Composition containing bacterial-derived minicells and method of using the same

Abstract

To provide pharmaceutical compositions for treating cancer.SOLUTION: An anti-cancer agent composition comprises: (a) a therapeutically effective dose of purified, intact, bacterially derived minicells comprising at least one anti-neoplastic agent comprising morpholinyl anthracycline; and (b) an interferon type II agonist selected from among C-glycoside-type α-galactosylceramide (α-C-GalCer), β-D-glucopyranosylceramide (β-GlcCer), 1,2-diacyl-3-galactosyl-sn-glycerol (BbGL-II), diacylglycerol containing glycolipids (Glc-DAG-s2), ganglioside (GD3), and the like.SELECTED DRAWING: None

Description

Reference to related applications

[0001] This application claims priority under 35 U.S.C. § 119 to U.S. Provisional Application 62 / 702,172 filed on 23 July 2018 and U.S. Provisional Application 62 / 788,265 filed on 4 January 2019, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Currently, most drugs used to treat cancer are administered systemically. While systemic delivery of cytotoxic anticancer drugs plays a crucial role in cancer treatment, it also presents serious problems. For example, systemic exposure of normal tissues / organs to administered drugs can cause severe toxicity. This is exacerbated by the fact that systemically administered cancer chemotherapy drugs often have to be delivered at very high doses to overcome the low bioavailability and large volume of distribution within the patient. Furthermore, systemic drug administration can be invasive, as it often requires the safe use of catheters in major blood vessels. Systemic drug administration often requires the use of either peripheral or central veins, potentially leading to local complications such as phlebitis. Extravasation of drugs can also result in blistering / tissue damage at the local administration site, as is commonly seen with the administration of vinca alkaloids and anthracyclines.

[0003] Another challenge in cancer treatment is endogenous or acquired clinical tumor resistance to chemotherapy. In tumors that do not respond to first-line chemotherapy, endogenous resistance is present at diagnosis. Acquired resistance occurs in tumors that may respond well to initial treatment but exhibit a resistance phenotype upon recurrence. Such tumors acquire resistance to both previously used drugs and new drugs, including those with different structures and mechanisms of action. The term MDR (multidrug resistance) refers to the phenomenon in which tumor cells, after exposure to one drug, develop cross-resistance to several structurally unrelated drugs. The mechanisms of multidrug resistance are complex and multifactorial, primarily due to the high levels of genomic instability and mutation in cancer cells. Exemplary mechanisms include drug inactivation, drug efflux by cell membrane pumps, reduced drug influx, mutations in drug targets, and impaired initiation of apoptosis. Bredel, 2001; Chen et al., 2001; Sun et al., 2001; and White & McCubrey, 2001.

[0004] The interaction between the immune system and malignant cells also plays a crucial role in tumorigenesis. If the immune system is unable to detect and reject transformed cells, it can lead to cancer development. Tumors employ multiple mechanisms to evade immune-mediated rejection. Many of these mechanisms are now known at the cellular and molecular levels. Despite these findings, cancer immunotherapy is not yet an established treatment in clinical practice.

[0005] Therefore, there remains a great need for a delivery system that can provide targeted drug delivery that can reduce drug resistance, promote apoptosis, induce an immune response, and simultaneously avoid the problems associated with systemic delivery of these drugs. The present invention satisfies these needs. [Overview of the project] [Means for solving the problem]

[0006] One embodiment of the present invention relates to (a) a therapeutically effective amount of purified, intact bacterial minicells comprising an antitumor agent, and (b) a composition comprising an interferon type I agonist, an interferon type II agonist, or a combination of an interferon type I agonist and an interferon type II agonist. The interferon type I agonist and / or interferon type II agonist may optionally be present in the intact bacterial minicells.

[0007] In one embodiment, the composition comprises (a) a therapeutically effective dose of purified, intact bacterial minicells containing an antitumor agent, and (b) a therapeutically effective dose of purified, intact bacterial minicells containing an interferon type I agonist. In another embodiment, the composition comprises (a) a therapeutically effective dose of purified, intact bacterial minicells containing an antitumor agent, and (b) a therapeutically effective dose of purified, intact bacterial minicells containing an interferon type II agonist. In further embodiments, the composition comprises (a) a therapeutically effective dose of purified, intact bacterial minicells containing an antitumor agent; (b) a therapeutically effective dose of purified, intact bacterial minicells containing an interferon type I agonist; and (c) a therapeutically effective dose of purified, intact bacterial minicells containing an interferon type II agonist.

[0008] In one embodiment, the antitumor agent and an interferon type I agonist, an interferon type II agonist, or a combination of an interferon type I agonist and an interferon type II agonist are packaged within two or more purified, intact bacterial minicells. In another embodiment, the antitumor agent and an interferon type I agonist, an interferon type II agonist, or a combination of an interferon type I agonist and an interferon type II agonist are packaged within three separate populations of purified, intact bacterial minicells.

[0009] In one embodiment, the composition comprises an antitumor agent, an interferon type I agonist, and an interferon type II agonist, wherein (a) the antitumor agent, interferon type I agonist, and interferon type II agonist are contained within the same mini-cell, (b) the antitumor agent and interferon type II agonist are contained within a second mini-cell, (c) the antitumor agent and interferon type II agonist are contained within a second mini-cell, (d) the antitumor agent is contained within a first mini-cell, or (e) the antitumor agent is contained within a second mini-cell. The type II interferon agonist is contained within a third mini-cell.

[0010] In one embodiment, the composition does not contain an interferon type I agonist.

[0011] In one embodiment, the antitumor agent is selected from the group consisting of radionuclides, chemotherapeutic agents, functional nucleic acids, and polynucleotides capable of transcribing functional nucleic acids. In one embodiment, the antitumor agent is a supertoxic chemotherapeutic agent. In one embodiment, the supertoxic chemotherapeutic agent is selected from the group consisting of morpholinyl anthracyclines, meitansinoids, ducalmycin, auristatin, calicheamicin (DNA damaging agent), α-amanitin (RNA polymerase II inhibitor), centamicin (LiNK), pyrrolobenzodiazepine (LiNK), streptonigtin, nitrogen mustard, nitrosolair, alkanesulfonates, pyrimidine analogs, purine analogs, antimetabolites, folic acid analogs, anthracyclines, taxanes, vinca alkaloids, topoisomerase inhibitors, hormones, and combinations thereof. In one embodiment, the morpholinyl anthracycline is selected from the group consisting of nemorubicin, PNU159682, idarubicin, daunorubicin, camiomycin, and oxorubicin. In one embodiment, the supertoxic chemotherapeutic agent is PNU159682.

[0012] In one embodiment, the functional nucleic acid is selected from the group consisting of siRNA, miRNA, shRNA, lincRNA, antisense RNA, and ribozymes. In one embodiment, the functional nucleic acid inhibits genes that promote tumor cell proliferation, angiogenesis, or resistance to chemotherapy, and / or inhibit apoptosis or cell cycle arrest. In some embodiments, siRNA inhibits ribonucleotide reductase M1 (RRM1) expression. In some embodiments, siRNA inhibits polo-like kinase 1 (Plk1) expression. In some embodiments, miRNA is miRNA16a.

[0013] In one embodiment, the interferon type I agonist, the interferon type II agonist, or a combination of the interferon type I agonist and the interferon type II agonist is an oligonucleotide. In one embodiment, the oligonucleotide comprises a sequence of at least about 40 nucleotides, at least about 50 nucleotides, or at least about 60 nucleotides. In some embodiments, the oligonucleotide is a polynucleotide product of PNPase1, poly(I:C), polyICLC, imiquimod, imidazochiolinereskimod, cGAMP, or CpG oligodeoxynucleotide.

[0014] In one embodiment, the interferon type I agonist is selected from the group consisting of double-stranded RNA (dsRNA), poly(dA:dT)DNA, double-stranded ZDNA and BDNA, DNA longer than 36 bp and DNARNA hybrid (dsDNA), bacterial second messenger cyclic GMP, TLR3, TLR4, TLR7, TLR8 and TLR9 agonists, STING agonists, and combinations thereof.

[0015] In one embodiment, the interferon type II agonist is C-glycoside type α-galactosylceramide (α-C-GalCer), α-galactosylceramide (α-GalCer), 12-carbon acyl-type galactosylceramide (β-GalCer), β-D-glucopyranosylceramide (β-GlcCer), 1,2-diacyl-3-galactosyl-sn-glycerol (BbGL-II), and diacylglycerol (Glc-DAG-) containing glycolipids. The following are selected from the group consisting of s2), ganglioxide (GD3), gangliotriceramide (Gg3Cer), glycosylphosphatidylinositol (GPI), α-glucuronosylceramide (GSL-1 or GSL-4), isoglobotrihexosylceramide (iGb3), lipophosphoglycan (LPG), phosphatidylcholine (LPC), α-lactosylceramide analog (OCH), slaytolceramide, and combinations thereof. In one embodiment, the interferon type II agonist is α-galactosylceramide (α-GalCer).

[0016] In one embodiment, the composition further comprises a bispecific ligand bound to minicells containing an antineoplastic agent. In one embodiment, the composition further comprises a bispecific ligand bound to minicells containing a type I interferon agonist. In one embodiment, the composition further comprises a bispecific ligand bound to minicells containing a type II interferon agonist.

[0017] In one embodiment, the bispecific ligand comprises a first arm having specificity for a minicell surface structure and a second arm having specificity for a non-phagocytotic mammalian cell surface receptor. In one embodiment, the minicell surface structure is the O-polysaccharide component of lipopolysaccharide on the minicell surface. In one embodiment, the non-phagocytotic mammalian cell surface receptor can activate receptor-mediated endocytosis of the minicell.

[0018] In one embodiment, the bispecific ligand comprises a bispecific antibody or an antibody fragment. In one embodiment, the antibody or antibody fragment comprises a first multivalent arm having specificity for a bacterial-derived mini cell surface structure and a second multivalent arm having specificity for a cancer cell surface receptor, and the cancer cell surface receptor can activate receptor-mediated endocytosis of the mini cell.

[0019] In one embodiment, the composition comprises less than about 1 contaminating parental bacterial cell per 10 7 mini cells, less than about 1 contaminating parental bacterial cell per 10 8 mini cells, less than about 1 contaminating parental bacterial cell per 10 9 mini cells, less than about 1 contaminating parental bacterial cell per 10 10 mini cells, or less than about 1 contaminating parental bacterial cell per 10 11 mini cells.

[0020] In one embodiment, the composition further comprises a pharmaceutically acceptable carrier. In one embodiment, the mini cells have a diameter of about 400 nm. In one embodiment, the composition does not contain parental bacterial cell contamination removable by 200 nm filtration.

[0021] In one embodiment, the composition comprises the following amounts of mini cells or killed bacterial cells: (a) at least about 10 9 ; (b) at least about 1×10 9 ; (c) at least about 2×10 9 ; (d) at least about 5×10 9 ; (e) at least 8×10 9 ; (f) about 10 11 or less; (g) about 1×10 11 or less; (h) about 9×10 10 or less; or (i) about 8×10 10 or less. One embodiment of the present invention relates to a method of treating a subject in need thereof, the method comprising administering to the subject an effective amount of the composition disclosed herein. In one embodiment, the subject is a human, non-human primate, dog, cat, cow, sheep, horse, rabbit, mouse, or rat. In one embodiment, the subject is a human. In one embodiment, the subject has cancer. In one embodiment, the cancer is selected from the group consisting of lung cancer, breast cancer, brain cancer, liver cancer, colon cancer, anal cancer, pancreatic cancer, and bladder cancer. In one embodiment, the cancer is acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, appendiceal cancer, astrocytoma, atypical teratoma / rhabdoid tumor, basal cell carcinoma, bladder cancer, brainstem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, cancer of unknown primary site, carcinoid tumor, cancer of unknown major site, atypical teratoma-like / rhabdoid tumor of the central nervous system, germ cell tumor of the central nervous system, cervical cancer. Cancer, childhood cancer, chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, colorectal cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, endocrine pancreatic islet cell tumor, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, sensory neurosarcoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, bile duct cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal cell tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic tumor, glioma, hairy cell leukemia, Cardiac tumors, head and neck cancers, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip cancer, liver cancer, malignant fibrous bone cancer, liposarcoma, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of unknown primary origin, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasmacytic neoplasm, mycosis fungoides, myelodysplasia Symptoms, nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, oral cancer, oral carcinoma, oropharyngeal cancer, osteosarcoma, other brain and spinal cord tumors, ovarian germ cell tumors, low-grade ovarian tumors, pancreatic cancer, papillomatosis, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, pharyngeal cancer, pineal parenchymal cell tumors showing intermediate differentiation, pineoblastoma, pituitary tumors, plasma cell neoplasms / multiple myeloma, pleuropulmonary blastoma, Primary central nervous system (CNS) lymphoma, primary hepatocellular carcinoma, pleuroblastoma, primary hepatocellular carcinoma, prostate cancer, renal cell carcinoma, renal cell carcinoma, renal cell carcinoma, retinoblastoma, retinoblastoma, rhabdomyosarcoma, Sézary syndrome, salivary gland cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, cervical cancer, gastric (stomach) cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, testicular cancer, pharyngeal cancer, thymic carcinoma, thymoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, chorioblastoma, The group is selected from ureteral cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor. In one embodiment, brain cancer or brain tumor is selected from the group consisting of brainstem glioma, atypical teratomatoid / rhabdoid tumor of the central nervous system, germ cell tumor of the central nervous system, astrocytoma, craniopharyngioma, ependymoblastoma, ependymodium, medulloblastoma, medullary epithelioma, intermediately differentiated pineal parenchymal tumor, supratentorial primitive neuroectodermal tumor, and pineoblastoma. In one embodiment, the composition is administered at least once a week over several weeks. In another embodiment, the composition is administered at least once a week over several weeks to several months. In one embodiment, the composition is administered at least once a week for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks or more. In one embodiment, the composition is administered about twice a week. In one embodiment, the composition is administered twice a week for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks or more. The above summary and the following description and detailed description of the drawings are illustrative and descriptive. They are intended to provide further details of the invention, but should not be construed as limiting. Other purposes, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the invention. [Brief explanation of the drawing]

