmid transfection. However, six of these seven patients demonstrated robust TGFβ1 knockdown. Additional product release results are shown in Table 8. TGFβ1 knockdown results were inconclusive in seven patients (six developed sufficient GMCSF expression), but 66 patients (73%) achieved 90% or greater TGFβ1 knockdown, demonstrating robust activity associated with Furin bi-shRNAi knockdown. Baseline TGFβ1 production was detectable in all patients before plasmid transfection, demonstrating a median TGFβ1 expression of 166 pg / 10 6 In both groups, BRCA1 / 2-wt TGFβ1 expression was found to be a median of 212 (mean 255, range 71–662) pg / 10 cells (mean 241, range 52–882). 6 BRCA1 / 2-mu TGFβ1 expression was found to be a median of 122 (mean 231, range 52-882) pg / 106 It was revealed that the number of cells was 100 (Table 9). [Table 8] [Table 9]

[0365] Effectiveness Of 91 evaluable patients, 62 patients had a BRCA1 / 2 molecular profile by local site analysis. The subgroup of BRCA1 / 2-wt patients demonstrated an RFS benefit in the Vigil® cohort compared with the placebo cohort. Results are shown in Figures 10A-10B. Median RFS, calculated from the time of surgery / harvesting, was not reached for BRCA-wt ovarian patients in the Vigil® cohort compared with 14.8 months in the placebo cohort (HR = 0.49, 90% CI, 0.25-0.97, one-sided p = 0.038). Similarly, when RFS was calculated from the time of randomization, median RFS in the BRCA-wt subgroup was 19.4 months in the Vigil® cohort compared with 8.0 months in the placebo group (HR = 0.51, 90% CI, 0.26-1.01, one-sided p = 0.050). Thirty-eight percent of BRCA1 / 2-wt patients treated with Vigil® experienced relapsed disease compared to 71% of placebo BRCA1 / 2-wt patients (chi-squared = 0.021) (Figure 11A). 62% of Vigil® patients remained BRCA-wt and remained relapse-free to date (October 31, 2019).

[0366] RFS calculated from the time of cytoreductive surgery and the time of randomization, regardless of BRCA1 / 2 status, is shown in Figures 10C-10D. Median RFS calculated from the time of randomization was 13.67 months (416 days) in the Vigil® cohort compared with 8.38 months (255 days) in the placebo cohort (HR = 0.69, one-sided log-rank p = -.054). RFS calculated from the time of cytoreductive surgery improved, with a HR of 0.64 (one-sided p = 0.054). Relapse was reported in 48% of patients treated with Vigil® compared with 73% of placebo patients (chi-square p = 0.013) (Figure 11B). Other factors associated with a trend toward benefit for Vigil® response were younger age ≤65 years (p 0.057), late stage IIIb-IV disease (p 0.075), and microscopic residual disease (<10 mm) / no evidence of disease (p=0.066) (Figure 12).

[0367] 3. Discussion BRCA1 / 2-wt patients demonstrated a significant RFS advantage (calculated from the time of cytoreductive surgery) with Vigil® over placebo (not reached vs. 14.8 months; HR=0.49, one-sided p=0.038) and had a lower incidence of post-treatment relapse (38% vs. 71%, p=0.021). These results supported Vigil® being considered maintenance in patients with BRCA1 / 2-wt ovarian cancer after complete cytoreductive surgery and adjuvant or neoadjuvant chemotherapy. Safety analyses demonstrated no evidence of toxic effects with Vigil® over placebo.

[0368] Increasing evidence suggests that GMCSF is involved in increasing tumor antigen presentation by dendritic cells (DCs). GMCSF has been shown to induce a subset of DCs that are superior in phagocytosis of apoptotic tumor cells. GMCSF induces higher levels of costimulatory molecules (which are characteristic of greater functional maturation and more efficient T cell stimulation), thereby expanding the arsenal of lymphocyte effector mechanisms induced by DCs. GMCSF also promotes lipid antigen presentation by DCs, which in turn leads to the activation of natural killer T cells (NKT cells), a lymphocyte population that may be important in the innate and therapeutic response to tumors. DCs initiate antigen-specific immune responses and express various receptors that enable antigen recognition and capture in peripheral tissues such as the dermis. DCs efficiently process this material, route it down the MHC class I and II presentation pathway, upregulate costimulatory molecules upon maturation, and migrate to secondary lymphoid tissues, where they present antigens to T cells. Vigil® GMCSF protein was upregulated, 1806 / 826 pg / 10 6 This resulted in a robust mean / median of 100 cells. In 84 of the 91 (91%) patients in whom this was achieved, this was related to GMCSF plasmid transfer alone.

[0369] It has also been previously shown that expression of TGF-β in ovarian malignant cells is significantly higher in malignant ovarian cancer tissues than in non-malignant ovarian cancer tissues. A gene expression meta-analysis conducted on over 1,500 ovarian cancer patients identified high- and low-risk groups based on the expression of several genes, and the results were validated by IHC or qRT-PCR. Pathway analysis of gene signatures demonstrated enrichment of TGF-β in patients with poor prognosis. TGF-β signals are transmitted through binding to the serine / threonine kinase receptors TGF-βRI and II, leading to the phosphorylation and activation of the intracellular effectors Smad2 and Smad3. Smad2 / 3 form a transcriptional complex with Smad4, translocate to the nucleus, and regulate the expression of TGF-β-regulated genes. TGF-β is involved in the progression of non-invasive serous ovarian tumors to invasive serous ovarian tumors. TGF-β can be activated by both Smad-dependent and Smad-independent pathways and is thought to promote tumor metastasis in ovarian cancer. A correlation between overexpression of TGFβ and tumor cell proliferation and metastasis has also been described in prostate, colon, and renal cell carcinomas.

[0370] TGF-β secreted by ovarian cancer cells induces the proliferation of immunosuppressive Treg cells (CD4+CD25+) in the tumor microenvironment. This has been shown to be associated with poor outcomes in patients with high-grade serous ovarian cancer treated with frontline cytoreductive surgery / chemotherapy. TGF-β inhibits the maturation of bone marrow-derived dendritic cells (DCs) and the induction of GMCSF-mediated expression of MHC class II molecules and costimulatory molecules. TGF-β inhibits activated macrophages, including their antigen-presenting function, and inhibits the expression of PD-L1 ligands by ovarian cancer and tumor-associated myeloid cells, which is further associated with poor overall survival in ovarian cancer. Evidence reveals that modulation of antitumor immunity can restore TGF-β-related immune surveillance.

[0371] TGF-β1 has also been shown to downregulate the miR181 / BRCA1 axis, thereby modulating DNA repair dysregulation and inducing "BRCA-ness" in breast cancers with wild-type BRCA genes. The term "BRCA-ness" has been used to identify sporadic tumors with clinicopathological and molecular features similar to those associated with BRCA1 / 2 germline mutations (DNA repair inhibition). Additionally, increased TGF-β signaling in low-HRD tumors has been demonstrated to induce NF-κB activation and elicit antitumor immune responses in lymphocytes. Therefore, in addition to BRCA molecular signaling, homologous recombination repair dysregulation (HRD) scores may also be relevant in predicting outcome and response to TGF-β-modulating immunotherapy.

[0372] The improved RFS results in the BRCA-wt population in this study further support the relationship between TGFβ inhibition and immune responsiveness, thereby supporting the use of Vigil® in BRCA-wt patients and other cancer subpopulations, possibly with other histological types, that have high TGFβ expression (e.g., prostate cancer, renal cell carcinoma, and colorectal cancer). High levels of furin mRNA and protein are also widely expressed in ovarian cancer and many other human tumors where gene expression differs between malignant (high) and non-malignant (low) cell populations. Expression of furin, a converting enzyme of TGFβ1 and TGFβ2, appears to be inversely correlated with survival and likely contributes significantly to the maintenance of TGFβ-mediated peripheral immune tolerance toward tumors. Downregulation of furin using bi-shRNAi induced a significant reduction in TGFβ1 expression, as validated in this study. Notably, 73% of the constructed Vigil® vaccines had 90% or greater TGFβ1 knockdown compared to untransfected tumor cells from the same patients.

[0373] BRCA-wt, HRD-low ovarian cancers also exhibit elevated tumor-infiltrating lymphocyte (TIL) rates, high PD-L1 expression, high type 1 IFN-gamma signaling activity in mononuclear cells, and high perforin-1 expression in the tumor microenvironment, which may suggest a generally improved immune response. However, several attempts to enhance both frontline and relapsed / refractory ovarian responses to checkpoint inhibitor therapy have failed. Hypothetically, this may also be related to the high TGF-β suppressive effect within the local tumor microenvironment, as previously described.