[0022] [Figure 1] This is a graph plot of EnGeneIC Dream Vehicles (EDVs) (e.g., bacterial mini-cells) loaded with the anticancer drug PNU159682 (anthracycline analog), which contains bispecific antibodies against O-polysaccharides and human epidermal growth factor receptor antigen. [Figure 2] This graph shows the EnGeneIC Dream Vehicle (EDV), which contains O-polysaccharides on its surface and is loaded with immunomodulatory 60-mer double-stranded DNA. [Figure 3]This graph shows the EnGeneIC Dream Vehicle (EDV), which contains O-polysaccharides on its surface and is loaded with immunomodulatory α-galactosylceramide (αGC). [Figure 4] This is a graphical overview of clinical trials evaluating EGFR targeting and miRNA16a-loaded EDV for the treatment of mesothelioma patients. [Figure 5] This figure shows the cytotoxic effects of the indicated chemotherapy drugs against the A549 lung cancer cell line. Figure 4A compares the effects of the indicated chemotherapy drugs with those of the highly toxic drug PNU159682. Figure 4B compares the effects of doxorubicin and PNU159682. [Figure 6] This figure shows the effects of the chemotherapeutic agents on adrenocortical cancer cell lines ACC01 (Figure 6A) and ACC07 (Figure 6B). [Figure 7] This figure shows the effects of chemotherapeutic drugs on the MDAMB468 breast cancer cell line. [Figure 8] This figure shows the effects of chemotherapeutic drugs on human colorectal cancer cell lines Caco2 (Figure 8A) and HCT116 (Figure 8B). [Figure 9] This figure shows the effects of the indicated chemotherapy drugs on the glioblastoma cell line U87MG. [Figure 10] This figure shows the effects of chemotherapeutic agents on the human pancreatic cell line MiaPaca2 (Figure 10A) and gemicitabine-resistant MiaPaca2 GemR cells (Figure 10B). [Figure 11] This figure shows the effect of EGFR-targeted EDVs loaded with PNU-159682 (EGFREDV682™) or Adriamycin (EGFREDVDox™) on the growth of A549 xenograft tumors in mice. Negative controls are saline alone or untargeted EDV loaded with PNU-159682 (EDV682™). Arrows indicate when mice were treated with the indicated saline or EDV composition. Asterisks indicate when mice initially treated with saline were controlled with the EGFREDV682™ composition. [Figure 12]This figure shows the expression levels of GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (G), KSP (kinesin spindle protein), Plk1 (polo-like kinase 1) (P), and RRM1 (ribonucleotide reductase enzyme 1) (R) compared to GAPDH expression in the NSCLC cell line shown. [Figure 13] This figure shows the effect of delivering EGFR-targeted siRRM1-packaged EDV to mesothelioma cell lines (MSTO, Figure 13A) or adrenocortical carcinoma cell lines (H295R, Figure 13B). [Figure 14] This figure shows the effect of delivery of EGFR-targeted miRNA16a (EGFREDVmiRNA16a™) or EGFR-targeted siRRM1-packaged EDV (EGFREDVsiRRM1™) on mesothelioma xenograft tumor growth in Balb / c nu / nu mice. Negative controls were saline loaded with scrambled siRNA or EGFR-targeted EDV. [Figure 15] This figure compares tumors isolated from mesothelioma xenograft Balb / c nu / nu mice treated with EGFR-targeted miRNA16a (EGFREDVmiRNA16a™) or EGFR-targeted siRRM1-packaged EDV (EGFREDVsiRRM1™) with those treated with scrambled siRNA-loaded saline-treated EDV or EGFR-targeted EDV. [Figure 16] This figure shows apoptosis induced in adrenocortic carcinoma cells (ACC01) by EGFR-targeted EDV loaded with siRNAs targeting polo-like kinase 1 (EGFREDV™ siPLK™) and ribonucleotide reductase enzyme 1 (EGFREDV siRRM1™), based on the measurement of cellular debris count (Figure 16A) and the ratio of annexin 5 to propidium iodide (PI)-positive cells (Figure 16B). The figure includes apoptosis in untreated ACC01 cells treated with EDV loaded with an unrelated siRNA (EGFREDV™ siluciferase™), and unloaded EDV (EGFREDV™) as a negative control. [Figure 17]This figure shows sub-G1 arrest induced in adrenocortical carcinoma cells (ACC01) by EGFR-targeted EDV loaded with siRNAs targeting polo-like kinase 1 (EGFREDV™ siPLK™) (Figure 17D) and ribonucleotide reductase enzyme 1 (EGFREDV™ siRRM1™) (Figure 17D) in adrenocortical carcinoma cells (ACC01) (Figure 17D), based on the number of cell debris and the ratio of annexin 5 to propidium iodide (PI)-positive cells (Figure 17D). Apoptosis of untreated ACC01 cells (Figure 17A), ACC01 cells treated with EDV loaded with unrelated siRNA (EGFREDV™ siluciferase™) (Figure 17C), and unloaded EDV (EGFREDV™) (Figure 17B) are included as negative controls. [Figure 18] This figure shows the effects of A549 (lung cancer) xenograft tumor growth in Balb / c nu / nu mice treated with (i) black triangles = EGFREDVPNU-159682 TM + EDV40mer TM, (ii) black circles = EGFREDVPNU-159682 TM, (iii) white squares = EGFREDVPNU-159682 TM + EDV, (iv) white triangles = EGFREDVPNU-159682 TM + EDV50mer TM, and (v) black squares = saline. As indicated by the upward arrows, mice were treated with these EDV combinations on days 24, 27, 29, 31, 34, 36, and 38 after xenograft transplantation. On days 36 and 38, mice in the saline group with a tumor volume of approximately 650 mm³ were treated with EGFREDVPNU-159682™ + EDV50mer™ as indicated by the downward arrow. [Figure 19] This figure shows the effect of treating Balb / c nu / nu mice with EDV containing 40mers (EGFREDV40mers™, which is EDV combined with PNU159682 (EGFREDVPNU™)) on xenograft tumor growth of A549 (lung cancer). The triangles indicate the number of treatment days. [Figure 20]This figure shows the effects of physiological saline (negative control), IFN-γ (0.5 × 10⁴ IU per dose), doxorubicin (EGFREDVDox™)-loaded EGFR-targeted EDV, and EGFREDVDox™ + IFN-γ-treated Balb / c nu / nu mice on A549 (lung cancer) xenograft tumor growth. Triangles indicate the number of treatment days. [Figure 21] This shows the effects on MDA-MB468 (breast cancer) xenograft tumor growth in Balb / c nu / nu mice treated with saline (negative control), IFN-γ (0.5 × 10⁴ IU per dose), doxorubicin (EGFREDVDox™), and EGFREDVDox™ + IFN-γ. Triangles indicate the number of treatment days. [Figure 22] Another study is shown demonstrating the effects of MDA-MB468 (breast cancer) xenograft tumor growth in Balb / c nu / nu mice treated with physiological saline (negative control), IFN-γ (0.5 × 10⁴ IU per dose), doxorubicin (EGFREDVDox™), and EGFREDVDox™ + IFN-γ. Triangles indicate the number of treatment days. [Figure 23] This study shows the effects of treatment with physiological saline (negative control, Group 1), doxorubicin-loaded EGFR-targeted EDV (EGFREDVDox™, Group 2), EGFREDVDox™ IFNγ (0.75 × 10⁴ IU per dose) (Group 3), and EGFREDVDox™ + IFNγ (0.5 × 10⁴ IU per dose) (Group 4) on doxorubicin-resistant A549 xenograft tumor growth in Balb / c nu / nu mice. Mice in Groups 1-3 were treated twice a week, as indicated by the black triangles. Mice in Group 4 were treated three times a week, as indicated by the white triangles. [Figure 24-1]This figure shows the cytokine profiles of patients from a human first-dose study administered with various doses of paclitaxel-loaded EGFREDVTM. (Figure 24A) = pg / mL of IL6 measured for each of the 5 doses; (Figure 24C) = pg / mL of IL10 measured for each of the 5 doses; (Figure 24D) = pg / mL of TNFα measured for each of the 5 doses; (Figure 24F) = pg / mL of IFNγ measured for each of the 5 doses; (Figure 24G) = pg / mL of IL-1β measured for each of the 5 doses; (Figure 24H) = pg / mL of IL2 measured for each of the 5 doses; (Figure 24I) = pg / mL of IL4 measured for each of the 5 doses; (Figure 24J) = pg / mL of IL12 measured for each of the 5 doses. Finally, (Figure 24K) shows the 5 doses tested. [Figure 24-2] This figure shows the cytokine profiles of patients from a human first-dose study administered with various doses of paclitaxel-loaded EGFREDVTM. (Figure 24A) = pg / mL of IL6 measured for each of the 5 doses; (Figure 24C) = pg / mL of IL10 measured for each of the 5 doses; (Figure 24D) = pg / mL of TNFα measured for each of the 5 doses; (Figure 24F) = pg / mL of IFNγ measured for each of the 5 doses; (Figure 24G) = pg / mL of IL-1β measured for each of the 5 doses; (Figure 24H) = pg / mL of IL2 measured for each of the 5 doses; (Figure 24I) = pg / mL of IL4 measured for each of the 5 doses; (Figure 24J) = pg / mL of IL12 measured for each of the 5 doses. Finally, (Figure 24K) shows the 5 doses tested. [Figure 24-3]This figure shows the cytokine profiles of patients from a human first-dose study administered with various doses of paclitaxel-loaded EGFREDVTM. (Figure 24A) = pg / mL of IL6 measured for each of the 5 doses; (Figure 24C) = pg / mL of IL10 measured for each of the 5 doses; (Figure 24D) = pg / mL of TNFα measured for each of the 5 doses; (Figure 24F) = pg / mL of IFNγ measured for each of the 5 doses; (Figure 24G) = pg / mL of IL-1β measured for each of the 5 doses; (Figure 24H) = pg / mL of IL2 measured for each of the 5 doses; (Figure 24I) = pg / mL of IL4 measured for each of the 5 doses; (Figure 24J) = pg / mL of IL12 measured for each of the 5 doses. Finally, (Figure 24K) shows the 5 doses tested. [Figure 24-4] This figure shows the cytokine profiles of patients from a human first-dose study administered with various doses of paclitaxel-loaded EGFREDVTM. (Figure 24A) = pg / mL of IL6 measured for each of the 5 doses; (Figure 24C) = pg / mL of IL10 measured for each of the 5 doses; (Figure 24D) = pg / mL of TNFα measured for each of the 5 doses; (Figure 24F) = pg / mL of IFNγ measured for each of the 5 doses; (Figure 24G) = pg / mL of IL-1β measured for each of the 5 doses; (Figure 24H) = pg / mL of IL2 measured for each of the 5 doses; (Figure 24I) = pg / mL of IL4 measured for each of the 5 doses; (Figure 24J) = pg / mL of IL12 measured for each of the 5 doses. Finally, (Figure 24K) shows the 5 doses tested. [Figure 25-1]This figure shows the cytokine profiles of patients from a human first-dose study administered with various doses of EGFREDVTM loaded with doxorubicin. (Figure 25A) = pg / mL of IL6 measured for each of the 8 doses; (Figure 25C) = pg / mL of IL10 measured for each of the 8 doses; (Figure 25D) = pg / mL of TNFα measured for each of the 8 doses; (Figure 25F) = pg / mL of IFNγ measured for each of the 8 doses; (Figure 25G) = pg / mL of IL-1β measured for each of the 8 doses; (Figure 25H) = pg / mL of IL2 measured for each of the 8 doses; (Figure 25I) = pg / mL of IL4 measured for each of the 8 doses; (Figure 25J) = pg / mL of IL12 measured for each of the 8 doses. Finally, (Figure 25K) shows the three additional doses that were tested, while the first five doses are the same as those shown in (Figure 24K). [Figure 25-2] This figure shows the cytokine profiles of patients from a human first-dose study administered with various doses of EGFREDVTM loaded with doxorubicin. (Figure 25A) = pg / mL of IL6 measured for each of the 8 doses; (Figure 25C) = pg / mL of IL10 measured for each of the 8 doses; (Figure 25D) = pg / mL of TNFα measured for each of the 8 doses; (Figure 25F) = pg / mL of IFNγ measured for each of the 8 doses; (Figure 25G) = pg / mL of IL-1β measured for each of the 8 doses; (Figure 25H) = pg / mL of IL2 measured for each of the 8 doses; (Figure 25I) = pg / mL of IL4 measured for each of the 8 doses; (Figure 25J) = pg / mL of IL12 measured for each of the 8 doses. Finally, (Figure 25K) shows the three additional doses that were tested, while the first five doses are the same as those shown in (Figure 24K). [Figure 25-3]This figure shows the cytokine profiles of patients from a human first-dose study administered with various doses of EGFREDVTM loaded with doxorubicin. (Figure 25A) = pg / mL of IL6 measured for each of the 8 doses; (Figure 25C) = pg / mL of IL10 measured for each of the 8 doses; (Figure 25D) = pg / mL of TNFα measured for each of the 8 doses; (Figure 25F) = pg / mL of IFNγ measured for each of the 8 doses; (Figure 25G) = pg / mL of IL-1β measured for each of the 8 doses; (Figure 25H) = pg / mL of IL2 measured for each of the 8 doses; (Figure 25I) = pg / mL of IL4 measured for each of the 8 doses; (Figure 25J) = pg / mL of IL12 measured for each of the 8 doses. Finally, (Figure 25K) shows the three additional doses that were tested, while the first five doses are the same as those shown in (Figure 24K). [Figure 25-4] This figure shows the cytokine profiles of patients from a human first-dose study administered with various doses of EGFREDVTM loaded with doxorubicin. (Figure 25A) = pg / mL of IL6 measured for each of the 8 doses; (Figure 25C) = pg / mL of IL10 measured for each of the 8 doses; (Figure 25D) = pg / mL of TNFα measured for each of the 8 doses; (Figure 25F) = pg / mL of IFNγ measured for each of the 8 doses; (Figure 25G) = pg / mL of IL-1β measured for each of the 8 doses; (Figure 25H) = pg / mL of IL2 measured for each of the 8 doses; (Figure 25I) = pg / mL of IL4 measured for each of the 8 doses; (Figure 25J) = pg / mL of IL12 measured for each of the 8 doses. Finally, (Figure 25K) shows the three additional doses that were tested, while the first five doses are the same as those shown in (Figure 24K). [Figure 26]This figure illustrates the signaling pathways of cytosolic DNA sensors involving DNA challenge. Many cytosolic DNA sensors have been defined to detect intracellular double-stranded DNA. RNA polymerase III transcribes AT-rich DNA into RNA recognized by the RNA sensor RIG-I, followed by activation of STING and IRF3. DNA sensors DAI, IFI16, DDX41, and LSm14A directly sense dsDNA and activate STING for type I IFN production. In the presence of dsDNA, cGAS catalyzes the synthesis of cGAMP, a potent activator of STING. Along with dsDNA, LRRFIP1 initiates STING-dependent β-catenin and IRF3 activation. Other DNA sensors prime the immune response independently of STING. After dsDNA recognition, Sox2 induces activation of the Tab2 / TAK1 complex in neutrophils. Upon detection by dsDNA, DHX9 / 36 activates NFκB and IRF7 via MyD88. The DNA sensor Ku70 induces the activation of IRF1 and IRF7. AIM2 initiates inflammasome activation via ASCs with DNA binding. [Figure 27-1]This figure shows the activation of RAW264.7 cells and bone marrow-derived dendritic cells (BMDCs) in response to EDV treatment. (Figure 27A) CD86 expression in RAW cells directly incubated with 1 μg / ml LPS, Ep-EDV, Ep-EDV682, or 682. (Figure 27B) CD86 expression in RAW cells co-cultured with 4T1 or CT26Ep12.1 cells treated with EpEDV, EpEDV682, or 682. RAW cells co-cultured with Ep-EDV682-treated cancer cells showed a significant increase in CD86 expression. (Figure 27C) TNFα production in a source cell / tumor cell co-culture showing a significant increase in TNFα production by source cells incubated with EDV-treated tumor cells. (Figure 27D) IL-6 production in a source cell / tumor cell co-culture shows a significant increase in IL-6 production by source cells incubated with EDV-treated tumor cells. (Figure 27E) Quantification of IFNα and IFNβ expression in BMDC / 4T1 co-culture. (Figure 27F) PCR quantification of IFNα and IFNβ expression in BMDC / CT26Ep12.1 co-culture. (Figure 27G) Quantification of CD86Hi and MHC class IIHi expression and (Figure 27H) CD80Hi expression in BMDC / tumor cell co-culture. (Figure 27I) Flow cytometry density plot of MHC class II vs CD86 expression in BMDC co-culture with EDV and drug-treated CT26Ep12.1 cells. (Figure 27J) ELISA analysis of TNFα (Figure 27K) IL12p40 and (Figure 27L) IL6 from the supernatant of BMDC / tumor cell co-culture. Data represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison test. [Figure 27-2]This figure shows the activation of RAW264.7 cells and bone marrow-derived dendritic cells (BMDCs) in response to EDV treatment. (Figure 27A) CD86 expression in RAW cells directly incubated with 1 μg / ml LPS, Ep-EDV, Ep-EDV682, or 682. (Figure 27B) CD86 expression in RAW cells co-cultured with 4T1 or CT26Ep12.1 cells treated with EpEDV, EpEDV682, or 682. RAW cells co-cultured with Ep-EDV682-treated cancer cells showed a significant increase in CD86 expression. (Figure 27C) TNFα production in a source cell / tumor cell co-culture showing a significant increase in TNFα production by source cells incubated with EDV-treated tumor cells. (Figure 27D) IL-6 production in a source cell / tumor cell co-culture shows a significant increase in IL-6 production by source cells incubated with EDV-treated tumor cells. (Figure 27E) Quantification of IFNα and IFNβ expression in BMDC / 4T1 co-culture. (Figure 27F) PCR quantification of IFNα and IFNβ expression in BMDC / CT26Ep12.1 co-culture. (Figure 27G) Quantification of CD86Hi and MHC class IIHi expression and (Figure 27H) CD80Hi expression in BMDC / tumor cell co-culture. (Figure 27I) Flow cytometry density plot of MHC class II vs CD86 expression in BMDC co-culture with EDV and drug-treated CT26Ep12.1 cells. (Figure 27J) ELISA analysis of TNFα (Figure 27K) IL12p40 and (Figure 27L) IL6 from the supernatant of BMDC / tumor cell co-culture. Data represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison test. [Figure 27-3]This figure shows the activation of RAW264.7 cells and bone marrow-derived dendritic cells (BMDCs) in response to EDV treatment. (Figure 27A) CD86 expression in RAW cells directly incubated with 1 μg / ml LPS, Ep-EDV, Ep-EDV682, or 682. (Figure 27B) CD86 expression in RAW cells co-cultured with 4T1 or CT26Ep12.1 cells treated with EpEDV, EpEDV682, or 682. RAW cells co-cultured with Ep-EDV682-treated cancer cells showed a significant increase in CD86 expression. (Figure 27C) TNFα production in a source cell / tumor cell co-culture showing a significant increase in TNFα production by source cells incubated with EDV-treated tumor cells. (Figure 27D) IL-6 production in a source cell / tumor cell co-culture shows a significant increase in IL-6 production by source cells incubated with EDV-treated tumor cells. (Figure 27E) Quantification of IFNα and IFNβ expression in BMDC / 4T1 co-culture. (Figure 27F) PCR quantification of IFNα and IFNβ expression in BMDC / CT26Ep12.1 co-culture. (Figure 27G) Quantification of CD86Hi and MHC class IIHi expression and (Figure 27H) CD80Hi expression in BMDC / tumor cell co-culture. (Figure 27I) Flow cytometry density plot of MHC class II vs CD86 expression in BMDC co-culture with EDV and drug-treated CT26Ep12.1 cells. (Figure 27J) ELISA analysis of TNFα (Figure 27K) IL12p40 and (Figure 27L) IL6 from the supernatant of BMDC / tumor cell co-culture. Data represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison test. [Figure 27-4]This figure shows the activation of RAW264.7 cells and bone marrow-derived dendritic cells (BMDCs) in response to EDV treatment. (Figure 27A) CD86 expression in RAW cells directly incubated with 1 μg / ml LPS, Ep-EDV, Ep-EDV682, or 682. (Figure 27B) CD86 expression in RAW cells co-cultured with 4T1 or CT26Ep12.1 cells treated with EpEDV, EpEDV682, or 682. RAW cells co-cultured with Ep-EDV682-treated cancer cells showed a significant increase in CD86 expression. (Figure 27C) TNFα production in a source cell / tumor cell co-culture showing a significant increase in TNFα production by source cells incubated with EDV-treated tumor cells. (Figure 27D) IL-6 production in a source cell / tumor cell co-culture shows a significant increase in IL-6 production by source cells incubated with EDV-treated tumor cells. (Figure 27E) Quantification of IFNα and IFNβ expression in BMDC / 4T1 co-culture. (Figure 27F) PCR quantification of IFNα and IFNβ expression in BMDC / CT26Ep12.1 co-culture. (Figure 27G) Quantification of CD86Hi and MHC class IIHi expression and (Figure 27H) CD80Hi expression in BMDC / tumor cell co-culture. (Figure 27I) Flow cytometry density plot of MHC class II vs CD86 expression in BMDC co-culture with EDV and drug-treated CT26Ep12.1 cells. (Figure 27J) ELISA analysis of TNFα (Figure 27K) IL12p40 and (Figure 27L) IL6 from the supernatant of BMDC / tumor cell co-culture. Data represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison test. [Figure 27-5]This figure shows the activation of RAW264.7 cells and bone marrow-derived dendritic cells (BMDCs) in response to EDV treatment. (Figure 27A) CD86 expression in RAW cells directly incubated with 1 μg / ml LPS, Ep-EDV, Ep-EDV682, or 682. (Figure 27B) CD86 expression in RAW cells co-cultured with 4T1 or CT26Ep12.1 cells treated with EpEDV, EpEDV682, or 682. RAW cells co-cultured with Ep-EDV682-treated cancer cells showed a significant increase in CD86 expression. (Figure 27C) TNFα production in a source cell / tumor cell co-culture showing a significant increase in TNFα production by source cells incubated with EDV-treated tumor cells. (Figure 27D) IL-6 production in a source cell / tumor cell co-culture shows a significant increase in IL-6 production by source cells incubated with EDV-treated tumor cells. (Figure 27E) Quantification of IFNα and IFNβ expression in BMDC / 4T1 co-culture. (Figure 27F) PCR quantification of IFNα and IFNβ expression in BMDC / CT26Ep12.1 co-culture. (Figure 27G) Quantification of CD86Hi and MHC class IIHi expression and (Figure 27H) CD80Hi expression in BMDC / tumor cell co-culture. (Figure 27I) Flow cytometry density plot of MHC class II vs CD86 expression in BMDC co-culture with EDV and drug-treated CT26Ep12.1 cells. (Figure 27J) ELISA analysis of TNFα (Figure 27K) IL12p40 and (Figure 27L) IL6 from the supernatant of BMDC / tumor cell co-culture. Data represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison test. [Figure 28-1]This figure shows tumor response and macrophage activation in response to EDV treatment. (Figure 28A) Tumor growth in response to EpEDV and EpEDV682 treatment in Balb / c mice with 4T1 or (Figure 28B) CT26Ep12.1 tumors. (Figure 28C) Tumor growth in response to EDV-682 and EDV-EGFR682 in BALB / C nude mice with T84 xenografts, and (Figure 28D) Tumor growth in response to EDV-682, EDV-EGFRDox and EDV-EGFR682 in BALB / C nude mice with A549 / MDR xenografts. Green arrows indicate the location where EDVEGFR682 treatment was initiated in mice formally treated with saline. Data (Figures 28A-D) represent mean ± sem and are analyzed by two-way ANOVA and Tukey's multiple comparison studies (Figure 28E) of CD11b+ cells isolated from 4T1 tumors and co-cultured with 4T1 cells in a 5:1 (E:T) ratio by xCELLigence RTCA. Plots represent normalized cell indices correlated with cell adhesion and proliferation / death time. CD11b+ cells from 4T1 tumors experience an initial adhesion and sedimentation phase, as indicated by an increase in cell indices, followed by proliferation or death, as indicated by an increase or decrease in cell indices. (Figure 28F) Ratio of M1(CD86+):M2(CD206+) macrophages in 4T1 tumors of treated mice. (Figure 28G) CD11b+ xCELLigence RTCA isolated from CT26Ep12.1 tumors and co-cultured with CT26Ep12.1 cells in a 5:1 (E:T) ratio. (Figure 28H) Ratio of M1(CD86+):M2(CD206+) macrophages in CT26Ep12.1 tumors of treated mice. Data (Figures 28F and 28H) represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison test. [Figure 28-2]This figure shows tumor response and macrophage activation in response to EDV treatment. (Figure 28A) Tumor growth in response to EpEDV and EpEDV682 treatment in Balb / c mice with 4T1 or (Figure 28B) CT26Ep12.1 tumors. (Figure 28C) Tumor growth in response to EDV-682 and EDV-EGFR682 in BALB / C nude mice with T84 xenografts, and (Figure 28D) Tumor growth in response to EDV-682, EDV-EGFRDox and EDV-EGFR682 in BALB / C nude mice with A549 / MDR xenografts. Green arrows indicate the location where EDVEGFR682 treatment was initiated in mice formally treated with saline. Data (Figures 28A-D) represent mean ± sem and are analyzed by two-way ANOVA and Tukey's multiple comparison studies (Figure 28E) of CD11b+ cells isolated from 4T1 tumors and co-cultured with 4T1 cells in a 5:1 (E:T) ratio by xCELLigence RTCA. Plots represent normalized cell indices correlated with cell adhesion and proliferation / death time. CD11b+ cells from 4T1 tumors experience an initial adhesion and sedimentation phase, as indicated by an increase in cell indices, followed by proliferation or death, as indicated by an increase or decrease in cell indices. (Figure 28F) Ratio of M1(CD86+):M2(CD206+) macrophages in 4T1 tumors of treated mice. (Figure 28G) CD11b+ xCELLigence RTCA isolated from CT26Ep12.1 tumors and co-cultured with CT26Ep12.1 cells in a 5:1 (E:T) ratio. (Figure 28H) Ratio of M1(CD86+):M2(CD206+) macrophages in CT26Ep12.1 tumors of treated mice. Data (Figures 28F and 28H) represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison test. [Figure 28-3]This figure shows tumor response and macrophage activation in response to EDV treatment. (Figure 28A) Tumor growth in response to EpEDV and EpEDV682 treatment in Balb / c mice with 4T1 or (Figure 28B) CT26Ep12.1 tumors. (Figure 28C) Tumor growth in response to EDV-682 and EDV-EGFR682 in BALB / C nude mice with T84 xenografts, and (Figure 28D) Tumor growth in response to EDV-682, EDV-EGFRDox and EDV-EGFR682 in BALB / C nude mice with A549 / MDR xenografts. Green arrows indicate the location where EDVEGFR682 treatment was initiated in mice formally treated with saline. Data (Figures 28A-D) represent mean ± sem and are analyzed by two-way ANOVA and Tukey's multiple comparison studies (Figure 28E) of CD11b+ cells isolated from 4T1 tumors and co-cultured with 4T1 cells in a 5:1 (E:T) ratio by xCELLigence RTCA. Plots represent normalized cell indices correlated with cell adhesion and proliferation / death time. CD11b+ cells from 4T1 tumors experience an initial adhesion and sedimentation phase, as indicated by an increase in cell indices, followed by proliferation or death, as indicated by an increase or decrease in cell indices. (Figure 28F) Ratio of M1(CD86+):M2(CD206+) macrophages in 4T1 tumors of treated mice. (Figure 28G) CD11b+ xCELLigence RTCA isolated from CT26Ep12.1 tumors and co-cultured with CT26Ep12.1 cells in a 5:1 (E:T) ratio. (Figure 28H) Ratio of M1(CD86+):M2(CD206+) macrophages in CT26Ep12.1 tumors of treated mice. Data (Figures 28F and 28H) represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison test. [Figure 28-4]This figure shows tumor response and macrophage activation in response to EDV treatment. (Figure 28A) Tumor growth in response to EpEDV and EpEDV682 treatment in Balb / c mice with 4T1 or (Figure 28B) CT26Ep12.1 tumors. (Figure 28C) Tumor growth in response to EDV-682 and EDV-EGFR682 in BALB / C nude mice with T84 xenografts, and (Figure 28D) Tumor growth in response to EDV-682, EDV-EGFRDox and EDV-EGFR682 in BALB / C nude mice with A549 / MDR xenografts. Green arrows indicate the location where EDVEGFR682 treatment was initiated in mice formally treated with saline. Data (Figures 28A-D) represent mean ± sem and are analyzed by two-way ANOVA and Tukey's multiple comparison studies (Figure 28E) of CD11b+ cells isolated from 4T1 tumors and co-cultured with 4T1 cells in a 5:1 (E:T) ratio by xCELLigence RTCA. Plots represent normalized cell indices correlated with cell adhesion and proliferation / death time. CD11b+ cells from 4T1 tumors experience an initial adhesion and sedimentation phase, as indicated by an increase in cell indices, followed by proliferation or death, as indicated by an increase or decrease in cell indices. (Figure 28F) Ratio of M1(CD86+):M2(CD206+) macrophages in 4T1 tumors of treated mice. (Figure 28G) CD11b+ xCELLigence RTCA isolated from CT26Ep12.1 tumors and co-cultured with CT26Ep12.1 cells in a 5:1 (E:T) ratio. (Figure 28H) Ratio of M1(CD86+):M2(CD206+) macrophages in CT26Ep12.1 tumors of treated mice. Data (Figures 28F and 28H) represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison test. [Figure 29-1]This figure shows the NK cell response to EDV treatment. (Figure 29 A) xCELLigence RTCA of NK cells isolated from the spleen of mice with 4T1 tumors, co-cultured with 4T1 cells in a 20:1 (E:T) ratio. The plots represent cell viability calculated from time-normalized cell indices. (Figure 29 B) % viability of 4T1 cells co-cultured with NK cells derived from saline, EpEDV, or EpEDV682, 70 hours after NK addition when mice were treated. (Figure 29 C) xCELLigence RTCA of NK cells isolated from the spleen of mice with CT26Ep12.1 tumors, co-cultured with CT26Ep12.1 cells in a 20:1 (E:T) ratio. (Figure 29D) The viability % of CT26Ep12.1 cells co-cultured with NK cells from saline, EpEDV, or EpEDV682-treated mice 50 hours after NK cell addition (Figure 29E) shows NKG2D expression in NK cells (CD45+, CD11b+, DX5+) within 4T1 tumors and increased NKG2D expression in EpEDV682-treated mice. (Figure 29F) RANTES and (Figure 29G) TNFα production occurs in co-culture of NK cells isolated from the spleen of EDV-treated mice with 4T1 tumors and 4T1 cells. (Figure 29H) Quantification of NKG2D ligands rae1, H60a, and MULT1 on the surface of four different mouse tumor cell lines. (Figure 29I) xCELLigence RTCA of NK cells isolated from the spleen of EpEDV682-treated mice with 4T1 tumors, co-cultured with 4T1 cells in a 20:1 (E:T) ratio in the presence of RAE1 and / or H60a inhibitory antibodies, demonstrates that both are important in NK tumor cell lysis. (Figure 29J) Quantification of NK cell lysis 80 hours after NK cell addition shows significant inhibition of NK cell lysis with H60a antibody alone compared with RAE inhibitory antibody. Data (Figures 29B, 29D, 29EG, and 29J) represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison test. [Figure 29-2]This figure shows the NK cell response to EDV treatment. (Figure 29 A) xCELLigence RTCA of NK cells isolated from the spleen of mice with 4T1 tumors, co-cultured with 4T1 cells in a 20:1 (E:T) ratio. The plots represent cell viability calculated from time-normalized cell indices. (Figure 29 B) % viability of 4T1 cells co-cultured with NK cells derived from saline, EpEDV, or EpEDV682, 70 hours after NK addition when mice were treated. (Figure 29 C) xCELLigence RTCA of NK cells isolated from the spleen of mice with CT26Ep12.1 tumors, co-cultured with CT26Ep12.1 cells in a 20:1 (E:T) ratio. (Figure 29D) The viability % of CT26Ep12.1 cells co-cultured with NK cells from saline, EpEDV, or EpEDV682-treated mice 50 hours after NK cell addition (Figure 29E) shows NKG2D expression in NK cells (CD45+, CD11b+, DX5+) within 4T1 tumors and increased NKG2D expression in EpEDV682-treated mice. (Figure 29F) RANTES and (Figure 29G) TNFα production occurs in co-culture of NK cells isolated from the spleen of EDV-treated mice with 4T1 tumors and 4T1 cells. (Figure 29H) Quantification of NKG2D ligands rae1, H60a, and MULT1 on the surface of four different mouse tumor cell lines. (Figure 29I) xCELLigence RTCA of NK cells isolated from the spleen of EpEDV682-treated mice with 4T1 tumors, co-cultured with 4T1 cells in a 20:1 (E:T) ratio in the presence of RAE1 and / or H60a inhibitory antibodies, demonstrates that both are important in NK tumor cell lysis. (Figure 29J) Quantification of NK cell lysis 80 hours after NK cell addition shows significant inhibition of NK cell lysis with H60a antibody alone compared with RAE inhibitory antibody. Data (Figures 29B, 29D, 29EG, and 29J) represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison test. [Figure 29-3]This figure shows the NK cell response to EDV treatment. (Figure 29 A) xCELLigence RTCA of NK cells isolated from the spleen of mice with 4T1 tumors, co-cultured with 4T1 cells in a 20:1 (E:T) ratio. The plots represent cell viability calculated from time-normalized cell indices. (Figure 29 B) % viability of 4T1 cells co-cultured with NK cells derived from saline, EpEDV, or EpEDV682, 70 hours after NK addition when mice were treated. (Figure 29 C) xCELLigence RTCA of NK cells isolated from the spleen of mice with CT26Ep12.1 tumors, co-cultured with CT26Ep12.1 cells in a 20:1 (E:T) ratio. (Figure 29D) The viability % of CT26Ep12.1 cells co-cultured with NK cells from saline, EpEDV, or EpEDV682-treated mice 50 hours after NK cell addition (Figure 29E) shows NKG2D expression in NK cells (CD45+, CD11b+, DX5+) within 4T1 tumors and increased NKG2D expression in EpEDV682-treated mice. (Figure 29F) RANTES and (Figure 29G) TNFα production occurs in co-culture of NK cells isolated from the spleen of EDV-treated mice with 4T1 tumors and 4T1 cells. (Figure 29H) Quantification of NKG2D ligands rae1, H60a, and MULT1 on the surface of four different mouse tumor cell lines. (Figure 29I) xCELLigence RTCA of NK cells isolated from the spleen of EpEDV682-treated mice with 4T1 tumors, co-cultured with 4T1 cells in a 20:1 (E:T) ratio in the presence of RAE1 and / or H60a inhibitory antibodies, demonstrates that both are important in NK tumor cell lysis. (Figure 29J) Quantification of NK cell lysis 80 hours after NK cell addition shows significant inhibition of NK cell lysis with H60a antibody alone compared with RAE inhibitory antibody. Data (Figures 29B, 29D, 29EG, and 29J) represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison test. [Figure 29-4]This figure shows the NK cell response to EDV treatment. (Figure 29 A) xCELLigence RTCA of NK cells isolated from the spleen of mice with 4T1 tumors, co-cultured with 4T1 cells in a 20:1 (E:T) ratio. The plots represent cell viability calculated from time-normalized cell indices. (Figure 29 B) % viability of 4T1 cells co-cultured with NK cells derived from saline, EpEDV, or EpEDV682, 70 hours after NK addition when mice were treated. (Figure 29 C) xCELLigence RTCA of NK cells isolated from the spleen of mice with CT26Ep12.1 tumors, co-cultured with CT26Ep12.1 cells in a 20:1 (E:T) ratio. (Figure 29D) The viability % of CT26Ep12.1 cells co-cultured with NK cells from saline, EpEDV, or EpEDV682-treated mice 50 hours after NK cell addition (Figure 29E) shows NKG2D expression in NK cells (CD45+, CD11b+, DX5+) within 4T1 tumors and increased NKG2D expression in EpEDV682-treated mice. (Figure 29F) RANTES and (Figure 29G) TNFα production occurs in co-culture of NK cells isolated from the spleen of EDV-treated mice with 4T1 tumors and 4T1 cells. (Figure 29H) Quantification of NKG2D ligands rae1, H60a, and MULT1 on the surface of four different mouse tumor cell lines. (Figure 29I) xCELLigence RTCA of NK cells isolated from the spleen of EpEDV682-treated mice with 4T1 tumors, co-cultured with 4T1 cells in a 20:1 (E:T) ratio in the presence of RAE1 and / or H60a inhibitory antibodies, demonstrates that both are important in NK tumor cell lysis. (Figure 29J) Quantification of NK cell lysis 80 hours after NK cell addition shows significant inhibition of NK cell lysis with H60a antibody alone compared with RAE inhibitory antibody. Data (Figures 29B, 29D, 29EG, and 29J) represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison test. [Figure 30-1]This figure shows stromal tumor cytokine / chemokine production and cytokine production in splenocyte / tumor cell cocultures in response to EDV treatment. (Figure 30A) ELISA analysis of stromal cytokines and chemokines produced in response to EpEDV and EpEDV682 treatment in Balb / c mice with 4T1 or (Figure 30B) CT26Ep12.1 tumors. Ep-EDV-682 treatment mainly increases Th1 cytokines. Data are expressed as mean ± standard deviation. Individual cytokine data analyzed by one-way ANOVA and Tukey's multiple comparison test. (Figure 30C) TNF-alpha (Figure 30D) IL-2 (Figure 30E) IL-1β (Figure 30F) IFN-gamma and (Figure 30G) IL-10 ELISA analysis was obtained from the supernatant of cocultures of splenocytes isolated from 4T1 and CT26Ep12.1 tumor-treated mice with EpEDV-682 and their corresponding tumor cells in saline, Ep-EDV, and Ep-EDV-682-treated mice. Data represent mean ± standard deviation. One-way ANOVA and Tukey's multiple comparisons were used to compare groups of + or - tumor cells. T-studies are used to compare individual treatments with and without tumor cells. [Figure 30-2]This figure shows stromal tumor cytokine / chemokine production and cytokine production in splenocyte / tumor cell cocultures in response to EDV treatment. (Figure 30A) ELISA analysis of stromal cytokines and chemokines produced in response to EpEDV and EpEDV682 treatment in Balb / c mice with 4T1 or (Figure 30B) CT26Ep12.1 tumors. Ep-EDV-682 treatment mainly increases Th1 cytokines. Data are expressed as mean ± standard deviation. Individual cytokine data analyzed by one-way ANOVA and Tukey's multiple comparison test. (Figure 30C) TNF-alpha (Figure 30D) IL-2 (Figure 30E) IL-1β (Figure 30F) IFN-gamma and (Figure 30G) IL-10 ELISA analysis was obtained from the supernatant of cocultures of splenocytes isolated from 4T1 and CT26Ep12.1 tumor-treated mice with EpEDV-682 and their corresponding tumor cells in saline, Ep-EDV, and Ep-EDV-682-treated mice. Data represent mean ± standard deviation. One-way ANOVA and Tukey's multiple comparisons were used to compare groups of + or - tumor cells. T-studies are used to compare individual treatments with and without tumor cells. [Figure 30-3]This figure shows stromal tumor cytokine / chemokine production and cytokine production in splenocyte / tumor cell cocultures in response to EDV treatment. (Figure 30A) ELISA analysis of stromal cytokines and chemokines produced in response to EpEDV and EpEDV682 treatment in Balb / c mice with 4T1 or (Figure 30B) CT26Ep12.1 tumors. Ep-EDV-682 treatment mainly increases Th1 cytokines. Data are expressed as mean ± standard deviation. Individual cytokine data analyzed by one-way ANOVA and Tukey's multiple comparison test. (Figure 30C) TNF-alpha (Figure 30D) IL-2 (Figure 30E) IL-1β (Figure 30F) IFN-gamma and (Figure 30G) IL-10 ELISA analysis was obtained from the supernatant of cocultures of splenocytes isolated from 4T1 and CT26Ep12.1 tumor-treated mice with EpEDV-682 and their corresponding tumor cells in saline, Ep-EDV, and Ep-EDV-682-treated mice. Data represent mean ± standard deviation. One-way ANOVA and Tukey's multiple comparisons were used to compare groups of + or - tumor cells. T-studies are used to compare individual treatments with and without tumor cells. [Figure 30-4]This figure shows stromal tumor cytokine / chemokine production and cytokine production in splenocyte / tumor cell cocultures in response to EDV treatment. (Figure 30A) ELISA analysis of stromal cytokines and chemokines produced in response to EpEDV and EpEDV682 treatment in Balb / c mice with 4T1 or (Figure 30B) CT26Ep12.1 tumors. Ep-EDV-682 treatment mainly increases Th1 cytokines. Data are expressed as mean ± standard deviation. Individual cytokine data analyzed by one-way ANOVA and Tukey's multiple comparison test. (Figure 30C) TNF-alpha (Figure 30D) IL-2 (Figure 30E) IL-1β (Figure 30F) IFN-gamma and (Figure 30G) IL-10 ELISA analysis was obtained from the supernatant of cocultures of splenocytes isolated from 4T1 and CT26Ep12.1 tumor-treated mice with EpEDV-682 and their corresponding tumor cells in saline, Ep-EDV, and Ep-EDV-682-treated mice. Data represent mean ± standard deviation. One-way ANOVA and Tukey's multiple comparisons were used to compare groups of + or - tumor cells. T-studies are used to compare individual treatments with and without tumor cells. [Figure 31-1]This figure shows T cell function and phenotype in response to EDV treatment. (Figure 31A) xCELLigence RTCA of CD8+ T cells isolated from the spleen of mice with 4T1 tumors, co-cultured with 4T1 cells in a 30:1 (E:T) ratio. The plots represent normalized cell indices correlated with cell adhesion and proliferation / death time. (Figure 31B) % viability of 4T1 cells co-cultured with CD8+ T cells from saline, Ep-EDV, or Ep-EDV-682 treated mice 30 hours after the addition of CD8+ T cells. (Figure 31C) xCELLigence RTCA of CD8+ T cells isolated from the spleen of mice with CT26Ep12.1 tumors, co-cultured with CT26Ep12.1 cells in a 30:1 (E:T) ratio. The plots represent normalized cell indices correlated with cell adhesion and proliferation / death time. (Figure 31D) The survival rate % of CT26Ep12.1 cells co-cultured with CD8+ T cells from saline, EpEDV, or EpEDV682-treated mice was 20 hours after treatment with additional CD8+ T cells. (Figure 31E) The percentage of CD8+ T-cells (defined as CD45+, CD3+, CD8+) was detected in the form of 4T1. (Figure 31F) The percentage of T-regs (defined as CD45+, CD3+, CD4, CD25+) was detected in the form of 4T1. (Figure 31G) The number of T cells in the tumor-discharging lymph nodes of mice with 4T1 tumors is shown as a percentage of total cells. (Figure 31H) % CD80 / MHC class II expression in dendritic cells in the tumor-discharging lymph nodes of mice with 4T1 tumors. (Figure 31I) Confocal image of interactions between CD8+ T cells isolated from Ep-EDV-682 mice treated with 4T1 cells. Red - actin, green - perforin, blue (dark) - DAPI; scale bar 10 μm. Data (Figures 31B, 31D, and 31EH) represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison tests (Figures 31B, 31D, and 31EG) or t-test (Figure 31H). [Figure 31-2]This figure shows T cell function and phenotype in response to EDV treatment. (Figure 31A) xCELLigence RTCA of CD8+ T cells isolated from the spleen of mice with 4T1 tumors, co-cultured with 4T1 cells in a 30:1 (E:T) ratio. The plots represent normalized cell indices correlated with cell adhesion and proliferation / death time. (Figure 31B) % viability of 4T1 cells co-cultured with CD8+ T cells from saline, Ep-EDV, or Ep-EDV-682 treated mice 30 hours after the addition of CD8+ T cells. (Figure 31C) xCELLigence RTCA of CD8+ T cells isolated from the spleen of mice with CT26Ep12.1 tumors, co-cultured with CT26Ep12.1 cells in a 30:1 (E:T) ratio. The plots represent normalized cell indices correlated with cell adhesion and proliferation / death time. (Figure 31D) The survival rate % of CT26Ep12.1 cells co-cultured with CD8+ T cells from saline, EpEDV, or EpEDV682-treated mice was 20 hours after treatment with additional CD8+ T cells. (Figure 31E) The percentage of CD8+ T-cells (defined as CD45+, CD3+, CD8+) was detected in the form of 4T1. (Figure 31F) The percentage of T-regs (defined as CD45+, CD3+, CD4, CD25+) was detected in the form of 4T1. (Figure 31G) The number of T cells in the tumor-discharging lymph nodes of mice with 4T1 tumors is shown as a percentage of total cells. (Figure 31H) % CD80 / MHC class II expression in dendritic cells in the tumor-discharging lymph nodes of mice with 4T1 tumors. (Figure 31I) Confocal image of interactions between CD8+ T cells isolated from Ep-EDV-682 mice treated with 4T1 cells. Red - actin, green - perforin, blue (dark) - DAPI; scale bar 10 μm. Data (Figures 31B, 31D, and 31EH) represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison tests (Figures 31B, 31D, and 31EG) or t-test (Figure 31H). [Figure 31-3]This figure shows T cell function and phenotype in response to EDV treatment. (Figure 31A) xCELLigence RTCA of CD8+ T cells isolated from the spleen of mice with 4T1 tumors, co-cultured with 4T1 cells in a 30:1 (E:T) ratio. The plots represent normalized cell indices correlated with cell adhesion and proliferation / death time. (Figure 31B) % viability of 4T1 cells co-cultured with CD8+ T cells from saline, Ep-EDV, or Ep-EDV-682 treated mice 30 hours after the addition of CD8+ T cells. (Figure 31C) xCELLigence RTCA of CD8+ T cells isolated from the spleen of mice with CT26Ep12.1 tumors, co-cultured with CT26Ep12.1 cells in a 30:1 (E:T) ratio. The plots represent normalized cell indices correlated with cell adhesion and proliferation / death time. (Figure 31D) The survival rate % of CT26Ep12.1 cells co-cultured with CD8+ T cells from saline, EpEDV, or EpEDV682-treated mice was 20 hours after treatment with additional CD8+ T cells. (Figure 31E) The percentage of CD8+ T-cells (defined as CD45+, CD3+, CD8+) was detected in the form of 4T1. (Figure 31F) The percentage of T-regs (defined as CD45+, CD3+, CD4, CD25+) was detected in the form of 4T1. (Figure 31G) The number of T cells in the tumor-discharging lymph nodes of mice with 4T1 tumors is shown as a percentage of total cells. (Figure 31H) % CD80 / MHC class II expression in dendritic cells in the tumor-discharging lymph nodes of mice with 4T1 tumors. (Figure 31I) Confocal image of interactions between CD8+ T cells isolated from Ep-EDV-682 mice treated with 4T1 cells. Red - actin, green - perforin, blue (dark) - DAPI; scale bar 10 μm. Data (Figures 31B, 31D, and 31EH) represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison tests (Figures 31B, 31D, and 31EG) or t-test (Figure 31H). [Figure 31-4]This figure shows T cell function and phenotype in response to EDV treatment. (Figure 31A) xCELLigence RTCA of CD8+ T cells isolated from the spleen of mice with 4T1 tumors, co-cultured with 4T1 cells in a 30:1 (E:T) ratio. The plots represent normalized cell indices correlated with cell adhesion and proliferation / death time. (Figure 31B) % viability of 4T1 cells co-cultured with CD8+ T cells from saline, Ep-EDV, or Ep-EDV-682 treated mice 30 hours after the addition of CD8+ T cells. (Figure 31C) xCELLigence RTCA of CD8+ T cells isolated from the spleen of mice with CT26Ep12.1 tumors, co-cultured with CT26Ep12.1 cells in a 30:1 (E:T) ratio. The plots represent normalized cell indices correlated with cell adhesion and proliferation / death time. (Figure 31D) The survival rate % of CT26Ep12.1 cells co-cultured with CD8+ T cells from saline, EpEDV, or EpEDV682-treated mice was 20 hours after treatment with additional CD8+ T cells. (Figure 31E) The percentage of CD8+ T-cells (defined as CD45+, CD3+, CD8+) was detected in the form of 4T1. (Figure 31F) The percentage of T-regs (defined as CD45+, CD3+, CD4, CD25+) was detected in the form of 4T1. (Figure 31G) The number of T cells in the tumor-discharging lymph nodes of mice with 4T1 tumors is shown as a percentage of total cells. (Figure 31H) % CD80 / MHC class II expression in dendritic cells in the tumor-discharging lymph nodes of mice with 4T1 tumors. (Figure 31I) Confocal image of interactions between CD8+ T cells isolated from Ep-EDV-682 mice treated with 4T1 cells. Red - actin, green - perforin, blue (dark) - DAPI; scale bar 10 μm. Data (Figures 31B, 31D, and 31EH) represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison tests (Figures 31B, 31D, and 31EG) or t-test (Figure 31H). [Figure 32-1]This figure shows prognostic indicators and immunophenotypic analysis of patient peripheral blood mononuclear cells (PBMCs), revealing evidence of enhanced antigen presentation by dendritic cells and monocytes and increased cytotoxic CD8+ T cell content at dose 12. Prognostic indicators (Figure 32A) CA199 and (Figure 32B) C-reactive protein serum levels. (Figure 32C) Analysis of PBMCs using the Duraclon immunophenotyping panel for monocyte and (Figure 32D) intermediate (CD14+CD16++) antigen-presenting monocyte subtypes. Expressed as % leukocytes. (Figure 32E) Myeloid dendritic (Clec9A+) cells (mDC) and (Figure 32F) Dendritic cell subtypes including plasmacytoid and myeloid dendritic (professional antigen-presenting DCs) that control the CD8+ effector T cell response. Expressed as %DC or %mDC as shown. (Figure 32G) CD8+ T cell subtypes. Cytotoxic CD8+ T cells include effector and depleted (PD1+) subtypes. [Figure 32-2] This figure shows prognostic indicators and immunophenotypic analysis of patient peripheral blood mononuclear cells (PBMCs), revealing evidence of enhanced antigen presentation by dendritic cells and monocytes and increased cytotoxic CD8+ T cell content at dose 12. Prognostic indicators (Figure 32A) CA199 and (Figure 32B) C-reactive protein serum levels. (Figure 32C) Analysis of PBMCs using the Duraclon immunophenotyping panel for monocyte and (Figure 32D) intermediate (CD14+CD16++) antigen-presenting monocyte subtypes. Expressed as % leukocytes. (Figure 32E) Myeloid dendritic (Clec9A+) cells (mDC) and (Figure 32F) Dendritic cell subtypes including plasmacytoid and myeloid dendritic (professional antigen-presenting DCs) that control the CD8+ effector T cell response. Expressed as %DC or %mDC as shown. (Figure 32G) CD8+ T cell subtypes. Cytotoxic CD8+ T cells include effector and depleted (PD1+) subtypes. [Figure 32-3]This figure shows prognostic indicators and immunophenotypic analysis of patient peripheral blood mononuclear cells (PBMCs), revealing evidence of enhanced antigen presentation by dendritic cells and monocytes and increased cytotoxic CD8+ T cell content at dose 12. Prognostic indicators (Figure 32A) CA199 and (Figure 32B) C-reactive protein serum levels. (Figure 32C) Analysis of PBMCs using the Duraclon immunophenotyping panel for monocyte and (Figure 32D) intermediate (CD14+CD16++) antigen-presenting monocyte subtypes. Expressed as % leukocytes. (Figure 32E) Myeloid dendritic (Clec9A+) cells (mDC) and (Figure 32F) Dendritic cell subtypes including plasmacytoid and myeloid dendritic (professional antigen-presenting DCs) that control the CD8+ effector T cell response. Expressed as %DC or %mDC as shown. (Figure 32G) CD8+ T cell subtypes. Cytotoxic CD8+ T cells include effector and depleted (PD1+) subtypes. [Figure 33]This figure schematic illustrates how EDV first creates an immunogenic tumor microenvironment through the delivery of cytotoxic substances to the tumor, then directly or indirectly stimulates the innate immune system toward an antitumor phenotype, and finally produces an adaptive response resulting in tumor-specific cytotoxic T cells (Figure 33A). EpEDV682 enters the tumor microenvironment via the leaky vascular system, resulting in tumor cell apoptosis and the release of immune-activating DAMP. Interaction of macrophages within the tumor microenvironment by direct engulfment of apoptotic cells or even EDV leads to M1 macrophage polarization and the release of inflammatory cytokines TNF-α and IL-6 (Figure 33C). M1 macrophages can further lyse tumor cells and release MIP-1α, which can mobilize further immune cells (Figure 33D). Immature dendritic cells engulfed apoptotic cell bodies and released tumor antigens produced in response to Ep-EDV-682 treatment and mature-release type 1 interferon, TNF-α, IL-12p40, and IL-6. (Figure 33E) Mature DCs then migrate to lymph nodes for antigen presentation to T cells. (Figure 33F) NK cell activation also occurs in the tumor microenvironment, where IFN attracts further immune cells, resulting in the release of γ and TNFα as well as RANTES. Furthermore, activated NK cells effectively lyse tumor cells. (Figure 33G) The release of RANTES and MIP-1α replenishes the tumor with further T cells, NK cells, and macrophages, where (Figure 33H) tumor-specific CD8+ T cells then contribute to the response through tumor cell lysis. (Figure 33I) All of these steps combine to create an effective anti-tumor immune response. [Figure 34-1]This figure shows the activation of RAW264.7 and JAWSII cells in response to EDV treatment. (Figure 34A) TNFα production by RAW264.7 cells directly incubated with EDV. (Figure 34B) IL6 production by RAW264.7 cells directly incubated with EDV. (Figure 34C) Flow cytometry histogram overlay of CD86 and MHC class II expression in co-culture of JAWSII cells with untreated CT26Ep12.1 and 4T1 cells or cells treated with EpEDV, EpEDV682, or 682 alone. (Figure 34D) Quantification of CD86 expression as determined by flow cytometry on JAWSII cells co-cultured with treated tumor cells. (Figure 34E) Quantification of MHC class II expression as determined by flow cytometry on JAWSII cells co-cultured with treated tumor cells. Data represent mean ± sem and are analyzed by one-way ANOVA and Tukey's multiple comparison test. [Figure 34-2] This figure shows the activation of RAW264.7 and JAWSII cells in response to EDV treatment. (Figure 34A) TNFα production by RAW264.7 cells directly incubated with EDV. (Figure 34B) IL6 production by RAW264.7 cells directly incubated with EDV. (Figure 34C) Flow cytometry histogram overlay of CD86 and MHC class II expression in co-culture of JAWSII cells with untreated CT26Ep12.1 and 4T1 cells or cells treated with EpEDV, EpEDV682, or 682 alone. (Figure 34D) Quantification of CD86 expression as determined by flow cytometry on JAWSII cells co-cultured with treated tumor cells. (Figure 34E) Quantification of MHC class II expression as determined by flow cytometry on JAWSII cells co-cultured with treated tumor cells. Data represent mean ± sem and are analyzed by one-way ANOVA and Tukey's multiple comparison test. [Figure 34-3]This figure shows the activation of RAW264.7 and JAWSII cells in response to EDV treatment. (Figure 34A) TNFα production by RAW264.7 cells directly incubated with EDV. (Figure 34B) IL6 production by RAW264.7 cells directly incubated with EDV. (Figure 34C) Flow cytometry histogram overlay of CD86 and MHC class II expression in co-culture of JAWSII cells with untreated CT26Ep12.1 and 4T1 cells or cells treated with EpEDV, EpEDV682, or 682 alone. (Figure 34D) Quantification of CD86 expression as determined by flow cytometry on JAWSII cells co-cultured with treated tumor cells. (Figure 34E) Quantification of MHC class II expression as determined by flow cytometry on JAWSII cells co-cultured with treated tumor cells. Data represent mean ± sem and are analyzed by one-way ANOVA and Tukey's multiple comparison test. [Figure 35-1]Figure 35A) CT26Ep12.1, Figure 35B) 4T1, Figure 35C) T84, and Figure 35D) % change in body weight in mice with A549 / MDR tumors. Weight loss of less than 5% was observed after the first dose, followed by recovery and stabilization with subsequent doses. Data represent mean ± standard deviation. Figure 35E) M1 / M2 (CD86:CD206) ratio of macrophages in A549 / MDR and T84 tumors in EDV-treated mice. Figure 35G) xCELLigence RTCA of CD11b+ isolated from A549 / MDR tumors and co-cultured with A549 / MDR cells in a 5:1 (E:T) ratio. Plots represent normalized cell indices correlated with cell adhesion and proliferation / death time. (Figure 35H) % cytolysis of A549 / MDR cells co-cultured with CD11b+ cells derived from tumors of EGFREDV682-treated mice was observed 6.5 hours after treatment with the addition of CD11b+ cells. (Figure 35I) Production of MIP1α in co-culture of CD11b+ cells isolated from 4T1 tumors treated with 4T1 cells. Data (Figures 35E, 35F, 35H, and 35I) represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison tests (Figures 35E, 35H, 35I) or t-test (Figure 35F). [Figure 35-2]Figure 35A) CT26Ep12.1, Figure 35B) 4T1, Figure 35C) T84, and Figure 35D) % change in body weight in mice with A549 / MDR tumors. Weight loss of less than 5% was observed after the first dose, followed by recovery and stabilization with subsequent doses. Data represent mean ± standard deviation. Figure 35E) M1 / M2 (CD86:CD206) ratio of macrophages in A549 / MDR and T84 tumors in EDV-treated mice. Figure 35G) xCELLigence RTCA of CD11b+ isolated from A549 / MDR tumors and co-cultured with A549 / MDR cells in a 5:1 (E:T) ratio. Plots represent normalized cell indices correlated with cell adhesion and proliferation / death time. (Figure 35H) % cytolysis of A549 / MDR cells co-cultured with CD11b+ cells derived from tumors of EGFREDV682-treated mice was observed 6.5 hours after treatment with the addition of CD11b+ cells. (Figure 35I) Production of MIP1α in co-culture of CD11b+ cells isolated from 4T1 tumors treated with 4T1 cells. Data (Figures 35E, 35F, 35H, and 35I) represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison tests (Figures 35E, 35H, 35I) or t-test (Figure 35F). [Figure 35-3]Figure 35A) CT26Ep12.1, Figure 35B) 4T1, Figure 35C) T84, and Figure 35D) % change in body weight in mice with A549 / MDR tumors. Weight loss of less than 5% was observed after the first dose, followed by recovery and stabilization with subsequent doses. Data represent mean ± standard deviation. Figure 35E) M1 / M2 (CD86:CD206) ratio of macrophages in A549 / MDR and T84 tumors in EDV-treated mice. Figure 35G) xCELLigence RTCA of CD11b+ isolated from A549 / MDR tumors and co-cultured with A549 / MDR cells in a 5:1 (E:T) ratio. Plots represent normalized cell indices correlated with cell adhesion and proliferation / death time. (Figure 35H) % cytolysis of A549 / MDR cells co-cultured with CD11b+ cells derived from tumors of EGFREDV682-treated mice was observed 6.5 hours after treatment with the addition of CD11b+ cells. (Figure 35I) Production of MIP1α in co-culture of CD11b+ cells isolated from 4T1 tumors treated with 4T1 cells. Data (Figures 35E, 35F, 35H, and 35I) represent mean ± sem and were analyzed by one-way ANOVA and Tukey's multiple comparison tests (Figures 35E, 35H, 35I) or t-test (Figure 35F). [Figure 36]This figure shows the NK cell response to EDV treatment. (Figure 36A) xCELLigence RTCA of NK cells isolated from the spleen of Balb / c nude mice with T84 tumors, co-cultured with T84 cells in a 10:1 (E:T) ratio. The plots represent cell viability calculated from time-normalized cell indices. (Figure 36B) Granzyme B production in co-cultures of NK cells isolated from the spleen of EGFREDV682-treated mice and T84 cells in saline. Data represent mean ± sem and were analyzed by t-test. (Figure 36C) xCELLigence RTCA of NK cells isolated from the spleen of Balb / c nude mice with A549 / MDR tumors, co-cultured with A549 / MDR cells in a 10:1 (E:T) ratio. The plots represent cell viability calculated from normalized cell indices over time (saline n=5; EGFREDV682 n=4). [Figure 37] This figure shows receptor expression and drug sensitivity screening of patient-derived pancreatic ductal adenocarcinoma cells. (Figure 37A) Quantification of EGFR surface receptors in cells derived from tumor heads. (Figure 37B) Quantification of EGFR surface receptors in cells from tumor tails. (Figure 37C) Drug sensitivity and IC50 of first and second line chemotherapy drug / drug combinations compared to sensitivity 682. [Figure 38]This figure shows that single cells from the entire PBMC pool (FSC v SSC) were gated based on forward scattering width (FSCW) versus forward scattering area (FSCA), and then analyzed for CD45 staining (CD45+ gated over FSC v CS45+). Cell viability was 96% (Count and viability kit #C00162, Beckman Coulter, data not shown), and dead cells were excluded based on 80 FSC threshold discriminants. All dendritic cells (DCs) were gated over leucoid (CD45+) and defined as HLA-DR+ and Lineage (#353351, Beckman Coulter). Lineage-negative markers consisted of a pool of antibodies conjugated with the same fluorophores (PE) produced against CD3, CD14, CD19, CD20, and CD56, respectively, used for negative selection of T cells, monocytes, B cells, and NK cells. The remaining cells that were HLA-DR+ were gated as dendritic cells and subdivided into plasmacytoid DCs (CD11c-CD123+) or antigen-presenting myeloid DCs (CD11c+CD123-). Myeloid DCs (mDCs) were divided into three major subsets: CD1c+mDC1, CD141+mDC2 (Clec9A+ shown here), and CD16+mDC. CD14+ expression defined gated monocytes (#B93604, Beckman Coulter) on leukocytes, which were then subdivided into classical (CD14+CD16-), intermediate (CD14+CD16+), and non-classical (CD14+CD16+). T cells (#B53328, Beckman Coulter) were CD3+ and gated on lymphocytes (CD45+ low SSC levels). Next, we defined CD3+ T cell subsets, T helper (CD4+), and cytotoxic T (CD8+). PD1+ CD8+ cytotoxic T cell gates were plotted against SSCs and gated against CD8+ T cells. All FSC and SSC axes are linear, while the fluorescence channel axis (all CD markers) is logarithmic or bi-exponential ("logicle", Kaluza software, Beckman Coulter). [Figure 39]The following shows flow cytometry analysis of the purity of cell isolates used in the xCelligence RTCA experiment. (Figure 39A) Isolation of CD11b+ cells from mouse tumors. Density plots showing isotype control vs. FSC and CD11b vs. FSC. The sample had CD11b+ cells with approximately 80% purity. (Figure 39B) Isolation of NK cells from mouse spleen. Density plots showing isotype control against FSC, NKp46 against FSC, and CD11b against FSC. The sample had NK cells with approximately 90% purity. (Figure 39C) Isolation of CD8+ T cells from mouse spleen. Density plots showing isotype control against FSC, CD3e against FSC, and CD8 against FSC. The sample had T cells (CD3e+) with approximately 90% purity, and more than 98% of those T cells were CD8+. [Figure 40] This figure shows a combination of treatments using Ep minicell Dox and minicell α-GC in a syngeneic mouse model (EpCT26 colorectal tumor in Balb / c mice). [Figure 41] This figure shows that combined treatment with Ep minicell Dox and minicell α-GC is effective in reducing large tumors in Balb / c mice carrying CT26 allografts. [Figure 42] This study demonstrates the effects of Ep minicell Dox and minicell α-GC on tumor regression in Balb / c mice carrying CT26 allografts. [Figure 43] Figures 43A and 43B show Ep minicells Dox and minicells α-GC, Figures 43D and 43E show minicells α-GC only, Figure 43F shows Ep minicells Dox only, and Figure 43C shows CT26 isografts of various sizes treated with physiological saline. [Figure 44] This figure shows CT26 isografts of various sizes treated with Ep minicells Dox and minicells α-GC. [Figure 45] This figure shows the αGC-CD1D presentation of JAWSII cells after α-GC treatment of mini-cells at various time points (Figure 45A-E). [Modes for carrying out the invention]