[0374] PARP inhibitors not only block the catalytic activity of PARP proteins on the surface of DNA, but also bind to and capture the protein, leading to numerous double-stranded DNA breaks and ultimately cell death. Lynparza® (olaparib) was one of the first PARP inhibitors approved for patients with high-grade serous ovarian cancer who harbor a germline BRCA mutation. Previous studies have shown improved progression-free survival in BRCA1 / 2-mu ovarian cancer (HR 0.3, p<0.0001). A recent long-term follow-up study (Study 19) of olaparib in patients with platinum-sensitive recurrent ovarian cancer showed favorable OS results, with an HR of 0.73 (p = 0.0138) for all patients enrolled. However, activity appeared to be limited to the BRCA-mu population, with an HR of 0.62 (p = 0.02140), in contrast to the BRCA-wt population, which showed no benefit, with an HR of 0.84 (p = 0.34) (82). However, some BRCA-wt patients with relapsed or refractory disease and homologous recombination repair deficiency (HRD) may benefit from PARP inhibitors. Recent reports have shown that some HRD-high tumors are associated with a better response to niraparib. Furthermore, recent studies have demonstrated that BRCA-wt, HRD-low tumors have elevated immunogenic signals, including increased TIL abundance, thereby demonstrating increased IFN-gamma signaling. This is consistent with the observed benefit of Vigil® on RFS, which was most significantly demonstrated in the BRCA-wt population. Further clinical trials of PARP-inhibiting Vigil® in BRCA-wt ovarian cancer are warranted.

[0375] Bevacizumab is the recommended maintenance therapy for frontline-treated ovarian cancer, but no evidence of a recurrence-free or overall survival benefit has been observed with bevacizumab alone as maintenance therapy. Vascular endothelial growth factor (VEGF) inhibitors (i.e., bevacizumab) target the polymorphic growth factor VEGF, which is not only a key regulator of tumor angiogenesis but also suppresses the immune system. The hypoxic tumor microenvironment promotes the secretion of the proangiogenic factor VEGF-A (also known as VEGF), which binds to the receptor tyrosine kinases VEGF receptor (VEGFR)-1 (FLT1) and -2 (FLK1 / KDR) and the VEGFR co-receptors neuropilin (NRP) 1 and 2. This proangiogenic switch in the tumor microenvironment not only promotes tumor angiogenesis, tumor maturation, and metastatic dissemination, but also exerts immunosuppressive effects, such as inhibiting dendritic cell (DC) maturation, promoting regulatory T cell function and enhancing tumor-associated macrophage development, and accumulating myeloid-derived suppressor cells. PD-1 expression on the surface of CD8+ T cells is also increased. VEGF-A and its receptors VEGFR1, VEGFR2, and NRP1 are commonly upregulated in ovarian cancer.

[0376] However, the clinical efficacy of bevacizumab in ovarian cancer remains challenging not only due to its association with limited clinical responses and a moderate toxicity profile, but also due to the relatively rapid activation of anticancer resistance after the initiation of antiangiogenic therapy. However, a potential direction is being explored in the use of antiangiogenic agents: enhancing immunotherapeutic activity. There is evidence that combining bevacizumab with therapeutic vaccines may induce an infiltrating T lymphocyte response, shifting the balance of immunosuppression from T regulatory suppression to CD8 T cell activation, creating a T cell-infiltrated tumor microenvironment (a "hot tumor") that may be more immunologically responsive to immunotherapy.

[0377] In conclusion, Vigil® demonstrated compelling RFS benefit and low toxicity as monotherapy maintenance therapy in frontline ovarian (stage III / IV) cancer patients with a BRCA1 / 2-wt molecular profile. Further investigation as a single agent and in combination with antiangiogenic agents, PARP inhibitors, and checkpoint inhibitors is also warranted.

[0378] Example 4 - Homologous Recombination Proficient (HRP) Ovarian Cancer: Gemogenovatucel-T (Vigil®) is an option in the search for an efficient maintenance therapy A recent meta-analysis of ovarian cancer patients (Xu et al., Oncotarget, 8(1):285-302, 2017) confirmed that the resulting inefficient repair mechanisms in BRCA-mutated (-m) tumors predict improved response rates to platinum-based chemotherapy compared with BRCA-wildtype (-wt) tumors. Counterintuitively, genomic instability as a result of germline mutations in BRCA and other homologous recombination (HR) genes significantly increases the probability of developing ovarian cancer. However, patients with HR-repair-deficient (HRD) cancers (both BRCA-m and HR-m) also exhibit improved survival. This is due not only to an impaired ability to repair chemotherapy-induced DNA damage, but also to disruption of BRCA-regulated autophagy, with subsequent effects on cancer stem cell maintenance and drug resistance. The therapeutic application of PARP inhibition (PARP-i) represents a significant positive shift in ovarian cancer therapy, leveraging synthetic lethality predicted by network biomolecular analysis in HRD cancers. Despite its significant contribution to the treatment paradigm, PARP-i selectively benefits patients with HRD cancers, with less survival benefit in patients with HR-proficient (HRP) ovarian cancer (Gonzalez-Martin et al., New England Journal of Medicine, 381(25):2391-2402, 2019). Problematically, PARP inhibitors induce moderate toxicity, narrowing the therapeutic index for long-term maintenance therapy with some agents, with up to 65% of patients experiencing grade 3 / 4 adverse events. Additionally, a 71% reduction rate and a 12% discontinuation rate have been observed, resulting in a more unfavorable therapeutic index (toxicity:benefit ratio), especially for patients with HRP tumors (Gonzalez-Martin et al., N Engl J Med, 381(25):2391-2402, 2019). Consequently, HRP tumors remain a subset of ovarian cancer with less effective first-line and maintenance treatments for survival.

[0379] Bevacizumab has also been demonstrated to provide statistically improved progression-free survival (PFS) in ovarian cancer patients with recurrent disease and newly diagnosed patients with resectable stage III / IV disease as consolidation and maintenance therapy after cytoreductive surgery, although long-term follow-up failed to demonstrate an OS benefit. Interestingly, regarding the limited recognition of the relationship between HRD and HRP and bevacizumab activity, the GOG-0218 trial (NCT00262847) demonstrated that the BRCA-wt / HRP ovarian cancer cohort had a nearly 20-month shorter survival compared with the BRCA-wt / HRD cohort with or without bevacizumab for both consolidation and maintenance therapy, further supporting the limited efficacy of bevacizumab, including in BRCA-wt / HRP ovarian cancer (Tewari et al., J Clin Oncol, 37(26):2317-2328, 2019).

[0380] We previously described the use of a novel autologous tumor cell vaccine, Gemogenovatucel-T (Vigil®), composed of tissue harvested during cytoreductive surgery, as maintenance therapy in newly diagnosed ovarian cancer (Oh et al., Gynecol Oncol, 143(3):504-510, 2016). Vigil® incorporates a multigene plasmid encoding the human immunostimulatory GMCSF gene and a bifunctional short hairpin RNA construct that specifically knocks out the proprotein convertase furin and its downstream targets, TGF-β1 and TGF-β2 (Maples et al., BioProcessing Journal, 8:4-14, 2010; Senzer et al., Mol Ther, 20(3):679-86, 2012; and Senzer et al., Journal of Vaccines & Vaccination, 4(8):209, 2013). Vigil® is designed to enhance the expression of cancer-associated neoantigens through upregulation of MHC-II and processing in dendritic cells, thereby increasing the afferent immune response and generating a systemic anti-tumor immune response.

[0381] In a 25-center, randomized, double-blind, placebo-controlled, phase 2 maintenance trial, Vigil® further demonstrated a favorable therapeutic index, documenting efficacy and no serious, therapy-related grade 3 / 4 adverse events (Rocconi et al., Lancet Oncology, 2020). A statistically non-significant improvement in the primary endpoint of recurrence-free survival (RFS) was determined for all patients receiving Vigil® compared with placebo: 11.5 and 8.4 months, respectively (n=91, HR=0.688, 90% CI 0.443-1.068; p=0.078). However, a prospective secondary endpoint, the preplanned measurement of RFS in BRCA-wt patients, revealed a hypothesis-generating improvement of RFS of 12.7 vs. 8.0 months (n=67, HR=0.514; 90% CI 0.3-0.88; p=0.020). Additionally, in a planned subset analysis of BRCA-wt patients, median overall survival (OS) was not reached compared with 41.4 months for placebo (n=67, HR=0.493; CI 0.24-1.009; p=0.049). Furthermore, only 21 (52%) of BRCA-wt patients had relapsed disease after Vigil® at the time of analysis compared with 20 (78%) of 27 placebo BRCA-wt patients (p=0.021, Fisher's exact probability). Two-year RFS rates from randomization were observed for Vigil® (33%) vs. placebo (14%) (p=0.045, one-sided Z-test), and a two-year OS advantage for Vigil® (90%) compared with placebo (67%) (p=0.020, one-sided Z-test) was also demonstrated.

[0382] Because the enhanced immune response to Vigil® immunotherapy in the BRCA-wt population is associated with limited benefit of standard therapy in the HRP population, we characterized the homologous recombination status (HRD vs. HRP) of all patients enrolled and treated in a recently published phase 2 trial (Rocconi et al., Lancet Oncology, 2020). This trial evaluated Vigil® versus placebo as maintenance therapy in newly diagnosed ovarian cancer patients with resectable stage III / IV disease who had undergone cytoreductive surgery and adjuvant treatment with platinum-based chemotherapy to determine the effect of Vigil® on the HRP population.

[0383] Recently, Vigil® demonstrated significant clinical benefit with improved progression-free survival and overall survival in pre-planned subgroup analyses in newly diagnosed stage III / IV ovarian cancer patients with a BRCA wild-type molecular profile. Here, we analyze the homologous recombination status of patients enrolled in the Phase 2b VITAL study to characterize the clinical benefit of Vigil® in patients with HRP.