[0023] I. Overview The present invention is based on the discovery that compositions comprising (i) an antitumor agent and a type I interferon agonist; (ii) an antitumor agent and a type II interferon agonist; or (iii) a combination of an antitumor agent, a type I interferon agonist, and a type II interferon agonist, wherein at least the antitumor agent is packaged within intact bacterial mini-cells, can synergistically improve cancer treatment strategies.

[0024] The combination of an activator and an immunomodulator, in which at least an antitumor agent, and optionally a type I and / or type II interferon agonist, is packaged within intact bacterial minicells, results in dramatic efficacy against cancer cells and a remarkable lack of drug resistance development in the target. The described compositions provide a synergistically improved cancer treatment strategy by avoiding the toxicity associated with systemic delivery of antitumor agents combined with immunomodulators such as type I and / or type II interferon agonists.

[0025] Recent advances in cancer immunotherapy have resulted in unprecedentedly durable clinical responses in certain cancers (Emens et al., 2017; Farkona et al., 2016; Oiseth and Aziz, 2017; Sharma et al., 2017; Ventola, 2017). However, current immunotherapy strategies have resulted in limited success rates across various tumor types, with a significant proportion of patients demonstrating accelerated recurrence-onset tumor regression (Emens et al., 2017; Mellman et al., 2011; Oiseth and Aziz, 2017; Sharma et al., 2017; Ventola, 2017).

[0026] The data described in Example 16 below elucidate the mechanism of the cell-immunotherapy function of tumor-targeted nanocell therapy, initiating a dual attack against the tumor via the delivery of hypercytotoxins combined with the engagement of multiple arms of the immune system. This approach also avoids some of the current pitfalls of immunotherapy by creating an immunogenic tumor microenvironment and thereby acting on multiple immune cell subsets to evade primary and / or adaptive resistance that may arise in the patient.

[0027] Furthermore, a subset of patients lack oncoimmunogenicity resulting from a lack of tumor cell antigens or immune cell infiltration, and therefore do not show an initial response to currently available strategies (Emens et al., 2017; Oiseth and Aziz, 2017; Sharma et al., 2017). Thus, the identification of novel and robust immunotherapy approaches remains a high-priority area, as it has the potential to lead to significant improvements in clinical outcomes.

[0028] To initiate an effective antitumor immune response, certain steps must be achieved, either spontaneously or therapeutically. Firstly, tumor cell antigens, which can be induced in situ via tumor cell death or delivered exogenously, must be taken up by dendritic cells (DCs) (Anguille et al., 2015; Emens et al., 2017; Jung et al., 2018; Mellman et al., 2011). Along with antigen uptake, DCs must receive appropriate maturation signals that promote antigen differentiation and enhanced processing and presentation, thereby promoting antitumor function opposite to resistance (Anguille et al., 2015; Emens et al., 2017; Jung et al., 2018; Mellman et al., 2011; Simmons et al., 2012). These mature tumor antigen-loaded DCs must then effectively generate an antitumor T cell response, which may occur through the production of tumor-specific cytotoxic T cells, the triggering of NK and / or NKT cell responses, and the enhancement of T-helper type 1 responses (Emens et al., 2017; Fang et al., 2017; Mellman et al., 2011; Sharma et al., 2017; Zitvogel et al., 2015). The antitumor T cells must ultimately enter the tumor microenvironment where immunosuppressive signals may be present and effectively perform their antitumor functions (Emens et al., 2017; Mellman et al., 2011). Problems arising in any of these steps can hinder the effectiveness of immunotherapy and lead to total treatment failure (Emens et al., 2017; Mellman et al., 2011; Sharma et al., 2017).

[0029] Currently, the most clinically significant immunotherapy strategies include immunological checkpoint inhibitors and chimeric antigen receptor T-cell therapy (CAR) (Emens et al., 2017; Mellman et al., 2011; Oiseth and Aziz, 2017; Sharma et al., 2017; Ventola, 2017). Checkpoint inhibitors such as cytotoxic T lymphocyte antigen 4 (CTLA4) and programmed cell death 1 / programmed cell death 1 ligand (PD / PDL) work by blocking the transmission of immunosuppressive signals and stimulating cytotoxic T lymphocytes to be activated within the tumor microenvironment (Dine et al., 2017; Jenkins et al., 2018; Sharpe, 2017). While inhibitors of these pathways have shown dramatic clinical outcomes in certain cancers, overall response rates across different cancers remain low (~15-25%), and immuno-related toxicity associated with these treatments may be high (Dine et al., 2017; Emens et al., 2017; Jenkins et al., 2018; Sharpe, 2017; Ventola, 2017). The continuous discovery of new checkpoints as potential immune targets clearly makes it possible to utilize a sophisticated and diverse set of immunosuppressive pathways (Dine et al., 2017; Emens et al., 2017; Farkona et al., 2016; Jenkins et al., 2018; Sharpe, 2017). Therefore, developing resistance to checkpoint inhibitors remains a challenge, and attempts have been made to overcome these problems by using combinations of multiple checkpoint inhibitors, but this often exacerbates the associated toxicity (Dine et al., 2017; Jenkins et al., 2018; Sharma et al., 2017; Ventola, 2017).

[0030] A second widely discussed treatment is CART cell therapy, which involves genetically modifying the patient's T cells to express membrane fusion receptors that have defined tumor antigen specificity and can induce potent T cell activation to initiate the killing of target tumor cells (D'Aloia et al., 2018'; Farkona et al., 2016; Mellman et al., 2011; Sharma et al., 2017). While this therapeutic approach has yielded unprecedented clinical results in the treatment of "liquid" hematological malignancies, to date it has not yielded comparable responses when targeting solid malignancies due to limitations associated with a lack of good specific antigen targets, poor tumor homing, poor tumor exudation, and lack of persistence within the adversarial tumor microenvironment (D'Aloia et al., 2018'; Sharma et al., 2017). There are also practical limitations related to the availability of lymphocytes from heavily pre-treated patients and the long manufacturing time, making it not a viable treatment option for patients with rapidly progressing disease (Oiseth and Aziz, 2017; Rezvani et al., 2017).

[0031] EnGeneIC Dream Vector (EDV) is a bacterial-derived delivery system consisting of non-viable nanocells with a diameter of 400 ± 20 nm, generated by reactivating the polarity sites of cell division in bacteria (MacDiarmid et al., 2007b). These nanocells can be packaged with cytotoxic drugs, siRNA, or miRNA and have been demonstrated to be specifically targeted to tumor cell surface receptors via the attachment of bispecific antibodies to the nanocell surface polysaccharides (MacDiarmid et al., 2009; MacDiarmid et al., 2007b; Reid et al., 2013). After intravenous administration in mouse and canine studies, they are retained in the vascular system due to their size, but subsequently, tumor-associated leakage occurs. It has been demonstrated that the cells rapidly spill into tumors via the vascular system (MacDiarmid et al., 2007b; Sagnella et al., 2018). Binding to tumor cell surface receptors via associated bispecific antibodies leads to macropinocytosis to endosomes and release of the payload via intracellular denatured red blood cells in lysosomes (MacDiarmid et al., 2009; MacDiarmid et al., 2007b; Sagnella et al., 2018). The safety of these nanocell therapies has been demonstrated in three Phase I clinical trials, with over 1000 doses administered to various terminally ill cancer patients, and 682 loaded EDVs currently being delivered to patients in Phase I trials, showing a promising safety profile to date (2017; Kao et al., 2015; Solomon et al., 2015; van Zandwijk et al., 2017; Whittle et al., 2015). A. Overview of bacterial mini-cell delivery methods The use of bacterial minicells to deliver chemotherapeutic agents to cancer cells has been previously described. This delivery method for treating cancer involves packaging toxic chemotherapeutic agents or drugs, or functional nucleic acids, into bacterial minicells, typically with a diameter of about 400 nm. Typically, the minicells possess antibodies that target specific cancer cells. These antibodies bind to the surface of cancer cells, and the minicells are taken up into the cells by the cancer cells. In this way, the toxic chemotherapeutic agents are not widely distributed throughout the body, and therefore the opportunity for side effects and intolerances is reduced as the toxic drug or compound is delivered into the cancer cells. Using antibody-targeted minicells as a delivery vehicle for toxic chemotherapeutic agents requires far less drug to kill cancer cells, thus improving the therapeutic index.

[0032] In fact, the inventors have shown that mini-cells (or EnGeneIC Dream Vehicle, EDV) can deliver chemotherapeutic agents such as paclitaxel or doxorubicin to xenograft tumors in mice (Example 1), dogs (Example 2), and monkeys (Example 3). Targeted delivery ensures that cancer cells receive the majority of the chemotherapeutic agent, resulting in low levels of toxicity. See Examples 1-3; also see MacDiarmid et al., 2007b; MacDiarmid et al., 2007a; MacDiarmid et al., 2009; and MacDiarmid et al., 2016. Furthermore, mini-cells do not induce a significant immune response in xenograft models, and mini-cells are well-tolerated (Example 4). Therefore, intact bacterial minicells are a well-acceptable vehicle for delivering anticancer drugs to patients, including examples such as doxorubicin targeting advanced solid tumors (Example 5), doxorubicin targeting glioblastoma (Example 6), and MicroRNA-16a targeting mesothelioma (Example 7).

[0033] However, these treatment strategies did not result in complete remission or cure of all cancers in all patients. Therefore, improvements in cancer treatment are needed. The inventors have discovered that using a combination of mini-cells with three different types of payloads yields remarkably dramatic and effective clinical results.

[0034] Specifically, the inventors discovered that mini-cells containing a chemotherapeutic agent (for example, in the following examples, the agent is PNU159682, a highly toxic chemotherapeutic agent) combined with mini-cells containing an interferon type I agonist and / or an interferon type II agonist produced a synergistic antitumor effect that was well tolerated by patients with advanced pancreatic cancer. See Example 12. In fact, patients with advanced pancreatic cancer showed a significantly improved quality of life after this treatment, which is remarkable for patients at that stage. This triple-of-Dur combination strategy synergistically improves cancer treatment. The inventors also discovered that mini-cells containing a chemotherapeutic agent combined with mini-cells containing an interferon type II agonist produced a synergistic antitumor effect.

[0035] Surprisingly, it was discovered that the combination of mini-cell packaged antitumor drugs with type II interferon agonists, and in the absence of type I interferon agonists, resulted in dramatic efficacy against large tumors. Such results have not been previously reported. It is hypothesized that in some patients, the combination of type I and type II interferon agonists may be counterproductive because the two interferon agonists may act competitively rather than synergistically. This data will be discussed in more detail below.

[0036] The following description outlines the present invention in relation to these discoveries, but does not limit the invention to the specific embodiments, methodologies, protocols, or reagents described herein. Similarly, the terms used herein describe only specific embodiments and do not limit the scope of the invention.

[0037] B. Summary of experimental results (i) Combination therapy with mini-cell packaged anti-cancer agents and mini-cell packaged type I interferon agonists In the first embodiment, compositions and methods relating to a combination of a bacterial minicell-packaged antitumor agent combined with a type I interferon agonist packaged in bacterial minicells are described.

[0038] Example 11 and Figure 18 describe data illustrating the results in a lung cancer xenograft model in mice treated with various mini-cell (EDV) compositions, as summarized in the table below. Animals in groups 1 and 5 were administered a combination of a chemotherapeutic agent (PNU159682) packaged in intact bacterial mini-cells and a type I interferon agonist (40-mer double-stranded DNA or 50-mer double-stranded DNA) packaged in intact bacterial mini-cells. All mini-cell compositions resulted in stabilization of tumor growth. However, the most dramatic results were obtained after treating the large tumor size resulting from saline treatment in Part 1 of the experiment. Subsequently, when the saline-treated control group was treated in Part 2 of the experiment with compositions including a combination of mini-cell packaged antitumor agent and mini-cell packaged type I interferon agonist, tumor size decreased by 62% over 5 days.

[0039] [Table 1] In the follow-up of Example 11, the addition of mini-cell packaged type I interferon agonists resulted in a dramatic reduction in tumor size, which was not observed when the mini-cell packaged antitumor agent was used in the absence of the type I interferon agonist immunostimulant. The results are summarized in the table below. These results clearly demonstrate the immunostimulatory effect of adding mini-cell packaged type I interferon agonists to mini-cell packaged antitumor agents.

[0040] [Table 2] (ii) Combination therapy with mini-cell packaged type I interferon agonists and, optionally, type II interferon agonists (not mini-cell packaged) and mini-cell packaged anti-cancer agents. The second embodiment relates to a method and composition using a mini-cell packaged anti-neoplastic agent that combines a mini-cell packaged type I interferon agonist or a mini-cell packaged anti-cancer agent with a mini-cell packaged type I interferon agonist and a type II interferon agonist (without bacterial mini-cells).

[0041] Further evidence of the dramatic and remarkable efficacy of the compositions of the present invention is reflected in the clinical results shown in Example 12. Specifically, Example 12 concerns what happened when patients with advanced solid tumors were treated with (1) a combination of a mini-cell packaged antitumor agent and a mini-cell packaged type I interferon agonist; and (2) a composition comprising a combination of a mini-cell packaged antitumor agent, a mini-cell packaged type I interferon agonist, and a type II interferon agonist.

[0042] In particular, the human clinical data detailed in Example 12 demonstrate the safety profiles of type I and type II IFN agonists used as immunostimulants in mini-cell packaged antitumor agents in human patients. Type I interferon agonists packaged in intact bacterial mini-cells contain 40-mer double-stranded DNA (EDV). 40mer ) or 60-mer double-stranded DNA (EDV) 60mer Please refer to the data in Table 3 below. Furthermore, the results were remarkable in a stage 4 pancreatic cancer patient who had exhausted all other treatment options. The patient's tumor marker (CA19-9) levels decreased by more than 90% after the first three doses, which was equivalent to just 10 days of treatment. After 10 doses, this decreased further, with tumor marker levels dropping by nearly 95%. This patient also showed significant weight gain and reported a remarkable improvement in quality of life, in contrast to the cachexia experienced by most patients with stage IV pancreatic cancer. These results are dramatic, especially considering the poor prognosis associated with advanced pancreatic cancer. In summary, there are 5 examples. EGFR(5) EDV PNU / Dox or EGFR(V) EDV PNU +EDV 40mer / 60mer Patients received a total of 69 treatments with (Type I IFN agonist) ± Imukin (Type II IFN agonist). The treatment was well-tolerated, and the addition of immunomodulatory immunostimulants did not appear to alter the safety profiles of monotherapy-loaded EDV and targeted EDV.

[0043] [Table 3] (iii) Combination therapy of mini-cell packaged anti-cancer agents with type II interferon agonists Example 13 describes the results of various studies conducted to evaluate the efficacy of combining mini-cell-packaged antineoplastic agents with type II interferon agonists, such as IFN-γ. The results show that the addition of type II interferon agonists enhances or enhances the anticancer effect of mini-cell-packaged antineoplastic agents in xenograft models of various cancers, including lung cancer and breast cancer. Furthermore, the data described in Example 13 and excerpted in Table 4 below demonstrate that the addition of type II interferon agonists to compositions containing mini-cell-packaged antineoplastic agents is essential for achieving tumor stabilization in the treatment of tumors that are typically resistant to antitumor agents alone. Therefore, drug resistance can be overcome by combining mini-cell-packaged antitumor agents with type II interferon agonists.

[0044] [Table 4] (iv) Triple combination therapy: mini-cell packaged antitumor agent, mini-cell packaged type I interferon, and type II interferon agonist (either alone or in mini-cell package form). The inventors also discovered that a triple combination of a mini-cell packaged antitumor agent, a mini-cell packaged type I interferon agonist, and a type II interferon agonist (either alone or in mini-cell packaging) can produce dramatic anticancer effects. Specifically, Example 14 details the treatment of dogs with late-stage endogenous tumors (brain cancer, sarcoma, or melanoma) using a combination of a mini-cell packaged antitumor agent, a mini-cell packaged type I interferon agonist, and a type II interferon agonist. The results indicate that the combination composition was well-tolerated. Furthermore, disease stabilization occurred in 6 of the 7 evaluable animals (85.7%), while one dog achieved near-partial response (29.8% reduction in tumor size).

[0045] (v) Dual therapy with a mini-cell packaged antitumor agent and a mini-cell packaged type II interferon agonist in the absence of type I interferon. In another embodiment, the present invention relates to a composition based on the discovery that a combination of a mini-cell packaged antitumor drug and a mini-cell packaged type II interferon agonist, such as α-galactosylceramide (α-GC), exhibits remarkable anticancer effects in the absence of a type I interferon agonist.

[0046] In particular, Example 23 describes data demonstrating the efficacy of a dual combination of a mini-cell-containing therapeutic agent and a mini-cell-containing interferon type II agonist against tumors. These results demonstrate that compositions lacking interferon type I agonists can be used to effectively treat tumors. See also Figures 40 and 42. Experimental results were obtained using saline andEp Miniature cells Dox Compared to the treatment, Ep Miniature cells Dox + Mini Cells α-GC In the group receiving combination therapy (interferon type II agonist), a significant cessation of tumor progression was observed. Ep Miniature cells Dox mini cells α-GC Adding this treatment supports the theory of immunostimulatory effects.

[0047] Further data showed that saline-treated control tumors demonstrated dramatic tumor regression after treatment changes to drug and α-GCEDV-mediated dual combination therapy (Figure 41); for example, a combination of mini-cell packaged antitumor agents and mini-cell packaged type II interferon agonists. In particular, 800 mm 3 The tumor reached 600 in 3 days. 3 The tumor size decreased to less than mm, and a significant reduction in tumor size (~25%) was observed in a short period. The ability of the dual combination composition to dramatically reduce large tumors in a short period was unknown prior to this invention.

[0048] In one embodiment of the present invention, a dual combination composition (e.g., a mini-cell packaged antitumor agent combined with a mini-cell packaged interferon-II agonist) can reduce tumor size (including large tumor size) by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% over about 3 days, about 5 days, about 1 week, about 2 weeks, about 3 weeks, about 1 The decrease in cell size can be measured over any appropriate period of time, such as approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1.5 years, or more than 2 years.

[0049] C. Immunotherapy Data Example 16 details data demonstrating that mini-cell packaged antitumor agents targeted to tumor cell surface receptors function as cancer immunotherapy, e.g., cell-immunotherapy. In particular, the example illustrates the ability of bacterial mini-cells to activate cells of the innate immune system, including macrophages, NK cells, and dendritic cells. This is followed by dendritic cell maturation and antigen presentation, production of tumor-specific cytotoxic T cells, and an adaptive T-cell response that leads to the recruitment of further immune cells into the tumor microenvironment. This approach avoids some of the current pitfalls of immunotherapy by creating an immunogenic tumor microenvironment and acting on multiple immune cell subsets, thereby avoiding potential primary and / or adaptive resistance in patients.

[0050] Therefore, this example demonstrates the ability of bacterial minicells to deliver cytotoxic drugs into tumor cells while simultaneously inducing innate and adaptive immune responses that specifically target the tumor.

[0051] Further immunotherapy data are presented in Example 18, which describes data demonstrating that NK cells adopt an antitumor phenotype in vivo following treatment with targeted minicells containing antineoplastic agents. This is significant because NK cells are the major effector cells of the innate immune system and are tightly regulated by the balance between activating and inhibitory signals (Morvan and Lanier, 2016; Wallace and Smyth, 2005). Impairment of NK cell function is associated with increased tumorigenesis, proliferation, and metastasis, and therefore its importance in contributing to the antitumor immune response is well demonstrated (Fang et al., 2017; Morvan and Lanier, 2016; Rezvani et al., 2017; Wallace and Smyth, 2005).

[0052] Interestingly, Example 19 details data showing that a primarily Th1 cytokine response within the tumor microenvironment is exhibited after treatment with a minicell-encapsulated antitumor agent (e.g., PNU159682). Cytokine and chemokine production within the tumor microenvironment allows immune cells to effectively communicate with each other to generate a coordinated response that may be either tumor-promoting or tumor-suppressing (Belardelli and Ferrantini, 2002; Lee and Margolin, 2011). The effect of individual cytokines on the immune response depends on various factors, including local concentration, cytokine receptor expression patterns, and the activation state of surrounding cells (Lee and Margolin, 2011). Thus, it has been shown that many cytokines can elicit opposing effects against tumor growth (Dredge et al., 2002; Landskron et al., 2014; Lee and Margolin, 2011).