[0384] 1. Method 1.1 Patient population and study design Vigil® plasmid construction, cGMP manufacturing, tissue processing, and transfection were performed as previously described (Maples et al., BioProcessing Journal, 8:4-14, 2010; Senzer et al., Mol Ther, 20(3):679-86, 2012; and Oh et al., Gynecologic Oncology, 143(3):504-510, 2016). The VITAL study was a randomized, phase 2b, double-blind, placebo-controlled trial. The patient population and study design have been previously published (Rocconi et al., Lancet Oncology, 2020). Briefly, patients were in complete response after surgical cytoreduction and five to eight cycles of chemotherapy for stage III / IV high-grade serous, endometrioid, or clear cell ovarian cancer. At the time of enrollment, patients were required to have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 and adequate organ and bone marrow function. 7 Patients received monthly injections of cells / Vigil® or placebo for a minimum of four doses and a maximum of 12 doses. Treatment continued until product was exhausted or disease progression occurred. Adverse events were recorded after the first dose of treatment and continued for 30 days after the last treatment. Disease recurrence was assessed by a blinded independent reviewer and was defined as any measurable lesions on two consecutive measurements or an elevated CA-125 level of >35 U / mL.

[0385] 1.2 HRP analysis BRCA1 / 2 mutation status was determined as previously described (Rocconi et al., Lancet Oncology, 2020). Patients identified as BRCA1 / 2 wild-type were sent for homologous recombination repair deficiency testing using MyChoice® CDx (Myriad, Inc., Salt Lake City, UT). A score of 42 or greater per assay guideline was used to identify patients with HRD, and >42 was HRP.

[0386] 1.3 Statistical analysis The primary endpoint of the VITAL study was recurrence-free survival from the time of randomization. Post-hoc analyses of recurrence-free survival and overall survival for Vigil® versus placebo in patients with HRP were performed by Kaplan-Meier analysis. Hazard ratios and confidence intervals were calculated using 90% CI, and p values ​​were one-sided. Stratification factors included residual disease and chemotherapy schedule. A marginal mean survival time difference (RMST) analysis was performed as a sensitivity analysis with a cutoff point equal to the minimum of the longest follow-up time in each group and without adjustment for covariates.

[0387] 2.Results 2.1 Baseline characteristics and demographics In this evaluation, 67 patients with BRCA-wt tumors underwent HRD analysis (60% of patients in the Vigil® group and 40% in the placebo group). HR analysis of tumor tissue revealed that 62.5% (n=25) of the Vigil® group and 74.1% (n=20) of the placebo group had HRP tumors as determined by the assay [threshold <42 (Myriad MyChoice®)]. Patient demographics are listed in Table 10. Demographics revealed that a greater number of patients with poor performance status scores (ECOG 1) were randomized to receive Vigil®. [Table 10]

[0388] 2.2 Safety Adverse events in each treatment group are shown in Table 11. No patients in the Vigil® group reported grade 3 or higher adverse events. There were no treatment-related deaths, no patients withdrew from the study due to adverse events, and no dose modifications occurred. Patients received a mean of 7.12 doses of Vigil® (range 1.00-12.00) compared with 6.90 doses of placebo (range 3.00-12.00). [Table 11]

[0389] 2.3 Endpoint analysis Kaplan-Meier analysis showed a favorable improvement from randomization in both RFS HR=0.386 (90% CI 0.199-0.750), p=0.007, and OS HR=0.342 (90% CI 0.141-0.832), p=0.019, in the HRP Vigil®-treated subset (Figures 22 and 24). Further evaluation of OS and RFS using RMST, which provides an alternative assessment comparing exposure over a specified time period, unlike hazard proportionality, which compares relative risks based on the number of events over time, also supported a favorable benefit of Vigil® over placebo for both RFS (p=0.017) and OS (p=0.008) (Figures 25 and 26).

[0390] 3.Results The relationship of BRCA-wt to BRCA-m expression in malignant tissues (Morand et al., JNCI Cancer Spectrum, 2020) is associated with greater clonal neoantigen expression (McGranahan et al., Science, 351(6280):1463-9, 2016), potentially identifying more appropriate neoantigen targets for the induced immune effector response characterized by a "hot" tumor microenvironment with respect to immune activation induced by Vigil® (Kraya, AA, et al. Clin Cancer Res, 25(14):4363-4374, 2019). Higher tumor mutation burden and median neoantigen load are observed in high-grade ovarian cancers with HRD compared with HRP, but at the lower end of the pan-tumor spectrum. However, the high subclonal mutation rate (approximately 40%) and greater intratumor heterogeneity in HRD likely reduce the immunogenic effects described in high-clonal neoantigen tumors associated with a "hot" infiltrated tumor microenvironment and increased tumor-infiltrating cells (McGranahan et al., Science, 351(6280):1463-9, 2016). This is particularly true in the setting of high-neoantigen / high-human leukocyte antigen (HLA) expression HRP tumors. These HRP tumors are also enriched in effector memory T cells and enhance IFNγ responses, whereas BRCA1-mutated tumors are associated with reduced type I / II IFN responses (McGranahan et al., Science, 351(6280):1463-9, 2016). Evaluation of the mechanism of Vigil® supports the enhanced ability of circulating mononuclear cells to induce antitumor responses after Vigil® treatment, which correlates with clinical benefit as assessed by ELISPOT assays (Oh et al., Gynecol Oncol, 143(3):504-510, 2016). Furthermore, circulating CD3+ / CD8+ mononuclear cells have been shown to be systemically upregulated after Vigil® treatment, particularly in a small group of treated patients (Herron et al., Cancer Science and Research, 2020).Additionally, greater subclonal neoantigen expression in BRCA-m-expressing cancers may result in a larger proportion of malignant cell "nest" subpopulations, potentially reducing the visibility of clonal neoantigens (which are likely enhanced in the BRCA-wt population) and the efficacy of immune responses targeting them (McGranahan et al., Science, 351(6280):1463-9, 2016). Persistent increased ELISPOT activity has also been observed after Vigil®, with induced antitumor activity persisting for several months beyond Vigil® discontinuation (Senzer et al., Journal of Vaccines & Vaccination, 4(8):209, 2013). These results suggest the induction of memory T cells (Craig et al., Vaccines (Basel), 8(4), 2020).

[0391] As previously described, adding PARP-i to our therapeutic opportunities in ovarian cancer has had an impact. However, because the molecular profiles of individual ovarian cancers are diverse, the relationship of molecular profiles to RFS, PFS, and OS is currently being evaluated in detail across the cancer spectrum, including ovarian cancer. While several therapeutic indications have been funded based on this and other PARP-i reports for the treatment of newly diagnosed ovarian cancer and the continuum of second-line ovarian cancer therapy, the majority of benefit has been seen in ovarian cancer patients with BRCA-m HRD tumors or BRCA-wt HRD tumors.

[0392] Consideration of Vigil® in the HRP ovarian cancer population is warranted because of clinical challenges resulting from the moderate toxicity profile and limited efficacy of both antiangiogenic agents and PARP-i agents in the BRCA-wt / HRP population compared with the BRCA-m / HRD and BRCA-wt / HRD populations. PARP-i agents are associated with a significant rate of grade 3 / 4 drug-related toxicities, which can result in treatment discontinuation in as many as 75% of patients (Gonzalez-Martin et al., N Engl J Med, 381(25):2391-2402, 2019). While the toxicity profile is less pronounced with antiangiogenic agents, toxicities can be severe, if not fatal, and include bleeding, hypertension, intestinal perforation, and the risk of venous and arterial thrombosis. Considering the recent approval of niraparib as maintenance for "all willing," including patients with HRP tumors (Gonzalez-Martin et al., N Engl J Med, 381(25):2391-2402, 2019), and weighing the limited toxicity, both quantitative and qualitative, against the limited benefit in the HRP population, further development of Vigil® and other potentially effective broader therapeutic index approaches, as well as other sensitive molecular subsets, is justified.

[0393] Vigil® immunotherapy is the first randomized, proof-of-principle study of immunotherapy efficacy in HRP epithelial ovarian cancer (Rocconi et al., Lancet Oncology, 2020). The simplicity of monthly intradermal injections, combined with the achievement of both extended delay in recurrent disease and overall survival benefit with a wide therapeutic index, are key components of optimal maintenance therapy in clinically disease-free patients. The study of Vigil® vaccine maintenance therapy in primary HRP ovarian cancer corresponds to an area of ​​encouragement from NRG Oncology to develop and apply therapies targeted to this subset of ovarian cancer. Therefore, phase 3 studies of Vigil® are warranted, specifically in the HRP ovarian cancer population and potentially in other solid tumor populations with an HRP profile.

[0394] Example 5: Gemogenovatucel-T (Vigil®) Immunotherapy (VITAL) as Maintenance in Frontline Stage III / IV Ovarian Cancer: A Randomized, Double-Blind, Placebo-Controlled Phase 2b Study Evidence prior to this study We searched PubMed from January 1, 1999, to March 1, 2020, using the search term "current standard of care" to find clinical trials, studies, and research articles published in English.

[0395] The search terms included: "ovarian cancer maintenance," "PARP inhibitors," "BRCA mutation status ovarian cancer," and "homologous recombination repair deficiency." Our search yielded 21,159 articles. A review of the literature revealed several studies involving poly(ADP-ribose) polymerase (PARP) inhibitors demonstrating improved progression-free survival, but no benefit in recurrence-free or overall survival was observed in frontline ovarian cancer, including systemic maintenance therapy. There are few options for the management of advanced frontline ovarian cancer. Delayed but persistent relapse occurs in approximately 75% of cases within the first 2 years of management with first-line standard of care, either primary cytoreductive surgery followed by adjuvant chemotherapy or neoadjuvant chemotherapy followed by cytoreductive surgery. Consolidation or maintenance therapy did not demonstrate improved recurrence-free or overall survival compared with standard management. Although several studies have demonstrated a progression-free survival benefit in patients who are homologous recombination proficient, the survival benefit of ARP inhibitors has been preferentially associated with patients with germline and somatic BRCA mutations or homologous recombination repair deficiencies in their molecular profile. ARP inhibition has also shown little benefit in overall survival, with the greatest benefit observed in patients with BRCA mutations or homologous recombination repair deficiencies, and less benefit in patients who are homologous recombination proficient. Furthermore, toxic effects related to bone marrow suppression and rare, treatment-related leukemias limit dose administration. The 5-year survival rate for patients with ovarian cancer remains low, representing an unmet medical need. Therefore, supported by robust phase 1 safety and immune response mechanisms and significant evidence of benefit in a phase 2a clinical trial, we initiated the VITAL trial to evaluate the safety and efficacy of gemogenovatucel-T in newly diagnosed patients with stage III / IV ovarian cancer who are candidates for primary cytoreductive surgery.

[0396] Additional value of this study To our knowledge, the VITAL study is the first to examine the efficacy of a triple-mechanism immunomodulatory autologous tumor vaccine as a frontline maintenance treatment for ovarian cancer and provides the first evidence of immunotherapy benefit in this population. The results of this study demonstrate that gemogenovatucel-T is non-toxic and that treatment leads to improved recurrence-free and overall survival in patients with advanced ovarian cancer who are wild-type for BRCA1 and BRCA2 (BRCA). The majority of patients newly diagnosed with ovarian cancer are BRCA.

[0397] What all the available evidence means Gemogenovatucel-T is a breakthrough immunotherapy platform technology with single-agent activity. Future studies of gemogenovatucel-T are warranted in combination with checkpoint inhibitors, PARP inhibitors, and anti-angiogenic agents.

[0398] background Gemogenovatucel-T is an autologous tumor cell vaccine produced from harvested tumor tissue that specifically reduces the expression of furin and its downstream TGF-I31 and TGF-I32. The purpose of this study was to determine the safety and efficacy of gemogenovatucel-T in the maintenance of frontline ovarian cancer.

[0399] method This randomized, double-blind, placebo-controlled phase 2b trial involved 25 hospitals across the United States. Women aged 18 years or older with stage III / IV high-grade serous, endometrioid, or clear cell ovarian cancer who achieved a complete clinical response after surgery and five to eight cycles of chemotherapy, including carboplatin and paclitaxel, and whose Eastern Cooperative Oncology Group status was 0 or 1, were eligible for inclusion in the study. Patients were randomly assigned (1:1) to gemogenovatucel-T or placebo by an independent, third-party automated response system after successful screening in permuted random block sizes of 2 and 4, and were stratified by the degree of surgical debulking and neoadjuvant versus adjuvant chemotherapy. Gemogenovatucel-T (1 × 10 per injection) was administered. 7 Gemogenovatucel-T (1000 cells) or placebo (once monthly) was administered intradermally for a minimum of four doses and up to 12 doses. Patients, investigators, and clinical staff were blinded to patient allocation until statistical analysis was completed. The primary endpoint was recurrence-free survival, which was analyzed in the per-protocol population. All patients who received at least one dose of gemogenovatucel-T were included in the safety analysis. This study is registered under ClinicalTrials NCT02346747.

[0400] Knowledge Between February 11, 2015, and March 2, 2017, 310 patients consented to this study at 22 centers. 217 patients were excluded. 91 patients received gemogenovatucel-T (n=47) or placebo (n=44) and were analyzed for safety and efficacy. Median follow-up was 40.0 months (IQR 35.0-44.8) from the first dose of gemogenovatucel-T and 39.8 months (35.5-44.6) from the first dose of placebo. Recurrence-free survival was 11.5 months (95% CI 7.5-not reached) for patients assigned to gemogenovatucel-T compared with 8.4 months (7.9-15.5) for patients assigned to placebo (HR 0.69, 90% CI 0.44-1.07; one-sided p=0.078). There were no grade 3 or 4 toxic effects with gemogenovatucel-T. Two patients in the placebo group experienced five grade 3 toxic events, including joint pain, bone pain, global muscle weakness, syncope, and dyspnea. Seven patients (four in the placebo group and three in the gemogenovatucel-T group) experienced 11 serious adverse events. No treatment-related deaths were reported in either group.

[0401] Interpretation: Frontline use of gemogenovatucel-T immunotherapy as maintenance therapy was well tolerated, but the primary endpoint was not met. Further study of gemogenovatucel-T in patients stratified by BRCA mutation status is warranted.

[0402] Most women diagnosed with ovarian cancer present at an advanced stage. Optimal standard treatment can achieve 5-year survival rates that vary depending on the stage, ranging from 41% (stage IIIa) to 20% (stage IV). Standard treatment for newly diagnosed ovarian cancer (stage III or IV) involves primary cytoreductive surgery followed by adjuvant chemotherapy with paclitaxel and carboplatin, or neoadjuvant chemotherapy followed by cytoreductive surgery. While most patients achieve complete remission with either approach, approximately 75% will relapse within 2 years.

[0403] Several studies involving bevacizumab and poly(ADP-ribose) polymerase (PARP) inhibitors have attempted to improve outcomes in frontline-treated ovarian cancer by administering maintenance therapy after patients achieve a complete response. However, despite the benefit in progression-free survival, to our knowledge, no studies have demonstrated a significant benefit in recurrence-free survival or overall survival. Additionally, the moderate drug-related toxicities of both bevacizumab and PARP inhibitors limit their use. PARP inhibitors have provided clinicians with a new platform for frontline maintenance therapy in ovarian cancer, but activity has been seen primarily in patients with germline and somatic BRCA mutations. PARP inhibitors are also approved for relapsed platinum-sensitive maintenance therapy, regardless of BRCA status. However, the magnitude of benefit is greatest in patients with BRCA-mutated (BRCAmut) tumors or evidence of homologous recombination repair defects.

[0404] Gemogenovatucel-T is an autologous tumor cell vaccine manufactured from harvested tumor tissue and transfected in vitro with a multigene plasmid encoding the human granulocyte-macrophage colony-stimulating factor (GMCSF) gene, immune-stimulating cytokines, and bifunctional short hairpin RNA (bi-shRNA) constructs, which specifically downregulates furin and its downstream TGF-β1 and TGF-β2. Activation of TGF-β1 and TGF-β2 is not the only function of furin; cleavage of furin activates several other proteins, including growth factors, cytokines, hormones, and receptors. However, TGF-β protein expression is significantly higher in malignant ovarian tissue than in nonmalignant ovarian tissue. Pathway analysis of a five-gene signature demonstrated increased TGF-β signaling in patients with ovarian cancer with poor prognosis (6). TGF-β is involved in the progression of noninvasive serous ovarian tumors to invasive serous ovarian cancer.

[0405] Furthermore, overexpression of TGF-β is associated with germline and somatic BRCA mutations. PARP inhibitors increase tumor cell proliferation and metastasis, and are also approved for platinum-sensitive tumors that recur in patients with suboptimally debulked ovarian cancer.

[0406] In early clinical trials examining TGF-β DNA products, GMCSF DNA products, the magnitude of benefit was greatest in patients with BRCA (including a phase 1 trial of gemogenovatucel-T in mutated (BRCAmut) tumors) or with evidence of relapsed or refractory solid tumors, with enzyme-linked immunospot assays demonstrating safe intradermal administration of 1 × 10 per dose per month. 7 It has been shown that autologous peripheral blood mononuclear cells (PBMCs) upregulate autologous peripheral blood mononuclear cell gamma interferon responses in response to autologous tumor antigens (9-11). A recurrence-free survival benefit and a correlated treatment-related immune response were demonstrated in a phase 2a trial using gemogenovatucel-T as frontline maintenance therapy in patients with stage III or IV resectable ovarian cancer. Given the limitations of frontline treatment for advanced ovarian cancer, particularly in patients with BRCA wild-type (BRCA) disease, we initiated the VITAL study. The objective of this study was to determine the safety and efficacy of gemogenovatucel-T in frontline ovarian cancer maintenance therapy.