[0053] Furthermore, Example 20 details data showing that treatment with a minicell-encapsulated antitumor agent (e.g., PNU159682) results in the production of tumor-specific CD8+ T cells. The initial in vitro experiment showed that EDV treatment could lead to dendritic cell maturation in response to targeted EDV loaded with effective chemotherapeutic agents, either through direct interaction or as a result of cell death. Thus, this study suggests that this result could be translated into in vivo DC maturation and antigen presentation, and tumor-specific CD8+ T cells. + The aim was to test whether mini-cell treatment would induce the production of cytotoxic T cells. The resulting data showed that mini-cell treatment induced tumor-specific CD8 + We demonstrated successful induction of T cell production. Furthermore, a significant increase in total T cell count (CD3+), as well as significant increases in both CD4+ and CD8+ T cell counts, was observed in the lymph nodes of mice treated with minicell-encapsulated antitumor agents (e.g., PNU159682) (Figure 31G). A significant increase in mature dendritic cells was also detected in the lymph nodes of treated mice (Figure 31H), and CD8+ cells were isolated from mice treated with 4T1 cells. +Visualization of the interaction between T cells shows that these T cells can attach perforin (green) to tumor cells and excrete it there (Figure 31I).

[0054] Example 21 demonstrates the ability of targeted bacterial minicells loaded with an anti-tumor agent (e.g., the super cell toxin PNU159682) to not only effectively deliver this drug to the tumor site but also act as an immunotherapeutic agent by stimulating a number of immune cell subsets. The example demonstrates the ability of minicell-encapsulated anti-tumor agent treatment to push immune cell subsets, including macrophages, NK cells, and CD8 + T cells, towards an anti-tumor phenotype capable of effectively eliminating tumor cells. In combination with the effectiveness of the anti-tumor drug, this results in a dual attack on the tumor.

[0055] The concept of cancer immunotherapy dates back several decades, but its potential has only recently begun to be realized with the approval of many immunotherapies (Farkona et al., 2016; Ventola, 2017). Bacterial minicells are a unique complex cell immunotherapy that first creates an immunogenic tumor microenvironment through the delivery of direct cytotoxic agents to the tumor, where it directly or indirectly stimulates the innate immune system towards an anti-tumor phenotype. This innate immune activation then induces an adaptive response that results in tumor-specific cytotoxic T cells (Figure 33).

[0056] Following intravenous administration, the mini-cells spill into the tumor via the tumor's leaky vascular system, where more than 30% of the administered dose of targeted mini-cells with their toxic payloads are directly deposited into the tumor microenvironment within 2 hours (MacDiarmid et al., 2007b). The targeted bacterial mini-cells bind to receptors on tumor cells (4T1 and CT26Ep12.1 in Example 21), and then effectively deliver their load (antitumor agent) directly into the tumor cells. PNU159682 is a highly potent hypercytotoxic agent that induces rapid apoptosis within 24 hours of delivery to tumor cells (Figure 33A). Subsequently, the apoptotic cells and DAMP signaling produced by bacterial mini-cell (e.g., Ep-EDV-682) treatment interact with innate immune cells such as tumor-associated macrophages (TAMs), stimulating CD86 upregulation and the production of Th1 pro-inflammatory cytokines such as TNFα and IL-6 (Figure 33B). These changes are typical of M1 polarization in macrophages, which can lyse tumor cells and signal the release of cytokines to activate other immune cell subsets, and have therefore been shown to possess antitumor properties (Sawa-Wejksza and Kandefer-Szerszen, 2018; Yuan et al., 2015).

[0057] Furthermore, bacterial minicells themselves can directly interact with TAMs that produce similar M1 polarization, although this is expected to occur at very low levels in the current system. TAMs are generally the most abundant immune cells in the tumor microenvironment, and an increase in the number of TAMs has been demonstrated to be associated with poor prognosis and increased tumor growth (Sawa-Wejksza and Kandefer-Szerszen, 2018). This is due to the fact that the majority of TAMs consist of anti-inflammatory M2 macrophages, which have been shown to have tumor-promoting properties, while inflammatory M1 macrophages exhibit antitumor properties (Sawa-Wejksza and Kandefer-Szerszen, 2018; Yuan et al., 2015). Example 21 demonstrates the ability of bacterial minicell treatment to shift the M1:M2 balance in the tumor microenvironment in four different tumor models. Despite the differences in the degree of this shift in the different tumor models, the increase in M1 polarization was shown to translate to increased tumor cell lysis by TAMs isolated from tumors of mice treated with bacterial minicells. In addition to the phenotypic shift to M1, TAMs from bacterial minicell-treated mouse tumors also mobilize immune cells, particularly NK cells, CD4+ T cells, and CD8 + The cells secreted increased amounts of MIP-1α, a chemokine established to be involved in promoting tumor infiltration by T cells (Figure 33C) (Allen et al., 2018).

[0058] In addition to TAM activation, immature dendritic cells (DCs) directly interact with bacterial minicells, or more likely, interact with apoptotic cells and DAMP signaling produced by bacterial minicell-treated tumors, leading to dendritic cell maturation and migration to lymph nodes for antigen presentation. DCs are known to be the most effective antigen-presenting cells and constitute a bridge between the innate and adaptive immune systems, making them a potential target in cancer immunotherapy (Allen et al., 2018).

[0059] Modern strategies for DC-based immunotherapy involve ex vivo manipulation and priming of DCs or DC precursors, but the success of these strategies is limited by various factors, including the development of immune tolerance, induction of an insufficient number of CD8+ cytotoxic T cells (CTLs), or insufficient antitumor efficacy, and the suppressive nature of the tumor microenvironment (Anguille et al., 2015; Jung et al., 2018; Landskron et al., 2014; Oiseth and Aziz, 2017). Bacterial minicell treatment, in response to dying tumor cells (Figure 33D), enables in vivo priming and maturation of DCs within the tumor microenvironment, where immature DCs can take up DAMP and / or apoptotic tumor cell bodies produced in response to targeted and drug-loaded bacterial minicells. These DAMPs and dying tumor cells are then processed via MHC class I and II molecules for antigen presentation on the DC surface, while DC maturation occurs simultaneously. Upregulation of the costimulatory molecules CD86, CD80, and MHC class II, identified as markers of the DC maturation process, was shown to occur in DCs co-cultured with bacterial minicell-treated tumor cells, along with an increased percentage of mature DCs detected in tumor drainage lymph nodes of bacterial minicell-treated mice (Anguille et al., 2015; Cauwels et al., 2018; Simmons et al., 2012). During the maturation process, DCs migrate to tumor drainage lymph nodes for antigen presentation to T cells, thereby initiating tumor-specific CD8+ T helper cells and an adaptive immune response against the tumor. +This increases CTL production (Figure 33E). Subsequently, increased production of IFNα / β, TNFα, IL-12p40, and IL-6 was detected in DCs co-cultured with bacterial minicell-treated tumor cells, and a significant increase in IFNα concentration in the tumor microenvironment was observed in both 4T1 and CT26Ep12.1 tumor models (Example 21). Expression levels of type 1 IFN (IFNα / β) and IFN-stimulating genes within the tumor microenvironment have been shown to correlate with favorable disease outcomes and may even be necessary for the success of cancer treatment, including immunotherapy (Cauwels et al., 2018; Fitzgerald-Bocarsly and Feng, 2007; Zitvogel et al., 2015). The antitumor activity of type 1 IFN arises indirectly through the activation of immune cells such as DCs, T and B lymphocytes, NK cells, and macrophages (Cauwels et al., 2018; Fitzgerald-Bocarsly and Feng, 2007; Showalter et al., 2017; Zitvogel et al., 2015).

[0060] In addition to enhancing macrophage and DC antitumor function, treatment of bacterial minicells with antitumor agents can induce NK cell activation, leading to increased cytotoxicity (Figure 33F). Because NK cells possess an inherent ability to lyse non-antigen-mediated malignant cells, their activation and functional state must be strictly controlled to avoid potential adverse effects on the host. The ability to attract and activate NK cells within the tumor microenvironment is essential for their ability to exert antitumor function. Cytokines, including IL-2, IFNγ, and IFNα, which are significantly increased in the microenvironment of Ep-EDV-682-treated tumors, are known to activate NK cells toward both increased cytokine production and enhanced cytolytic function (Fang et al., 2017; Ferlazzo and Munz, 2004; Lee and Margolin, 2011; Morvan and Lanier, 2016; Rezvani et al., 2017). Indeed, evidence indicates that type 1 IFN is required for the activation of NK cytotoxicity (Ferlazzo and Munz, 2004; Muller et al., 2017). Furthermore, type 1 IFN can induce cellular senescence, followed by upregulation of NKG2D ligand expression in tumor cells, thereby promoting their elimination by NK cells (Muller et al., 2017). Upregulation of the NKG2D receptor was observed in NK cells within tumors of mice treated with Ep-EDV-682, and this receptor was demonstrated to significantly contribute to the cytolytic ability of NK cells isolated from Ep-EDV-682-treated mice. Furthermore, NK cells from immature, intermediate, and mature mice express both CCR1 and CCR5 chemokine receptors, which can bind to the chemokines MIP1α and RANTES, respectively, and both are upregulated by Ep-EDV-682-treated tumors, as well as by macrophages and NK cells from mice treated with Ep-EDV-682 (BeRNArdini et al., 2016).

[0061] Chemokines such as MIP1α and RANTES are involved in the further recruitment of helper and effector immune cells, including NK cells, macrophages, and T cells, into the tumor microenvironment (Figure 33G) (Allen et al., 2018; Bernardini et al., 2016; Zibert et al., 2004). Following the initial innate immune response by EDV treatment, which includes macrophages, NK cells, and DCs, an adaptive immune response is mounted, where tumor-specific CTLs and T-helper cells are produced and subsequently recruited to the tumor site (Figure 33H). Tumor-specific CTLs then target and lyse tumor cells, further contributing to a preferred anti-tumor environment created by other immune cell subsets in combination with targeted, drug-loaded EDV. Targeted drug-loaded EDV treatment primarily induces a Th1 response, as evidenced by the increase in Th1 cytokines (TNFα, IFNα, IFNγ, IL-2, and IL-6) within the tumor microenvironment. As previously mentioned, when activated, a subset of innate immune cells becomes a primary source of one or more of these specific cytokines. T cells can similarly produce all of the aforementioned cytokines (Belardelli and Ferrantini, 2002; Lee and Margolin, 2011). The release of these cytokines by either innate immune cells or T cells is involved in the simultaneous stimulation, activation, proliferation, and increased antigen presentation of further immune cells, creating a feedback loop that further enhances the antitumor activity of the immune system (Figure 33I) (Lee and Margolin, 2011).

[0062] Bacterial mini-cell therapy represents a unique cancer treatment strategy capable of directly delivering conventional and novel drug therapies to tumor sites, followed by the induction of an anti-tumor immune response. A dual attack on the tumor occurs, first through cell death in response to the delivered therapy, followed by innate immune cell activation that leads to an adaptive immune response. This type of therapy has certain advantages over current immunotherapy strategies in that immune cell activation occurs primarily at the tumor site, even in vivo, in a rapidly changing, dynamic environment. Furthermore, it induces effects on multiple immune cell subsets, avoiding the problem associated with patients who create an immunogenic tumor environment and show little adaptation to immune responses against those tumors or to therapies targeting only a single immune cell subset. The study described in Example 21 highlights the potential of bacterial mini-cells as a novel cancer immunotherapy, and future bacterial mini-cell formulations can further leverage their intrinsic immunogenicity, considering the versatility of this technology in terms of both payload and targeting capabilities (MacDiarmid et al., 2007a).

[0063] D. Highly toxic anti-cancer agents Example 17 details data demonstrating the effective delivery of a supertoxic antitumor agent, such as PNU159682, which cannot be delivered using conventional means due to the severe toxicity associated with the compound. Specifically, Example 17 details how PNU159682 is a supercytotoxin with an IC50 even in drug-resistant cancer cells in the pM range (Quintieri et al., 2005), which means the compound cannot be used clinically due to severe systemic toxicity (Staudacher and Brown, 2017). However, when encapsulated in bacterial minicells, supercytotoxins like PNU159682 can be effectively delivered to tumors with few side effects.

[0064] II. Components As described above, the compositions of the present invention comprise at least two different activators, an antitumor agent and a type I interferon agonist, a type II interferon agonist, or both a type I interferon agonist and a type II interferon agonist and an antitumor agent. The three different activators can be packaged in one, two, or three different minicells. The type II interferon agonist can also be included in the methods and compositions of the present invention without being packaged in minicells.

[0065] A. Antitumor agents or cytotoxic agents useful in the treatment of cancer "Antineoplastic agents" refer to pharmaceuticals that inhibit or block the proliferation, development, maturation, or spread of tumor cells, whether chemical or biological. "Antineoplastic agents" are interchangeable with "chemotherapeutic agents." In the context of this disclosure, the selection of an antitumor agent for treating a given brain tumor patient depends on several factors, consistent with conventional medical practice. These factors include, but are not limited to, the patient's age, Karnovski score, and any past treatments the patient may have received. See, in general, Principles and Practices of Neuro-Oncology, M. Mehta (Demos Medical Publishing 2011), and Principles of Neuro-Oncology, D.Schiff and P.O. Neill, eds. (McGraw-Hill 2005).

[0066] The composition may contain at most about 1 mg of an antitumor or chemotherapeutic agent. Alternatively, the amount of chemotherapeutic agent may be at most about 750 μg, about 500 μg, about 250 μg, about 100 μg, about 50 μg, about 10 μg, about 5 μg, about 1 μg, about 0.5 μg, or about 0.1 μg. In another embodiment, when the composition is used without being packaged in mini-cells, it may contain a chemotherapeutic agent in an amount less than about 1 / 1,000 of the therapeutically effective dose of the drug, or less than about 1 / 2,000, 1 / 5,000, 1 / 10,000, 1 / 20,000, 1 / 50,000, 1 / 100,000, 1 / 200,000, or 1 / 500,000. According to yet another embodiment of the present disclosure, the composition may contain at least about 1 nmol of chemotherapeutic agent. Accordingly, the disclosure also includes embodiments in which the amount of chemotherapeutic agent is at least about 2 nmol, about 3 nmol, about 4 nmol, about 5 nmol, about 10 nmol, about 20 nmol, about 50 nmol, about 100 nmol, or about 800 nmol, respectively.

[0067] In the context of this disclosure, the selection of an antitumor agent to treat a given tumor depends on several factors. These factors include, but are not limited to, the patient's age, the stage of the tumor, and any past treatments the patient may have received.

[0068] According to this disclosure, drugs may be selected from one of the classes detailed below for packaging into intact bacterial mini-cells. These drugs may also be synthetic analogues designed from drug design and inventive efforts. Any known chemotherapeutic agent may be used in the compositions of the present invention. Known chemotherapeutic agents include, but are not limited to, the following: (1) Mustard gas derivatives (mechloretamine, cyclophosphamide (cytoxane), chlorambucil (Leukeran), melphalan, ifosfamide, etc.), ethyleneimines (thiotepa (thioplex) and hexamethylmelamine), alkyl sulfonates (busulfan (milleran)), hydrazines and triazines (altretamine (hexalen), procarbazine (mathuran), dacarbazine (DTIC) and temozolomide), nitrosoureas (carmustine, lomustine and streptozosin), metal salts (carboplatin, cisplatin (platinol) and oxaliplatin), alkylating agents such as mechloretamine and melphalan (Alkeran); (2) (3) Antitumor drugs such as plant alkaloids, terpenoids and topoisomerase inhibitors, for example vinca alkaloids (vincristine (Oncovin), vinblastine (Verban), vindesine, and vinorelbine), taxanes (paclitaxel (Taxol) and docetaxel (Taxotere)), podophyllotoxins (etoposide and tenisopide), and camptotecan analogs (irinotecan and topotecan); (3) anthracyclines (doxorubicin (Adriamycin, Rubex, Doxil), daunorubicin, epirubicin, mitoxantrone, idarubicin, duocalmycin, and dactinomycin (Cosmegen)), chromomycin (dactinomycin and plicamycin (Mitramycin)), and others (mitomycin and bleomycin (Blenoxan));(4) Folic acid antagonists (methotrexate), pyrimidine antagonists (methotrexate), pyrimidine antagonists (5-fluorouracil, foxuridine, cytarabine, floururacil (5-FU), capecitabine and gemcitabine), purine antagonists (6-mercaptopurine (purinetol) and 6-thioguanine), 6-thiopurine, and antimetabolites such as adenosine deaminase inhibitors (cladribine (leustatin), fludarabine, nelarabine and pentostatin), azacitidine, thioguanine, and cytarabine (ara-C); (5) (6) Topoisomerase inhibitors such as topoisomerase I inhibitors (ironotecan, topotecan) and topoisomerase II inhibitors (amsacrine, etoposide, etoposide phosphate, teniposide); (7) Hormones exemplified by estrogen and androgen inhibitors (tamoxifen and flutamide), gonadotropin-releasing hormone agonists (leuprolide and goserelin (Zoladex)), and aromatase inhibitors (aminoglutethimide and anastrozole (Arimidex)); (8) DNA hypomethylating agents, e.g., azacitidine, decitabine; (9) Poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) pathway inhibitors such as iniparib, olaparib, and veliparib; (10) PI3K / Akt / mTOR pathway inhibitors, e.g., everolimus; Histone deacetylase (HDAC) inhibitors, e.g., vorinostat, entinostat (Sndx-275), mosetinostat (MGCD103), panobinostat (LBH589), romidepsin, valproic acid

[0049] ; cyclin-dependent kinase (CDK) inhibitors, e.g., flavopyridol, oromoucin, roscovitine, kaempaulon, AG-024322 (Pfizer), fascapricin, liuvidine, pluvalanol A, NU2058, BML-259, SU 9516, PD-0332991, P276-00

[0050] ; heat shock protein (HSP90) inhibitors, e.g., geldanamycin, tanespimycin, albespimycin, radicicol, degrin, and BIIB021;(11) Mouse double microchromosome 2 (MDM2) inhibitors, e.g., cis-imidazoline, benzodiazepine dione, spiro-oxindole, isoquinolinone, thiophene, 5-deazaflavin, tryptamine; (12) Anaplastic lymphoma kinase (ALK) inhibitors, e.g., aminopyridine, diaminopyrimidine, pyridoisoquinoline, pyrrolopyrazole, indolocarbazole, pyrrolopyrimidine, dianilinopyrimidine; (13) Poly[ADP-ribose] polymerase (PARP) inhibitors, exemplified by benzamide, phthalazinone, tricyclic indole, benzimidazole, indazole, pyrrolocarbazole, phthalazinone, isoindolinone; and (14) Other anticancer agents exemplified by ansacrine, asparaginase (El-spar), hydroxyurea, mitoxantrone (Novantrone), mitotane (Lytantrone), maytansinoids, retinoic acid derivatives, myeloproliferative factors (salgramostim and filgrastim), amiphostine, agents that disrupt folic acid metabolism, such as pemetrexed, ribonucleotide reductase inhibitors (hydroxyurea), corticosteroid inhibitors (mitotane), enzymes (asparaginase and pegasparagase), antimicrotubule agents (estramustine), and retinoids (bexarotene, isotretinoin, tretinoin (ATRA)).