[0407] method Study design and participants This phase 2b, randomized, double-blind, placebo-controlled trial involved 25 hospitals in the United States, of which 22 enrolled patients. (The other three did not enroll patients after Institutional Review Board approval.) Women aged 18 years or older with stage III / IV high-grade serous, endometrioid, or clear cell ovarian cancer who had achieved a complete clinical response after surgery and five to eight cycles of chemotherapy, including carboplatin and paclitaxel, were eligible for inclusion in the study. Patients had to have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 1 and normal organ and bone marrow function. Concurrent maintenance therapy with a PARP inhibitor or bevacizumab was not permitted as a protocol requirement. Patients were ineligible for study enrollment if they required a chronic steroid or immunosuppressive regimen and had or had a history of congestive heart failure, unstable angina, ventricular or hemodynamically significant atrial arrhythmias, cardiovascular disease, myocardial infarction, brain metastases, HIV, chronic hepatitis B or C infection, previous solid organ or bone marrow transplantation, or active autoimmune disease. Patients were also excluded if they had histologically confirmed papillary serous adenocarcinoma of the uterus, any disease involving the myometrium or endothelium, or brain metastases.

[0408] Tumor harvesting for vaccine production was performed during laparoscopic cytoreduction before neoadjuvant therapy or at the time of cytoreduction surgery in patients not receiving neoadjuvant therapy. All tumor harvesting procedures were performed before any chemotherapy.

[0409] A washout period of 3 weeks was required after chemotherapy, 4 weeks after surgery involving general anesthesia, radiation therapy, immunotherapy, or investigational drugs, and 14 days after immunosuppressive therapy. All patients provided written informed consent before tissue collection. A Drug Safety Monitoring Board (BSMB) was established before the start of the study to maintain the safety of all patients. No toxic effects of grade 3 or 4 or higher were observed. The DSMB's recommendation was to continue the study as planned. At each institution, written institutional review board and ethical approval of the protocol and written informed consent were required before patient enrollment was permitted.

[0410] Randomization and masking Eligible study participants were randomly assigned (1:1) to receive gemogenovatucel-T or placebo. Patients were randomly assigned throughout the study by Clinipace (Morrisville, NC, USA) through an automated response system (Tempo system; Clinipace; Morrisville, NC, USA) in random permuted block sizes of two and four. This occurred 3–8 weeks after chemotherapy completion. Randomization was stratified by the extent of surgical cytoreduction (microscopic or macroscopic) and neoadjuvant versus adjuvant chemotherapy. To ensure masking was maintained, site pharmacists were required to wrap concealment tape around the barrel of the syringe used for treatment administration to make the medication indistinguishable from placebo. Study staff, investigators, and patients were all blinded to study assignment. The sponsor's research team, except for quality assurance staff, was blinded to patients' treatment assignments.

[0411] procedure Gradalis (Carrollton, TX, USA) manufactured gemogenovatucel-T from harvested tumor tissue. Tumor harvest for vaccine production occurred before any chemotherapy. Placebo was manufactured at the same dose based on the number of gemogenovatucel-T vials. 10–30 g of tumor tissue was required for vaccine production. Lesions extending into the intestinal lumen were excluded due to the risk of bacterial infection. The placebo consisted of freezing medium, dispensed into sterile 2.0 mL borosilicate glass vials to a final fill volume of 1.2 mL. After slow freezing at −80°C, the vials were stored in vapor-phase liquid nitrogen during release testing for sterility and endotoxin. The placebo vial product matched the available product dose manufactured for the patient. Exceptions to protocol enrollment were granted based on partial plasmid-mediated increased GMCSF expression or decreased (knockdown) TGF-β1 expression, which provided sufficient evidence of plasmid transfection. Further details are provided in Table 12 below. [Table 12]

[0412] Patients received gemogenovatucel-T or placebo at 1 × 10 cells per intradermal injection monthly (within 8 weeks after their last chemotherapy treatment) for at least four doses and up to 12 doses. Protocol-defined treatment was discontinued after the following events: unacceptable (grade 3 or higher) toxic effects considered by the patient's physician to be related to treatment; grade 3 or 4 toxic effects unrelated to treatment that did not resolve within 4 weeks; grade 3 or higher allergic reaction to the study drug; grade 2 autoimmune reaction unless there was evidence of clinical benefit; grade 3 or higher autoimmune reaction; any illness that could affect the assessment of study endpoints; nonprotocol therapy (including chemotherapy); patient noncompliance; or withdrawal of consent. Criteria for dose modification included toxic effects of grade 1 or worse according to the National Cancer Institute Common Toxicity Criteria, but excluded injection-site reactions (grade 2 or 3) related to study treatment (at which point the dose would be reduced by 50%).

[0413] A treatment delay of up to 4 weeks was acceptable to allow for recovery. In the case of treatment delays due to other factors unrelated to toxic events, injections were given within 3 days. If the delay was due to infection or disease symptoms, a 2-week delay was acceptable.

[0414] Baseline testing consisting of complete blood cell differential count, C-125, and serum chemistry was performed along with CT of the chest, abdomen, and pelvis. Germline or somatic BRCA1 and BRCA2 molecular profiling data were collected on all tissue and blood samples and analyzed centrally using next-generation sequencing (Ocean Ridge Biosciences; Deerfield Beach, FL, USA).

[0415] Patients were monitored for disease progression by CT of the chest, abdomen, and pelvis every 3 months for the first 3 years and at the end of treatment or relapse. CT scans were performed every 6 months during follow-up, planned for up to 5 years. Patients remained on study treatment until disease progression, as defined by a masked independent central reviewer (World Care Clinical; Boston, Massachusetts, USA; using Response Evaluation Criteria in Solid Tumors version 1.1), or until gemogenovatucel-T or placebo supplies were exhausted (a minimum of four doses based on prior clinical immune response). All 12 laboratory assessments (complete blood cell differential count, C-125, and serum chemistry) were performed monthly while enrolled in the study. Laboratory assessments were repeated upon disease relapse or treatment termination and continued every 6 months thereafter during follow-up.

[0416] Adverse events were recorded after the first dose of gemogenovatucel-T and continued until 30 days after the last study treatment. Adverse events were graded and reported using the Common Toxicity Criteria for Adverse Events, version 4.03. All adverse events were recorded, regardless of whether they were related to study treatment.

[0417] Outcome The primary endpoint was recurrence-free survival from the time of randomization. Secondary endpoints in the priority order were recurrence-free survival from the time of tumor tissue collection for patients with BRCA disease, BRCA disease recurrence from the time of randomization, recurrence-free survival from the time of tumor tissue collection for all patients, overall survival from the time of randomization for all patients, and overall survival from the time of tumor tissue collection for all patients. The time of collection was the time of autologous tumor cell collection after surgery. Recurrence-free survival from the time of randomization or tumor tissue collection was defined as the time from the date of random assignment or collection to the date of the patient's first documented disease recurrence (even if the patient stopped treatment due to toxic effects) or the date of death if the subject died of any cause before disease recurrence. Disease recurrence was defined as the appearance of any measurable or evaluable disease or a subclinical CA-125 level >35 U / ml on two consecutive measurements at least one month apart. Overall survival was defined as the time from the date of random assignment or tumor tissue collection to the date of death.

[0418] statistical analysis Based on a sample size calculation, 54 recurrence-free survival events were required to achieve 90% efficacy, an estimated true hazard ratio (HR) of 0.45, and a one-sided alpha level of 0.05. After analyzing the primary endpoint, secondary endpoints will be examined using a hierarchical design according to the order listed in the statistical analysis plan. This design was used to adjust for multiplicity. The primary efficacy analysis was based on the per-protocol population, which included patients who were randomly assigned to receive at least one dose of their assigned study treatment, attended at least 80% of study visits, and had no major protocol deviations. All patients who received at least one dose of gemogenovatucel-T or placebo were included in the safety analysis. As a sensitivity analysis, recurrence-free survival and overall survival from randomization were also analyzed in the intention-to-treat population (a total of 92 randomly assigned patients). Distributions of recurrence-free survival and overall survival were estimated using the Kaplan-Meier method and compared using the stratified log-rank test. A one-sided p-value of 0.05 or less (stratified log-rank test stratified by the randomization stratification factors of residual disease and chemotherapy schedule) was considered significant. 90% CIs are shown for comparisons of treatment effects, and 95% CIs are shown for individual statistics. HRs were assessed by Cox proportional hazards models stratified by randomization stratification factors. The Grambsch and Therneau test was performed at the two-sided 0.05 significance level to check the proportional hazards assumption for the stratified Cox models.

[0419] Additional exploratory post-hoc analyses of 1- and 2-year recurrence-free and overall survival were estimated using the Kaplan-Meier method and compared using the asymptotic Z test. The variance of the difference in proportions was estimated using Greenwood's method. The bounded mean survival time difference was calculated as a sensitivity analysis (the cutoff point was equal to the minimum of the longest follow-up time in either study group). The bounded mean survival time difference was calculated without adjusting for covariates. Recurrence-free and overall survival from randomization or tumor tissue collection for patients with germline and somatic BRCA1 or BRCA2 mutations and patients with BRCA mutations (non-mutated group) were preplanned subgroup analyses. The inclusion of both survival endpoints more accurately reflected previous maintenance therapy trials that incorporated arms with and without combination therapy. The proportion of patients whose disease relapsed was analyzed using Fisher's exact t-test. Forest plots were constructed for planned subgroup analyses for all patients and for patients with BRCA disease (by age, disease stage, ECOG performance status, timing of chemotherapy, residual disease status, BRCA mutation status, TGF-β1 (% knockdown), granulocyte-macrophage colony-stimulating factor expression, survival, and number of vaccines produced). P interactions were calculated to assess effect modification between BRCA subgroups. To assess the effect of modification between BRCA subgroups on recurrence-free survival, a post-hoc analysis of the p-value of the interaction term between BRCA status and treatment was performed by including the interaction term in the Cox proportional hazards model.

[0420] All statistical analyses planned before blinding were performed independently (Stat Beyond Consulting, Irvine, CA, USA). All statistical analyses were performed in R version 4.0.0. This study is registered with ClinicalTrials.gov, NCT02346747.

[0421] result Between February 11, 2015, and March 2, 2017, 310 patients consented to the study at 22 centers, from which 309 samples were produced. 217 patients were excluded (Figure 34). 91 patients received gemogenovatucel-T (n=47) or placebo (n=44) and were analyzed for safety and efficacy. 67 patients were BRCA and 24 were BRCAmut (Appendix, p. 16). Patient baseline characteristics are shown in Table 13. [Table 13-1] [Table 13-2]

[0422] Eighty-four of 91 patients (92%) met all inclusion criteria. Median harvested tumor mass was 50 g (IQR 31-75). Median survival at inclusion was 88% (IQR 83-90; assessed in 91 patients). Seven of 91 patients (8%) (5 patients assigned to gemogenovatucel-T and 2 patients assigned to placebo) had GMCSF expression >10% after plasmid transfer. 6 None of these patients showed an increase of more than 30 pg / mL per cell. However, 6 of these 7 patients (86%) had sufficient TGF-β1 knockdown of at least 30%. The median evaluable GMCSF production was 10 6The median evaluable TGF-β1 level was 860 pg / mL per cell (IQR 254-2506, 84 patients; 7 patients were below the threshold), and the median evaluable TGF-β1 level was 100% (100-100, 84 patients; Table 12). TGF-β1 knockdown outcomes were indeterminate in 7 patients (6 with adequate GMCSF expression; 4 patients assigned to gemogenovatucel-T and 3 to placebo). 78 of 91 patients (86%) had at least 80% TGF-β1 knockdown. Because TGF-β1 signaling is downstream of Furin expression, these results support the robust activity associated with Furin bi-shRNA knockdown. Baseline TGF-β1 production before plasmid transfection was 10 6 The median TGF-β1 expression was 164 pg per cell (IQR 119-336; mean 241 [SD 162]). TGF-β1 expression in BRCA1 and BRCA2 wild-type (BRCA) patients was 10 6 The median TGF-β1 expression was 164 pg per cell (IQR 125-350; mean 249

[0154] ). 6 The median value was 146 pg per cell (IQR 115-267; mean 219

[0183] ).

[0423] The median follow-up was 40.0 months (IQR 35.0-44.8) from the first dose of gemogenovatucel-T and 39.8 months (35.5-44.6) from the first dose of placebo. The median time from surgery to random assignment was 7.0 months (IQR 6.5-7.4) for patients in the gemogenovatucel-T group and 6.7 months (IQR 5.9-7.1) for patients in the placebo group. The median time from the end of prior chemotherapy treatment to the start of treatment was 1.6 months (IQR 1.4-1.8) for patients in the gemogenovatucel-T group and 1.5 months (IQR 1.2-1.9) for patients in the placebo group. As of January 21, 2020, 59 recurrence events had been observed in this study by independent third-party radiographic review. The recurrence-free survival (RFS) calculated from the time of randomization for patients assigned to gemogenovatucel-T compared with patients assigned to placebo was 11.5 months (95% CI 7.5 to not reached) versus 8.4 months (7.9 to 15.5; HR 0.69, 90% CI 0.44 to 1.07; one-sided p = 0.078; Figure 2A). Twenty-six patients in the gemogenovatucel-T group and 33 patients in the placebo group experienced a recurrence.

[0424] The proportional hazards assumption was met based on the Grambsch and Thernau test. At the time of efficacy assessment, 33 of 44 patients (75%) in the placebo group had relapsed disease compared with 26 of 47 patients (55%) in the gemogenovatucel-T group. Post-hoc testing showed that the 1-year relapse-free survival rate from the time of random assignment was 49% (95% CI 36-68) for patients in the gemogenovatucel-T group compared with 39% (27-58) for patients in the placebo group (p=0.18). Post-hoc testing showed that the 2-year relapse-free survival rate from the time of random assignment was 32% (95% CI 19-53) for patients in the gemogenovatucel-T group compared with 25% (15-44) for patients in the placebo group (p=0.26).

[0425] Recurrence-free survival from the time of tissue collection in all patients was longer in patients receiving gemogenovatucel-T than in patients receiving placebo (Figure 35B).

[0426] The secondary endpoint of recurrence-free survival, calculated from the time of tissue collection in patients with BRCA disease, was significantly longer in patients receiving gemogenovatucel-T than in patients receiving placebo (Figure 35D). At the time of efficacy assessment, 21 of 40 patients with BRCA disease in the gemogenovatucel-T group (52%) had relapsed disease compared with 21 of 27 patients with BRCA disease in the placebo group (78%). In a post-hoc analysis, the 1-year recurrence-free survival rate from the time of surgery or tissue collection was 81% (95% CI 69-95) in patients with BRCA disease in the gemogenovatucel-T group compared with 63% (47-84) in patients with BRCA disease in the placebo group (p=0.056). The 2-year recurrence-free survival rate from the time of surgery or tissue collection for patients with BRCA disease was 42% (95% CI 28-64) for patients in the gemogenovatucel-T group compared with 24% (11-49) for patients in the placebo group (p=0.073). Recurrence-free survival from randomization for patients with BRCA disease who received gemogenovatucel-T was significantly longer than that for patients who received placebo (21 of 40 patients [52%] in the gemogenovatucel-T group had a recurrence-free survival event compared with 21 of 27 patients [78%] in the placebo group; Figure 35C). In patients with BRCA disease, in a post hoc analysis, the 1-year recurrence-free survival rate from the time of randomization was 51% (95% CI 36-71) for patients in the gemogenovatucel-T group compared with 28% (15-53) for patients in the placebo group (p=0.036). In a post hoc analysis, the 2-year recurrence-free survival rate from the time of randomization for patients with BRCA disease was 33% (95% CI 20-57) for patients in the gemogenovatucel-T group compared with 14% (5-39) for patients in the placebo group (p=0.048).

[0427] Post-hoc recurrence-free survival from the time of random assignment and the time of surgery or tissue harvest for patients with BRCA1 or BRCA2 mutant disease is shown in Figure 41A and Figure 41B. A preplanned intention-to-treat analysis calculated from the time of random assignment and the time of surgery or tissue harvest showed similar results to the per protocol analysis (Figures 37A-37D).

[0428] Overall survival from the time of randomization or harvest was not significantly longer with gemogenovatucel-T than with placebo (Figures 35E and 35F). Thirteen of 47 patients (28%) died in the gemogenovatucel-T group compared with 17 of 44 patients (39%) in the placebo group. The intention-to-treat analysis of overall survival calculated from the time of random assignment and the time of surgery or tissue harvest showed results similar to the per-protocol analysis. The proportional hazards assumption was met for all secondary endpoint analyses based on the Grambsch and Thernau test.

[0429] In planned subanalyses of patients with BRCA disease, we observed benefits in the vaccine group assigned to gemogenovatucel-T in overall survival from randomization (median overall survival not reached, 95% CI 35.6 to not reached vs. 41.4 months, 26.9 to not reached; HR 0.49, 90% CI 0.24 to 1.01; p=0.049) and overall survival from time of tissue collection (median overall survival not reached, 95% CI 41.6 to not reached vs. 48.3 months, 32.3 to not reached; HR 0.49, 90% CI 0.24 to 1.00; p=0.047). Post hoc testing showed that the 1-year overall survival rate from the time of randomization was 100% (95% CI 100-100) for patients with BRCA disease in the gemogenovatucel-T group compared with 89% (78-100) for patients with BRCA disease in the placebo group (p=0.033). Post hoc testing showed that the 2-year overall survival rate from the time of random assignment was 90% (95% CI 79-100) for patients with BRCA disease in the gemogenovatucel-T group compared with 67% (51-89) for patients with BRCA disease in the placebo group (p=0.021). For patients with BRCAmut disease, no difference in overall survival was observed between patients in the gemogenovatucel-T group and those in the placebo group (Figures 40A-40D). p = 0.001 between patient BRCA status and treatment to assess effect variation between BRCA subgroups. 相互作用 The values ​​were 0.173 for recurrence-free survival from the time of randomization, 0.072 for overall survival from the time of randomization, and 0.090 for overall survival from the time of tissue collection. Baseline demographics (including pre-specified stratification factors and product inclusion criteria) for disease effects (planned subanalyses) are shown for recurrence-free survival from the time of randomization for patients with BRCA disease and all patients (Figures 36A-36B) and overall survival from the time of randomization (Figures 38 and 39).

[0430] BRCA in the gemogenovatucel-T and placebo groups WTThe marginal mean survival time differences between patients with disease were 7.2 months (90% CI 0.8 to 13.5; p = 0.032) for recurrence-free survival from the time of random assignment and 5.6 months (0.2 to 11.5; p = 0.057) for overall survival from the time of random assignment. A censoring point equal to the minimum of the longest follow-up time in either study group was used in the marginal mean survival analysis.