[0069] Examples of chemotherapeutic drugs in the small molecule drug subcategory include actinomycin-D, Alkeran, Ara-C, anastrozole, BiCNU, bicalutamide, bleomycin, busulfan, carboplatin, capecitabine, carbomustine, CCNU, chlorambucil, cisplatin, cladribine, CPT-11, cyclophosphamide, cytarabine, cytoxane, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, DTIC, epirubicin, etoposide, fluorizine, fludarabine, flutamide, fotemustine, gemcitabine, hexamethylamine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, and mechloresamine. These include melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, procarbazine, steroids, streptozocin, STI-571, streptozocin, tamoxifen, temozolomide, teniposide, tetrazine, thioguanine, thioteva, thioguanine, tomodex, topotecan, treosulfan, trimethrexate, vinbalastine, vincristine, vindesine, vinorelbine, VP-16, and Xeloda.

[0070] Maytansinoids (molecular weight: ~738 daltons) are a group of chemical derivatives of maytansine and possess potent cytotoxicity. Although considered unsafe for use in human patients, due to toxicity concerns, maytansinoids are suitable for delivery to brain tumor patients via minicells, according to the present invention.

[0071] Duocalmycin (molecular weight: ~588 daltons) is a series of related natural products initially isolated from Streptomyces bacteria. They also possess potent cytotoxicity but are considered unsafe for human use. Similar to maytansinoids, duocalmycin is a suitable chemotherapeutic agent for use in this invention.

[0072] The subcategory of biological chemotherapy drugs is not limited to, but includes asparaginase, AIN-457, bapineozumab, belimunab, brentuximab, briakinumab, canakinumab, cetuximab, darotuzumab, densosumab, epratuzumab, estafenatox, farletuzumab, figtumumab, galiximab, gemtuzumab, gilentuzumab (WX-G250), herceptin, ibritumomab, inotuzumab, and These include polizumab, mepolizumab, muromonab-CD3, naptumomab, necitutumumab, nimotumumab, ocrelizumab, ofatumumab, otelixizumab, ozogamicin, pagimaxibumab, panitumumab, ramucirumab, pertuzumab, reslizumab, rituximab, REGN88, solanezumab, teprizumab, tiuxetan, tositumomab, trastuzumab, tremelimumab, vedolizumab, zaltumumab, and zanorimumab.

[0073] In some embodiments, the anti-cancer drug is actinomycin-D, Alkeran, ara-C, anastrozole, BiCNU, bicalutamide, bleomycin, busulfan, capecitabine, carboplatin, carboplatin, carmustine, CCNU, chlorambucil, cisplatin, cladribine, CPT-11, cyclophosphamide, cytarabine, cytoxin arabinoside, cytoxane, dacarbazine, dactinomycin, daunorubicin, dexorazoxane, docetaxel, doxorubicin, DTIC, epirubicin, ethyleneimine, etoposide, floxuridine, fluorouracil, flutamide, fotemustine, gemcitabine, hexamethylamine, hydroxyurea, ifosfamide, irino Tecan, Lomustine, Mechloretamine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitotane, Mitoxantrone, Oxaliplatin, Paclitaxel, Pamidotamine, Pentositatin, Plicamycin, Procarbazine, Steroids, Streptozocin, STI-571, Tamoxifen, Temozolomide, Teniposide, Tetrazine, Thioguanine, Thiotepa, Tomdex, Tomotecan, Treosulfan, Trimethrexate, Vinblastine, Vinkristine, Vindesine, Vinorelbine, VP-16, Xeroda, Asparaginase, AIN-457, Bapinuutumab, Belimumab, Brentuximab, Briakinumab, Canakinumab, Cetuximab, Darotuzumab, Denosumab epratuzumab, estafenatox, phaletuzumab, figtumumab, galiximabGemtutumab, Gilentuximab (WX-G250), Herceptin, Ibritumomab, Intotutumomab, Iprizumab, Mepolizumab, Muromonab-CD3, Naptumomab, Nesitumomab, Nimotutumomab, Ocrelizumab, Ofatumumab, Oterizumab, Ozogamicin, Padibaximab, Panitumumab, Partutzumab, Ramucirumab, Resurizumab, Rituximab, REGN88, Soranezumab, Tanezumab, Teprizumab, Tiuxetan, Trastutzumab, Tositumomab, Tremerimumab, Vedolizumab, Zaltumumab, Zanolimab, 5FC Accutan Hoffmann-La Roche, AEE788 Novartis, AMG-102, Antineoplaston, AQ4N (banoxantrone), AVANDIA (rosiglitazone maleate), Avastin (bevacizumab) Genetec, BCNU, biCNU carmustine, CCI-779, CCNU, CCNU lomustine, celecoxib (Systemic), chloroquine, sirengitide (EMD121974), CPT-11 (CAMPTOSAR, irinotecan), dasatinib (BMS-354825, Sprycel), dendritic cell therapy, etoposide (eposin, etopophos, bepecid), GDC-0449, Grevec (imatinib mesylate), Gliadelwafer, hydroxychloroquine, IL-13, IMC-3G3, immunotherapy Iressa (ZD-1839), Lavatinib (GW572016), Methotrexate for cancer (Systemic), Novocure, OSI-774, PCV, RAD001 Novatis (mTOR inhibitor), Rapamycin (Rapamne, Sirolimus), RMP-7, Simvastatin, Sirolimus, Sorafenib, SU-101, SU5416 Sugen, Sulfasalazine (Azulfidene), Sutent (Pfizer), TARCEVA (Erotonib HCl), Taxol, TEMODAR schering-plough, TGF-B antisense, Salomide (Thalidomide), Topotecan (Systemic), VEGF trap, VEGF-trap, Vorinostat (SAHA), XL765, XL184, XL765, Zarnestra (tipifarnib), ZOCOR (simbastin), cyclophosphamide (cytoxane), (Alkeran), chlorambucil (lyu, -Kerann), Thiopeta (Tioplex), Busulfan (Milleran), Procarbazine (Matsuran), Dacarbazine (DTIC), Altrethamine (Hexalen), Chlorambucil, Cisplatin (Platinol), Ifosfamide, Methotrexate (MTX), 6-Thioprince (Mercatopurine [6-MP], Thioguanine [6-TG]), Mercaptopurine (Prinnetol), Fludalacin Phosphate (Leustatin), Fluloracil (5-FU), Cytarabine (ara-C), Azacitidine, Vinblastine (Velban), Vincristine (Oncovin), podophyllotoxin (etoposide {VM-16} and teniposide VM {VM-26}), camptothecin (topotecan and irinotecan (Taxol)), taxanes such as paclitaxel (Taxol) and docetaxel (Taxotele), (Adriamycin, Rubex, Doxil)), dactinomycin (Cosmegen), plicamycin (Mizlamycin), mitomycin (Mutamicin), bleomycin (Blenoxan), estrogen and androgen inhibitors (Tamoxifen), gonadotropin-releasing hormone agonists (Leuprolide and goserelin (Zoladex), anastromatase inhibitors (aminoglutethimide and anastrozole (Arimidex)), amsacrin, asparaginase (El-spar), mitoxantrone (Novantrone), mitotane (Roisodren), retinoic acid derivatives, myelocyte growth factors (Surglamostim and filgrastim), amifostin, decitabine, iniparib, olaparib, veliparib, everolimus, vorinostat, entinostat (SNDX-275), mosetinostat (MGCD0103), panobi Nostat (LBH589), Romidepsin, Valproic acid, Flavopyridol, Oromoucin, Roscovitine, Kaempaulon, AG-024322 (Pfizer), Fascapridine, Leubidin, Pulvalanol A, NU2058, BML-259, SU9516, PD-0332991, P276-00, Gerdanamycin, Tanespimycin, Albespimycin, Radicicol, Deguerin, BIIB021, cis-Imidazolin, Benzodiazepine, Spiro-Oxindole, Isoquinolinone, Thiofen, 5-Deazaflavin,It contains a compound selected from the group consisting of tryptamine, aminopyrimidine, diaminopyrimidine, pyridoisoquinoline, pyrrolopyrazole, indolocarbazole, pyrrolopyrimidine, dianilinopyrimidine, benzamide, phthalazinone, tricyclic indole, benzimidazole, indazole, pyrrolocarbazole, isoindolinone, morpholinyl anthracycline, mitansineoid, duocarmycin, calicheamicin (DNA damaging agent), α - amanitin (RNA polymerase inhibitor), centamycin, pyrrolobenzodiazepine, nitrogen mustard, streptomycin, nitrosourea, alkanesulfonate, pyrimidine analog, purine analog, antimetabolite, folic acid analog, anthracycline, taxane, vinca alkaloid, topoisomerase inhibitor, hormonal agent, and any combination thereof., The active agents that can be used in accordance with the present disclosure are not limited to the drug classes or specific agents listed above. Different discovery platforms continue to produce new agents targeted at the unique molecular characteristics of cancer cells; indeed, thousands of such chemical and biological agents have been discovered, and only some of them are listed here. However, the surprising ability of intact bacterium - derived minicells and killed bacterial cells to accommodate the packaging of a wide variety of hydrophilic or hydrophobic active agents means that, in accordance with the discoveries of the present disclosure, essentially any such agent has the potential to treat cancer when packaged in minicells.

[0074] Examples of types of anti - tumor agents include radionuclides, chemotherapeutic agents, and functional nucleic acids including, but not limited to, regulatory RNAs. The members of the types are further discussed below.

[0075] i. Radionuclides A “radionuclide” is an atom with an unstable nucleus, i.e., an atom characterized by excess energy available to be conferred to either newly generated radioactive particles or atomic electrons within the nucleus. In this specification, radionuclides may also be referred to as “radioisotopes,” “radioimaging agents,” or “radiolabels.” Radionuclides can be used for imaging and / or therapeutic purposes. They may be contained within minicells or attached to ligands, peptides, or glycolipids on the extracellular surface of minicells. Binding can be direct or via a linker, using a linker containing a chelating moiety with a chelating agent such as mercaptoacetyltriglycine (MAG3), DOTA, EDTA, HYNIC, DTPA, or crown ether. The chelating agent can be attached directly to the minicell surface component or to the minicell via a linker. Many radionuclides are known in this field, and many are known to be suitable for medical applications, including yttrium-90, technetium-99m, iodine-123, iodine-124, iodine-125, iodine-131, rubidium-82, thallium-201, gallium-67, fluorine-18, xenon-133, and indium-111.

[0076] Therefore, in some embodiments, the radioactive isotopes include radioactive isotopes selected from the group consisting of yttrium 90, yttrium 86, terbium 152, terbium 155, terbium 149, terbium 161, technetium 99m, iodine 123, iodine 131, rubidium 82, thallium 201, gallium 67, fluorine 18, copper 64, gallium 68, xenon 133, indium 111, lutetium 177, and any combination thereof.

[0077] Radioisotopes useful for attaching to minicells for both imaging and therapeutic purposes include, for example, iodine-131 and lutetium-177, which are gamma and beta emitters. Therefore, these agents can be used for both imaging and therapeutic purposes.

[0078] Different isotopes of the same element, such as iodine-123 (gamma emitter) and iodine-131 (gamma and beta emitters), can also be used for both imaging and therapeutic purposes (Gerard and Cavalieri, 2002; Alzahrani et al., 2012).

[0079] New examples include yttrium-86 / yttrium-90 or terbium isotopes (Tb): 152 Tb (beta-plus emitter), 155 Tb (gamma emitter), 149 Tb (alpha emitter), and 161 This refers to Tb(beta-particle) (Muller et al., 2012; Walrand et al., 2015).

[0080] Nuclear imaging utilizes gamma and positron emitters (β+). Technetium-99m( 99m Tc) or Iodine-123 123 Gamma emitters such as I) can be positioned using a gamma camera (planar imaging) or SPECT (single-photon emission computed tomography) (Holman and Tumeh, 1990).

[0081] The tissue penetration of these particles is proportional to the energy of the radioactive isotope (Kramer-Marek and Capala, 2012). While β particles have a potential cytotoxic effect, they penetrate tissue only a few millimeters, thus leaving surrounding healthy tissue intact. Lutetium-177 is a β-emitter commonly used in routine nuclear oncology practices. 177 Lu, tissue penetration: 0.5-0.6 mm, maximum: 2 mm, 497 keV, half-life: 6.7 days) and Yttrium 90 ( 90 This includes Y (tissue penetration: average 2.5 mm, maximum: 11 mm, 935 keV, half-life: 64 hours) (Teunissen et al., 2005; Kwekkeboom et al., 2008; Ahmadzadehfar et al., 2010; Pillai et al., 2013; Ahmadzadehfar et al., 2016).

[0082] Radionuclides have found widespread use in nuclear medicine, particularly as beta-emitting emitters for damaging tumor cells. In some embodiments, radionuclides are appropriately used as antitumor agents.

[0083] Radionuclides can be associated with intact, bacterial-derived minicells by any known technique. Therefore, proteins or other minicell surface components (see below) can be labeled with radionuclides using commercially available labeling methods, such as the use of Pierce iodine-labeled reagents, commercially available from Pierce Biotechnology Inc (Rockford, Ill.) and detailed in Rice et al., Semin. Nucl. Med., 41, 265 (2011). Alternatively, radionuclides can be incorporated into proteins within minicells.

[0084] In the latter scenario, a bacterial strain that produces minicells is transformed with plasmid DNA encoding a foreign protein. When minicells are formed during asymmetric cell division, several copies of the plasmid DNA separate into the cytoplasm of the minicells. The resulting recombinant minicells are incubated in the presence of radiolabeled amino acids under conditions in which the foreign protein expressed within the minicells from the plasmid DNA is incorporated into the radionuclide-carrying amino acid. For example, recombinant minicells are incubated according to the protocol of Clark-Curtiss anDCurtiss, Methods Enzymol, 101:347-362 (1983). 35S The plasmid-coding protein was incubated in a minimal growth medium containing methionine, thereby allowing the newly expressed plasmid to be expressed. 35S Methionine is incorporated. Using a similar method, recombinant minicells can be packaged with other radioactive labels as desired.

[0085] Oligosaccharides on the surface of minicells can also be radiolabeled using well-established protocols, such as those described in Fukuda, Curr Protocols Molec. Biol. (Suppl. 26), 17.5.1-17.5.8 (1994). An example of such oligosaccharides specific to minicells is the O-polysaccharide component of lipopolysaccharide (LPS) found on the surface of minicells derived from Gram-negative bacteria (see below).

[0086] A preferred approach in this regard is to radiolabel bispecific antibodies used as tumor targeting ligands to target minicells to specific tumors. See U.S. Patent Publication 2007 / 0237744, the contents of which are incorporated herein by reference. That is, bispecific antibodies "coated" on minicells expose a considerable amount of additional surface proteins for radiolabeling. Thus, it is possible to achieve a higher specific activity of radiolabeling associated with antibody-coated minicells. In contrast, radiolabeling of uncoated minicells, i.e., when the radionuclide labels only endemic bacteria, may result in weaker labeling (lower specific activity). In one embodiment, this weaker labeling is thought to occur because the outer membrane-related proteins of minicells derived from Gram-negative bacteria are masked by LPS containing long chains of O-polysaccharides covering the minicell surface, as will be discussed further below.

[0087] To treat a tumor, the compositions of this disclosure are delivered in doses or multiple doses that provide a level of intratumoral irradiation sufficient to at least reduce the tumor mass, if not completely eliminate the tumor. The progress of treatment can be monitored on a case-by-case basis along this line. However, while the amount of radioactivity packaged in a composition is typically on the order of about 30 to about 50 Gy, the present invention also intends for higher amounts of radioactivity, such as about 50 to about 200 Gy, giving the full range of about 30 Gy to about 200 Gy.

[0088] In some cases, considering the highly efficient and specific delivery of mini-cell bone radionuclides to tumors, the amount of radioactivity packaged in the composition may be even lower than that described above. Thus, in one embodiment, the composition contains about 20 to about 40 Gy, or about 10 to about 30 Gy, or about 1 to about 20 Gy, or less than about 10 Gy.

[0089] Some tumor-targeting ligands may contain radioisotopes that have the function of delivering radiation to the tumor while the ligand is binding to tumor cells. In some embodiments, the ligands are Arg-Gly-Asp (RGD) peptide, bombesin (BBN) / gastrin-releasing peptide (GRP), cholecystokinin (CCK) / gastrin peptide, α-melanocyte-stimulating hormone (α-MSH), neuropeptide (NT), [ 68 Ga]Ga-PSMA-HBEDCC([ 68 Ga]Ga-PSMA-11[PET]), [ 177 Lu]Y-J591, [ 123 I]I-MIP-1072, 131 I]I-MIP-1095, 68 Ga or 177 Lu-labeled PSMA-I&T, 68 Ga or Lu-labeled DKFZ-PSMA-617 (PSMA-617), somatostatin (SST) peptide, substance P, tumor molecular target peptide 1 (TMTP1) containing T140, vasoactive intestinal peptide (VIP), or any combination thereof.

[0090] In some embodiments, the radioisotope is bound to the tumor targeting ligand. In some embodiments, the binding is via a linker. In some embodiments, the tumor targeting ligand includes a peptide containing a functional group for binding to the radioisotope or a chelating moiety that chelates the radioisotope. Functional groups of peptides available for binding include, but are not limited to, ε-amino groups on lysine side chains, guanidinium groups on arginine side chains, carboxyl groups on aspartic acid or glutamic acid, cysteine ​​thiols, and phenols on tyrosine. The most common conjugation reactions are carbodiimide / N-hydroxysuccinimidyl (EDC / NHS)-mediated carboxyl-amine coupling, maleimide conjugation to thiol groups, and diazonium modification of phenols on tyrosine. Representative chemistry for binding peptides to imaging moieties can be found in many reviews (Erathodiyil and Ying, 2011; Takahashi et al., 2008).

[0091] In some embodiments, radioisotopes function as radioactive contrast agents. Some radioisotopes are used for SPECT imaging. 99m Tc, 123 I, and 111 In, and for PET imaging 18 F, 64 Cu, and 68 It is used for peptide labeling containing ga (Chatalic et al., 2015). Generally, these radioisotopes are bound to peptides via chelating agents. Some widely used chelating agents are described (Sun et al., 2017). Most therapeutic radiopharmaceuticals are labeled with β-emitting isotopes.

[0092] The mini-cells of the present invention, which target tumor cells, also deliver targeted radiation from a radioisotope to the tumor cells to which the mini-cells are bound. In some embodiments, the radioisotope functions as a therapeutic radioactive agent, and the amount of radiation delivered by the radioisotope is sufficient to provide a therapeutic effect on the tumor. In some embodiments, the therapeutic effect is a reduction in tumor size. The tumor may be reduced in size by about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, or about 5%.

[0093] Radiolabeled phosphonates have high bone affinity and can be used for imaging and palliative treatment of painful bone metastases. Depending on the degree of bone metabolism, tracers accumulate via adhesion to bone, preferably osteoblastic bone metastases. Treatment planning should involve bone scintigraphy with technetium-99m-hydroxyethylidene diphosphonate (HEDP) to estimate the extent of metabolism and metastatic lesions. Bisphosphonate HEDP can be labeled for treatment with either rhenium-186 (β-emitter, half-life: 89 hours, 1.1 MeV maximum energy, maximum range: 4.6 mm) or rhenium-188 (β-emitter [85%, 2.1 MeV] and γ-emitter [15%, 155 keV], half-life: 16.8 hours, maximum range in soft tissue: 10 mm) (Palmedo, 2007). A new and promising radiopharmaceutical for bone palliative therapy is the radiolabeled conjugate of zoledronic acid. Zoledronic acid belongs to a new and highly potent generation of bisphosphonates with cyclic side chains. Zoledronic acid labeled with scandium-46 or lutetium-177 exhibits excellent osteoffinity absorption (98% compared to [177Lu]Lu-zoledronic acid, 82% compared to [46Sc]Sc-zoledronic acid), which is significantly higher than that of samarium-153-labeled bisphosphonates (maximum: 67%) (Majkowska et al., 2009). These bisphosphonates can be conjugated to intact mini-cells for use in the diagnosis or treatment of bone metastases.