[0431] A median of six (range 1-12, IQR 5-10) gemogenovatucel-T injections or six (3-12, 6-9) placebo injections were administered per patient. One patient in the placebo group experienced a treatment delay, possibly due to a drug-related pelvic infection. During the study, two deaths due to disease occurred in the gemogenovatucel-T group compared with eight deaths in the placebo group, and no treatment-related deaths occurred in either group. Adverse events and dose modifications were not reported, and no patients were withdrawn from the study. No major protocol deviations affecting patient safety were reported.

[0432] The number of adverse events by grade in each treatment group is reported in Table 14. Two patients in the placebo group experienced grade 3 treatment-related toxicity (one patient experienced arthralgia, and one patient experienced bone pain, global muscle weakness, syncope, and dyspnea [these were serious adverse events]). No grade 3 treatment-related adverse events were reported with gemogenovatucel-T. Seven patients (four in the placebo group and three in the gemogenovatucel-T group) experienced 11 serious adverse events. All but one of these events was reported as unlikely or unrelated to study treatment. [Table 14-1] [Table 14-2]

[0433] Consideration Gemogenovatucel-T did not demonstrate an improvement in the primary endpoint of recurrence-free survival. However, several secondary endpoints were considered hypothesis-generating, showing significant improvements in recurrence-free survival and overall survival with gemogenovatucel-T compared with placebo, particularly in patients with BRCA tumors. These results suggest that patients with BRCA ovarian cancer may be more sensitive to gemogenovatucel-T than patients with BRCA-mutated ovarian cancer.

[0434] The current landscape for frontline treatment of advanced-stage ovarian cancer has remained virtually unchanged over the past 25 years. Despite significant efforts, the addition of alternative approaches, either as combination therapy or maintenance therapy, has, for the most part, not led to an overall change in the standard of care for all patients with ovarian cancer. One exception, however, is the development of PARP inhibitors. For example, niraparib as maintenance therapy in patients with stage III or IV ovarian cancer demonstrated improvements in progression-free survival (HR 0.62, 95% CI 0.50-0.76; p<0.001) and 2-year overall survival compared with placebo (HR 0.70, 95% CI 0.44-1.11). However, concerns have been raised about the high rate of grade 3 or 4 drug-related toxicities and toxicity-related discontinuations.

[0435] GMCSF is involved in increasing tumor antigen presentation by dendritic cells, leading to greater concentrations of costimulatory molecules and more efficient cell stimulation. GMCSF also promotes lipid antigen presentation by dendritic cells, which in turn leads to the activation of natural killer T cells. TGF-β secreted by ovarian cancer cells induces the proliferation of immunosuppressive regulatory T cells (CD4+, CD25+) in the tumor microenvironment. (26) This response has been associated with poor prognosis in patients with frontline-treated high-grade serous ovarian cancer. TGF-β inhibits GMCSF-induced dendritic cell-derived myeloid maturation and the expression of MHC-II and costimulatory molecules. TGF-β inhibits activated macrophages, including their antigen-presenting function, and also inhibits PD-L1 expression by ovarian cancer and tumor-associated myeloid cells, which is associated with poor overall survival in ovarian cancer patients. Knockdown of TGF-β1, as shown in this study, may contribute to suppressing the effects of TGFβ, which may be more important in patients with BRCA disease.

[0436] Few studies have examined immunotherapy in ovarian cancer. More recently, there has been case reports and phase 1 / 2 trial evidence of responses in ovarian cancer with checkpoint inhibitors, but several trials involving the combination of avelumab in frontline and recurrent ovarian cancer failed to demonstrate benefit. However, in one trial, 11 of 38 women with relapsed ovarian cancer achieved an objective response with the combination of nivolumab plus bevacizumab. Further studies involving checkpoint inhibitor therapy in patients with ovarian cancer are ongoing.

[0437] Given its well-tolerated nature, ease of administration, and promising efficacy, gemogenovatucel-T represents an ideal maintenance therapy for patients with ovarian cancer. One strength of our study results is its safety in patients with BRCA disease and the observed clinical benefits in recurrence-free survival and overall survival. However, BRCA subsets were secondary endpoints, and the use of autologous tumor harvest as a component of product manufacturing imposes limitations on the timing and manufacturing success rate of the product. While insufficient tumor tissue was associated with manufacturing failure, there is no evidence to suggest that the number of vaccines manufactured correlates with the number of vaccines administered to patients or their health status. The failure rate due to insufficient cells increased with smaller tumor tissue weights resected.

[0438] Because only a small subset of patients with BRCAmut disease received gemogenovatucel-T, our conclusions regarding negative effects in such patients require further study. Further evaluation of gemogenovatucel-T in combination with bevacizumab or other PARP inhibitors is planned. Furthermore, combination with checkpoint inhibitor therapy may further increase gemogenovatucel-T immune responses.

[0439] In conclusion, gemogenovatucel-T demonstrated benefit as frontline maintenance therapy in patients with ovarian cancer harboring a BRCA tumor molecular profile, a finding that warrants further investigation.

[0440] Example 6: Supplementary Information Study design and participants Normal organ and bone marrow function was required for protocol compliance and defined as protocol compliance (absolute granulocyte count ≥ 1,500 / mm 3 Absolute lymphocyte count ≥ 500 / mm 3 ;platelets ≧75,000 / mm 3(Total bilirubin ≤ 2 mg / dL; AST(SGOT) / ALT(SGPT) ≤ 2 × institutional upper limit of normal; Creatinine < 1.5 mg / dL). All patients were required to be able to understand and willing to sign a written, protocol-specific consent form.

[0441] procedure Surgically resected tumor tissue was harvested and cut into ¼- to ½-inch sections, placed in specimen containers supplemented with gentamicin (Fresenius Kabi), and packaged on ice for overnight transport. On day 1, the sterility of the transport medium and tumor samples was checked (BacT / Alert 3D Microbial Identification System, BioMérieux). On day 1, tumor tissue was trimmed and dissociated with a scalpel, then enzymatically dissociated (type I collagenase solution) and incubated at 37°C to form a single-cell suspension. This cell suspension was filtered through a sterile 100 μm strainer (Corning) to separate cells from cellular debris. The freed cells were washed with PlasmaLyte (Baxter) supplemented with 1% human serum albumin (Octapharma) and manually counted using a hemocytometer (InCyto). Quality control (QC) samples were retained and removed for immune monitoring, and pre-transfection cultures were initiated to obtain baseline cytokine levels by ELISA for GMCSF (R&D Systems), TGFβ1 (R&D Systems), and TGFβ2 (R&D Systems). The suspension was adjusted to a concentration of 40 million cells / mL and electroporated using a Gene Pulser XL (BioRad) to facilitate plasmid insertion. The transfected cells were then transferred to sterile T-225cm tubes. 2 1 × 10 cells in X-VIVO 10 medium (Lonza) supplemented with gentamicin (Fresenius Kabi) in a 1 × 10 flask (Corning). 6The cells were plated at 100x the cell mass per mL and incubated overnight (14-22 hours) at 37°C in a 5% CO2 overlay (Sanyo) to allow the bi-shRNA Furin and GMCSF mRNA to be incorporated into the tumor cells.

[0442] On day 2, overnight cultures were harvested and resuspended in fresh X-VIVO medium. A QC sample and a minimum of 4 doses per patient were required before proceeding.

[0443] Two 1-mL mycoplasma samples containing cells were collected and frozen at -80°C. Cells were irradiated with 4 × 25 Gy cycles using a gamma irradiator to arrest replication / proliferation. Cells were washed with PlasmaLyte (Baxter) supplemented with 1% human serum albumin (Octapharma), and a QC sample was removed for retention. Cells were placed in freezing medium consisting of PlasmaLyte (Baxter) at pH 7.4 with 10% DMSO (dimethyl sulfoxide; Cryoserv USP; Mylan), 1% HSA (Octapharma), and frozen at 1 × 10 6 cells / mL or 1 x 10 7Cells were aseptically dispensed at 1.2 cells / mL into sterile 2.0 mL borosilicate glass vials (Algroup Wheaton Pharmaceutical and Cosmetic Packaging); the final fill volume was adjusted to 1.2 mL and sealed with butyl rubber stoppers coated with Flurotec® barrier files (West Pharmaceutical Services). Final product vials were frozen at a controlled rate using CoolCell® freezing containers (Biocision) placed in a -80°C freezer (Sanyo). Product release testing specifications are listed in Table 3 in the Appendix. After freezing, cells were stored in a vapor-phase liquid nitrogen tank during release testing. Frozen product vials were tested for sterility (USP 1000) by gelling (Limulus Amoebocyte Lysate, Lonza). <71> ) and endotoxin testing. Product release testing specifications for individual patients are summarized in Table 12 above. Assay validation for (GMCSF, TGFβ1, TGFβ2) was completed post-study, and the data shown were calculated using appropriately validated parameters. As a result, TGFβ2 knockdown is no longer used as a product release criterion.

[0444] The placebo consisted of a freezing medium consisting of Plasma-Lyte A (Baxter) at pH 7.4 containing 10% DMSO (Cryoserv USP; Mylan) and 1% HSA (Octapharma). The medium was slowly cooled to -80°C and frozen, after which vials were stored in vapor phase liquid nitrogen pending release testing for sterility and endotoxin. The placebo vial product matched the available product dose manufactured for the subjects.

[0445] Sample processing for NGS sequencing High-molecular-weight (HMW) DNA was isolated from cryogenically stored samples using the Qiagen MagAttract HMW DNA Kit. The HMW DNA was fragmented using dsDNA Fragmentase, which targets nucleic acids in the 200-300 nt range. DNA libraries were generated using the fragmented HMW DNA with an input of 5 ng using the SMARTer ThruPLEX DNA-Seq Kit. These libraries were combined into 5-6x pools and hybridized. Gene sequences were selected using the SeqCap EZ Human Oncology Panel (981 genes, 2.75 Mb of exonic region) in combination with the HyperCap Target Enrichment Kit. The gene-selected libraries were pooled and sequenced using the NextSeq 500 Mid Output v2.5 (300 cycles) kit.

[0446] Samples were run on a NextSeq 500. Variant cells were generated using GATK HaplotypeCaller v4.1.2.0. The .vcf files were split into SNP and indel portions using GATK SelectVariants v4.1.2.0, and each file was counted using GATK CountVariants v4.1.2.0. After extracting the variant list from the IVA software, the variants were further filtered to retain specific classes of polymorphisms based on the American College of Medical Genetics and Genomics (ACMGG) classification guidelines implemented in IVA (33) and supplemented by ClinVar1. The ACMGG classification guidelines group predicted deleterious variants into five possible categories: pathogenic, likely pathogenic, of uncertain significance, likely benign, and benign. Classification is based on missense predictions, splice site predictions, nucleotide conservation predictions, well-established functional studies, and many other factors. The predicted worsening column in Table IV includes the total number of BRCA and non-BRCA HRD mutations identified in each of the 17 samples belonging to all five IVA pathogenicity categories described above. This final classification assigned by ORB was based on selecting the most stringent classification from IVA or ClinVar for variants originally assigned by IVA as pathogenic, likely pathogenic, or of uncertain significance. Variants called likely benign or benign by IVA were retained in the ORB classification. Variants found in more than 20% of samples (both PBMC and tumor samples) were filtered out, along with variants appearing in more than 5% of the general population in the Allele Frequency Community (AFC), 1000 Genomes, EXac, or GnomAD databases. Variants were classified as germline or somatic by comparing the allele proportion of each variant in tumor samples with that in PBMC samples.A variant was classified as germline if it had a tumor sample allele fraction that was 0. If it had a tumor sample allele fraction greater than zero and a PBMC sample allele fraction that was zero, the variant was classified as somatic. If both allele fractions were greater than zero, the variant was classified as germline, but if the tumor sample allele fraction was greater than 10-fold, the variant was classified as somatic. [Table 15-1] [Table 15-2]

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[0480] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It should be understood that the present disclosure can be practiced utilizing various alternatives to the embodiments of the disclosure described herein. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A pharmaceutical composition for the treatment of cancer, the pharmaceutical composition comprising an expression vector, wherein the pharmaceutical composition is administered to an individual in need thereof, and wherein the expression vector comprises: a. a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; and b. a second insert comprising the sequence set forth in SEQ ID NO:4; Including, the individual is homologous recombination deficiency (HRD) negative, and / or The pharmaceutical composition, wherein the individual has a wild-type BRCA1 gene, a wild-type BRCA2 gene, or a combination thereof.

2. 2. The pharmaceutical composition of claim 1, wherein the cancer is selected from the group consisting of solid tumor cancer, ovarian cancer, adrenocortical carcinoma, bladder cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, glioblastoma, glioma, hepatocellular carcinoma, head and neck cancer, kidney cancer, leukemia, lymphoma, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, prostate cancer, sarcoma, gastric cancer, uterine cancer, thyroid cancer, and blood cancer.

3. 3. The pharmaceutical composition of claim 2, wherein the cancer is ovarian cancer, breast cancer, melanoma, or lung cancer.

4. 3. The pharmaceutical composition of claim 2, wherein the solid tumor cancer is selected from the group consisting of endometrial cancer, bile duct cancer, bladder cancer, hepatocellular carcinoma, gastric / esophageal cancer, ovarian cancer, melanoma, breast cancer, pancreatic cancer, colorectal cancer, glioma, non-small cell lung cancer, prostate cancer, cervical cancer, kidney cancer, thyroid cancer, neuroendocrine cancer, small cell lung cancer, sarcoma, head and neck cancer, brain cancer, renal clear cell carcinoma, skin cancer, endocrine tumors, thyroid cancer, tumors of unknown primary, and gastrointestinal stromal tumors.

5. 5. The pharmaceutical composition of any one of claims 2 to 4, wherein ovarian cancer is substantially eradicated in the individual, and wherein the pharmaceutical composition prevents or delays regrowth of the substantially eradicated ovarian cancer.

6. 6. The pharmaceutical composition of claim 5, wherein the substantially eradicated ovarian cancer is stage III or stage IV ovarian cancer.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the expression vector is administered into autologous cancer cells.

8. 1. A pharmaceutical composition for treating ovarian cancer that is homologous recombination repair deficient (HRD) negative and / or has a wild-type BRCA1 gene, a wild-type BRCA2 gene, or a combination thereof, wherein the pharmaceutical composition comprises autologous tumor cells transfected with an expression vector, and wherein the treatment of ovarian cancer comprises administering the autologous tumor cells to an individual in need thereof, wherein the expression vector comprises: a. a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; and b. A second insert comprising the sequence set forth in SEQ ID NO:

4.

9. 9. The pharmaceutical composition of claim 8, wherein the treatment of ovarian cancer comprises administering to the individual one or more doses of an autologous tumor cell vaccine comprising the autologous tumor cells.

10. 10. The pharmaceutical composition according to claim 8 or 9, wherein the autologous tumor cells are an autologous tumor cell vaccine.

11. 1. A pharmaceutical composition for preventing or delaying the recurrence of ovarian cancer in an individual in need thereof, wherein the pharmaceutical composition comprises an expression vector, wherein the pharmaceutical composition is administered to the individual, and wherein the expression vector comprises i. a first insert comprising a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) sequence; and b. A second insert comprising the sequence set forth in SEQ ID NO:4 Including, the individual is homologous recombination repair deficient (HRD) negative, and / or The pharmaceutical composition, wherein the individual has a wild-type BRCA1 gene, a wild-type BRCA2 gene, or a combination thereof.

12. 12. The pharmaceutical composition of any one of claims 8 to 11, wherein the individual is homologous recombination repair deficient (HRD) negative and has a wild-type BRCA2 gene; or the individual is homologous recombination repair deficient (HRD) negative and has a wild-type BRCA1 gene; or The pharmaceutical composition, wherein the individual is homologous recombination repair deficient (HRD) negative and has a combination of wild-type BRCA1 and wild-type BRCA2 genes.

13. The pharmaceutical composition according to any one of claims 8 to 12, wherein the ovarian cancer is stage III or stage IV ovarian cancer, and / or the ovarian cancer is refractory ovarian cancer.

14. The pharmaceutical composition of claim 13, wherein the refractory ovarian cancer is refractory to chemotherapy.

15. 15. The pharmaceutical composition of claim 14, wherein the chemotherapy comprises a platinum agent or a taxane.

16. 16. The pharmaceutical composition of claim 15, wherein the platinum agent comprises carboplatin and the taxane comprises paclitaxel.

17. The pharmaceutical composition of any one of claims 8 to 16, wherein an additional therapeutic agent is further administered.

18. 18. The pharmaceutical composition of claim 17, wherein the additional therapeutic agent is selected from the group consisting of an angiogenesis inhibitor, a PARP inhibitor, and a checkpoint inhibitor administered to the individual.

19. 19. The pharmaceutical composition of claim 18, wherein the angiogenesis inhibitor is a vascular endothelial growth factor (VEGF) inhibitor and the checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, and ipilimumab.

20. 20. The pharmaceutical composition of claim 19, wherein the VEGF inhibitor is selected from the group consisting of sorafenib, sunitinib, bevacizumab, pazopanib, axitinib, cabozantinib, and levatinib.

21. 21. The pharmaceutical composition of claim 20, wherein the VEGF inhibitor is bevacizumab and the PARP inhibitor is selected from the group consisting of niraparib, olaparib, rucaparib, niraparib, talazoparib, veliparib, and pamiparib.

22. The pharmaceutical composition of any one of claims 1 to 21, wherein the GM-CSF is a human GM-CSF sequence.

23. The pharmaceutical composition of any one of claims 1 to 22, wherein the expression vector further comprises a promoter.

24. 24. The pharmaceutical composition of claim 23, wherein the promoter is a cytomegalovirus (CMV) mammalian promoter.

25. 25. The pharmaceutical composition of claim 23 or 24, wherein the expression vector further comprises a CMV enhancer sequence and a CMV intron sequence.

26. 26. The pharmaceutical composition of any one of claims 23 to 25, wherein the first insert and the second insert are operably linked to the promoter.

27. 27. The pharmaceutical composition of any one of claims 1 to 26, wherein the expression vector further comprises a nucleic acid sequence encoding a picornaviral 2A ribosomal skipping peptide between the first nucleic acid insert and the second nucleic acid insert.

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