[0094] II. Chemotherapy Drugs The antitumor agents used in this disclosure may also be chemotherapeutic agents. In this specification, “chemotherapeutic agent,” “chemotherapeutic agent,” and “chemotherapeutic agent” are used interchangeably to mean a drug having the ability to kill or destroy tumor cells. A chemotherapeutic agent may be a small molecule drug or a biological drug, as will be further detailed below.

[0095] The “small molecule drugs” subcategory encompasses compounds characterized by (i) effects on biological processes and (ii) having a low molecular weight compared to protein or polymer macromolecules. Small molecule drugs are typically about 800 daltons or less, with a lower limit of about 150 daltons, as exemplified by Temodar® (temozolomide), which is about 194 daltons and used to treat glioblastoma and other types of brain cancer. In this context, “about” indicates that eligible molecular weight values ​​are subject to variations in measurement accuracy and experimental errors on the order of several daltons or tens of daltons. Thus, small molecule drugs can have molecular weights in the range of about 150 to about 400 daltons, for example, about 900 daltons or less, about 800 daltons or less, about 700 daltons or less, about 600 daltons or less, about 500 daltons or less, or about 400 daltons or less. More specifically, the small molecule drug may have a molecular weight of approximately 400 daltons or more, approximately 450 daltons or more, approximately 500 daltons or more, approximately 550 daltons or more, approximately 600 daltons or more, approximately 650 daltons or more, approximately 700 daltons or more, or approximately 750 daltons or more. In another embodiment, the small molecule drug packaged in mini-cells has a molecular weight of approximately 400 to approximately 900 daltons, approximately 450 to approximately 900 daltons, approximately 450 to approximately 850 daltons, approximately 450 to approximately 800 daltons, approximately 500 to approximately 800 daltons, or approximately 550 to approximately 750 daltons.

[0096] Specifically, suitable small molecule drugs include, but are not limited to, those listed above, such as nitrogen mustard, nitrosolar, ethyleneimines, alkanesulfonates, tetrazines, platinum compounds, pyrimidine analogs, purine analogs, antimetabolites, folic acid analogs, anthracyclines, taxanes, vinca alkaloids, and topoisomerase inhibitors. Therefore, small molecule drugs for use in the present invention may be selected from among the following: enegyoins (e.g., dynemycin A, uniramycin, calicheamicin γ1 and calicheamicin θ1); meamycin, synthetic analogs of FR901464; and benzosverene derivatives (e.g., Tanpure et al., Bioorg Med. Chem., 21: 8019-32). (as described in 2013); auristatin (e.g., auristatin E, monomethyl auristatin E (MMAE)) and auristatin F (these are synthetic analogs of drastatin); duocalmycin (e.g., duocalmycin SA and CC-1065); meitansine and its derivatives (meitansinoids) (e.g., DM1 and DM4); irinotecan (Camptosar®) and other topoisomerase inhibitors (e.g., topotecan, etoposide, mitoxantrone and teniposide); and yatakemycin (its synthesis is detailed by Okano et al., 2006).

[0097] More specifically, examples of specific small molecule drugs suitable for use in the present invention include: actinomycin D, Alkeran, C arabic, anastrozole, BiCNU, bicalutamide, bisanthren, bleomycin, busulfan, capecitabine (Xeloda®), carboplatin, carboplatinum, carmustine, CCNU, chlorambucil, cisplatin, cladribine, CPT11, cyclophosphamide, cytarabine, cytosine arabinoside, cytoxane, dacarbazine, dactinomycin, dacnorubicin, dexalazoxane, doxorubicin, DTIC, epirubicin, ethyleneimine, etoposide, floxylidine, fludara Vinblastine, fluorouracil, flutamide, fotemustine, gemcitabine, hexamethylamine, hydroxyurea, idarubicin, ifosfamide, irinotecan, irinotecan, lomustine, mechloretamine, melphalan, mercaptopurine, methoterexaate, mitomycin, mitotane, mitoxantrone, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, procarbazine, streptozocin, STI-571, tamoxifen, temozolomide, teniposide, tetrazine, thioguanine, thiotepa, topotecan, tomdex, treosulfan, trimethrexate, vinblastine, vincristine, vindesine, violerbin, and VP-16.

[0098] For the purposes of this specification, “biological drug” is defined, in contrast, as any biologically active macromolecule that can be produced by biological processes, excluding “functional nucleic acids” as discussed below, whose polypeptides qualify as small molecule drugs as defined above, depending on their size. Therefore, the “biological drug” subcategory excludes and does not overlap with the small molecule drug and functional nucleic acid subcategories. Examples of biological drugs include, for example, therapeutic proteins and antibodies, whether natural, recombinant, or synthetic, produced using medicinal chemistry and drug design tools.

[0099] iii. Supertoxic chemotherapeutic drugs Certain molecules designed for chemotherapy fail during preclinical or clinical trials due to unacceptable toxicity. The inventors have shown that when highly toxic or "hypertoxic" chemotherapeutic drugs are packaged in mini-cells and subsequently delivered systemically to tumor patients, the drugs are delivered to tumor cells. Furthermore, even after the tumor cells are destroyed and the drug-containing cytoplasm is released into nearby normal tissue, the result is not toxicity to normal tissue. This is because the drug has already bound to tumor cell structures such as DNA and can no longer attack normal cells. Therefore, the present invention is particularly useful for the delivery of highly toxic ("hypertoxic") chemotherapeutic drugs to cancer patients.

[0100] When cancer patients have exhausted all treatment options, the tumor is likely to have reached a stage of considerable heterogeneity with a high degree of resistance to conventional cytotoxic drugs. In this description, "highly toxic chemotherapeutic agents" or "supertoxic chemotherapeutic agents" refer to chemotherapeutic agents that can overcome resistance to conventional drugs because the lethal dose to normal cells is relatively low compared to the effective dose to cancer cells.

[0101] Therefore, in one embodiment, a highly toxic chemotherapeutic agent has a median effective dose (ED) for the target cancer. 50 A median lethal dose (LD) lower than ) 50 ) have. For example, highly toxic or supertoxic chemotherapy drugs have drug ED against the target cancer. 50 Approximately 500%, 400%, 300%, 250%, 200%, 150%, 120%, or less than 100% of LD 50 In another embodiment, a highly toxic or supertoxic chemotherapeutic agent may have a maximum sublethal dose (i.e., the highest dose that does not cause severe or irreversible toxicity) that is lower than its minimum effective dose, for example, about 500%, about 400%, about 300%, about 250%, about 200%, about 150%, about 120%, about 100%, about 90%, about 80%, about 70%, about 60%, or about 50% of the minimum effective dose. In one embodiment, the target cancer may be, for example, (1) the type of cancer on which the drug is designed, (2) the first type of cancer on which preclinical or clinical trials are conducted for the drug, or (3) the type of cancer on which the drug shows the highest efficacy among all cancers on which the drug has been tested.

[0102] Exemplary, non-limiting examples of highly toxic chemotherapeutic drugs include, but are not limited to, maytansinoids, duocalmycin, morpholinyl anthracyclines, and their derivatives. Maytansinoids (molecular weight: approximately 738 daltons) are a group of chemical derivatives of maytansine that possess potent cytotoxicity. Although considered unsafe for use in human patients, due to toxicity concerns, maytansinoids are suitable for delivery to tumor patients via minicells, according to the present invention. Duocalmycin (molecular weight: approximately 588 daltons) are a series of related natural products first isolated from Streptomyces bacteria. They are also, Although it possesses potent cytotoxicity, it is considered unsafe for use in humans. Similar to meitansinoids, duocalmycin is a suitable chemotherapeutic agent for use in this invention.

[0103] Similarly, examples include compounds of the class of morpholinyl anthracycline derivatives described in international patent application WO1998 / 002446. Such derivatives include nemorubicin (3'-deamino-3'-[2(S)-methoxy-4-morpholinyl]doxorubicin) (MMDX), its major metabolite PNU-159682 (3'-deamino-3'-anhydro-[2''(S)-methoxy-3''(R)-hydroxy-4''-morpholinyl]doxorubicin), and four other such derivatives described in U.S. Patent No. 8,470,948 (the contents of which are incorporated herein by reference): 3'-deamino-3''-4'-anhydro-[2''(S)-methoxy-3''(R)-hydroxy-4''-morpholinyl]idarubicin; 3'-deamino-3''-4'-anhydro-[2''(S)-methoxy-3''(R)-hydroxy-4''-morpholinyl]daurirubicin; 3'-deamino-3''-4'-anhydro-2'' (S)-Methoxy-3''(R)-hydroxy-4''-morpholinyl]-caminomycin; and 3'-deamino-3''-4'-anhydro-[2''(S)-ethoxy-3''(R)-hydroxy-4''-morpholinyl]d-oxorubicin.

[0104] In exemplary embodiments of this disclosure, mini-cells contain the supertoxic chemotherapeutic drug 3'deamino3'',4'anhydro[2''(S)methoxy3''(R)oxy4''morpholinyl]doxorubicin (PNU159682). We have found that PNU-159682 is a potent drug that appears to overcome drug resistance in many different tumor cell lines and is far more potent than a range of conventional chemotherapeutic drugs in cytotoxicity analyses against many different tumor cell lines. See Examples 8 and 9. Furthermore, in vivo mouse xenograft experiments have shown that doxorubicin-resistant human tumor xenografts can be effectively treated by IV administration of EGFR-targeted and PNU-159682-loaded EDV. See Example 11. Notably, the combination of PNU-159682-loaded EDV and a type I interferon agonist has been found to be well-tolerated and to provide a synergistic and improved anticancer effect in patients with advanced pancreatic cancer. See Example 12. Therefore, in one embodiment of the present invention, the composition comprises EGFR-targeted minicells containing PNU159682 as an active anticancer agent.

[0105] Other suitable cancer chemotherapy agents that may exhibit hypertoxic chemotherapeutic properties include auristatin, calicheamicin (DNA damage agent), alpha-amanitin (RNA polymerase II inhibitor), centanamycin, geldanamycin, pyrrolobenzodiazepines, streptonigtin, nitrogen mustard, nitrosolace, ethyleneimine, alkanesulfonates, tetrazine, platinum compounds, pyrimidine analogs, purine analogs, antimetabolites, folic acid analogs, anthracyclines, taxanes, vinca alkaloids, topoisomerase inhibitors, and hormonal agents.

[0106] iv. Biological chemotherapy drugs In another context, mini-cells may contain biological chemotherapy drugs. Examples of such drugs include asparaginase, AIN-457, bapineozumab, belimumab, brentuximab, briakinumab, canakinumab, cetuximab, darotuzumab, denosumab, epratuzumab, estrafenatox, phaletuzumab, figtumumab, galiximab, gemtuzumab, gilentuximab (WX-G250), ipritumomab, inotuzumab, ipilizumab, mepolizumab, muromonab-CD3, and naptumo Examples include, but are not limited to, mab, necitumumab, nimotumumab, ocrelizumab, ofatumumab, otelizumab, ozogamicin, padibaximab, panitumumab, pertuzumab, ramucirumab, reslizumab, rituximab, REGN88, soranezumab teprizumab, tanezumab, tiuxetan, tositumomab, trastuzumab (Herceptin®), tremelimumab, vedolizumab, zaltumumab, and zanorimumab.

[0107] v.Functional nucleic acid A "functional nucleic acid" is a nucleic acid molecule that, upon introduction into a host cell, specifically interferes with protein expression. In relation to cancer treatment, according to this disclosure, the functional nucleic acid payload delivered to cancer cells via intact bacterial mini-cells preferably inhibits genes that promote tumor cell proliferation, angiogenesis, or resistance to chemotherapy, and / or inhibit apoptosis or cell cycle arrest, i.e., "oncogenic genes."

[0108] Generally, the functional nucleic acid molecules used in this disclosure have the ability to reduce protein expression by interacting with protein transcripts. This category of mini-cell payloads for this disclosure includes, among other things, siRNA, shRNA, short RNA (typically less than 400 nucleotides in length), microRNA (miRNA), ribozymes and decoy RNA, antisense nucleic acids, and regulatory RNA such as LincRNA. A “ribozyme” is an RNA molecule that has enzymatic activity capable of repeatedly cleaving other RNA molecules in a sequence-specific manner; an “antisense oligonucleotide” is a nucleic acid molecule complementary to a portion of a particular gene transcript that can hybridize with the transcript to inhibit translation; antisense oligonucleotides can include RNA or DNA; and “LincRNA” or “long intergene non-coding RNA” encompasses non-protein-coding transcripts longer than 200 nucleotides. As discussed by Khalil et al., 2009, LincRNA can regulate gene transcription, splicing, and / or translation.

[0109] Each type of regulatory RNA inhibits tumorigenic genes as described above and can therefore be a source of functional nucleic acid molecules suitable for use according to this disclosure. In one embodiment of this disclosure, intact mini-cells possess siRNA molecules that mediate a post-transcriptional gene silencing RNA interference (RNAi) mechanism that can be utilized to target tumorigenic genes. See, for example, MacDiarmid et al., 2009 (Antibody-presenting mini-cells possess siRNAs that counteract the development of resistance to chemotherapy drugs), and Oh and Park, Advanced Drug Delivery Rev., 61:850-62 (2009) (Delivery of therapeutic siRNAs to treat breast cancer, ovarian cancer, cervical cancer, liver cancer, lung cancer, and prostate cancer, respectively).

[0110] "siRNA" generally refers to double-stranded RNA molecules about 10 to about 30 nucleotides long, named for their ability to specifically interfere with protein expression. Preferably, siRNA molecules are about 12 to about 28 nucleotides long, more preferably about 15 to about 25 nucleotides long, even more preferably about 19 to about 23 nucleotides long, and most preferably about 21 to about 23 nucleotides long. Thus, siRNA molecules may be, for example, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, or about 29 nucleotides long.

[0111] The length of one strand indicates the length of the siRNA molecule. For example, an siRNA called a 21-ribonucleotide-length (21-mer) can contain two opposing RNA strands that anneal for 19 consecutive base pairs. The remaining two ribonucleotides on each strand form an "overhang," and if the siRNA contains two strands of different lengths, the longer strand indicates the length of the siRNA. For example, a dsRNA with a 21-nucleotide-length single strand and a 20-nucleotide-length double strand constitutes a 21-mer.

[0112] Tools to assist in the design of siRNA-specific and regulatory RNAs are generally readily available. For example, computer-based siRNA design tools are available on the internet at www.dharmacon.com.

[0113] In another preferred embodiment, the intact mini-cells of this disclosure possess miRNAs that, like siRNAs, can mediate post-transcriptional gene silencing RNA interference (RNAi) mechanisms. Also, like siRNAs, the gene silencing effect mediated by miRNAs can be used to target tumor-promoting genes. See, for example, Kota et al., 2009 (delivery of miRNAs via transfection results in inhibition of cancer cell proliferation, tumor-specific apoptosis, and dramatic protection from disease progression in a mouse liver cancer model), and Takeshita et al., 2010 (delivery of synthetic miRNAs via transient transfection inhibits the proliferation of metastatic prostate tumor cells on bone tissue).

[0114] Both miRNAs and siRNAs mediate RNA interference, but there are differences between them. In this regard, "miRNA" generally refers to a class of single-stranded RNA molecules of approximately 17 to 27 nucleotides (rather than double-stranded RNA molecules like siRNAs). Therefore, miRNA molecules can be, for example, approximately 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides in length. Preferably, miRNA molecules are approximately 21 to 25 nucleotides in length.

[0115] Another difference between miRNA and siRNA is that the former generally does not fully complement the mRNA target. In contrast, siRNA must be perfectly complementary to the mRNA target. Therefore, siRNA generally results in the silencing of a single, specific target, while miRNA is messy.

[0116] Furthermore, although both siRNA and miRNA assemble into RISC (RNA-induced silencing complex), their initial processes before RISC assembly differ. These differences are described in detail in Chu et al., 2006; and Gregory et al., 2006. Numerous databases serve as miRNA repositories. See, for example, miRBase (www.mirbase.org) and http: / / diana.cslab.ece.ntua.gr / DianaToolsNew / index.php?r=tarbase / index). In conventional usage, miRNAs are usually named with the prefix "-mir" combined with a sequential number. For example, a new miRNA discovered after mouse mir-352 would be named mouse "mir-353". Again, tools to assist in the design of regulatory RNAs, including miRNAs, are readily available. In this regard, computer-based miRNA design tools are available on the internet at wmd2.Weigelworld.org / cgi / mirnatools.pl.

[0117] Our discovery is that miRNA16a can be administered to mesothelioma and adrenocortical carcinoma cells by targeted mini-cell mediated delivery. See Example 7. Once taken up by cancer cells, miRNA16a was found to potently inhibit cancer cell proliferation. Therefore, in some embodiments, the mini-cells of this disclosure contain miRNA16a. Other microRNAs useful for inhibiting tumor cell proliferation include the mir34 family and the let7 family.

[0118] As described above, the functional nucleic acids used in the compositions of the present invention can inhibit genes that promote tumor cell proliferation, angiogenesis, or resistance to chemotherapy. The inhibited genes themselves can also inhibit apoptosis and cell cycle arrest. Examples of genes that can be targeted by functional nucleic acids are provided below.

[0119] The functional nucleic acids of this disclosure preferably target genes or transcripts of proteins that promote drug resistance, inhibit apoptosis, or promote tumor phenotypes. While successful application of functional nucleic acid strategies in these contexts has been achieved in the art, the advantages of mini-cell vectors are not present. See, for example, Sioud, Trends Pharmacol. Sci., 2004; Caplen, Expert Opin. Biol. Ther., 2003; Nieth et al., 2003; Caplen and Mousses, 2003; Duxbury et al., 2004; Yague et al., 2004; and Duan et al., 2004.

[0120] Proteins that contribute to drug resistance constitute preferred targets for functional nucleic acids. These proteins may contribute to acquired or endogenous drug resistance. A resistance phenotype is acquired when affected cells, such as tumor cells, initially respond to drugs but become refractory during subsequent treatment cycles. Useful targets involved in acquired drug resistance include ATP-binding cassette transporters such as P-glycoproteins (P-gp, P-170, PGY1, MDR1, ABCB1, MDR-related proteins, multidrug resistance protein 1), MDR-2, and MDR-3. Growth factors such as MRP2 (multidrug resistance-associated protein), BCR-ABL (Abelson proto-oncogene), STI-571 resistance-associated protein, lung resistance-associated protein, cyclooxygenase-2, nuclear factor κ, XRCC1 (X-ray cross-complementarity group 1), ERCC1 (excision cross-complementarity gene), GSTP1 (glutathione S-transferase), mutant β-tubulin, and IL-6 are further targets involved in acquired drug resistance.

[0121] Particularly useful targets contributing to drug resistance include ATP-binding cassette transporters such as P-glycoprotein, MDR-2, MDR-3, BCRP, APT11a, and LRP. Useful targets also include proteins that promote apoptosis resistance. These include Bcl2 (B-cell leukemia / lymphoma), Bcl-XL, A1 / Bfl1, local adhesion kinases, dihydrodiol dehydrogenase, and p53 mutant proteins.

[0122] Useful targets also include oncogenic and mutagenic tumor suppressor proteins. Examples of these include β-catenin, PKC-α (protein kinase C), C-RAF, DP97 Deadbox RNA helicase 1, FLIP (DNA methyltransferase 1), C-Sfc, 53BPI, Polycomb group protein EZH2 (zeste homolog enhancer), ErbB1, HPV-16 E5 and E7 (human papillomavirus early 5 and early 7), FortiLin & MCI1P (myeloid cell leukemia 1 protein), DIP13α (DDC interaction protein 13a), MBD2 (methyl CpG binding domain), p21, KLF4 (Kruppel-like factor 4), tpt / TCTP (translation-regulating oncoprotein), SPK1 and SPK2 (sphingosine kinase), P300, PLK1 (polo-like kinase-1), Trp53, and ErbB1. VEGF (Vascular endothelial growth factor), BAG-1 (BCL2-associated athanogene 1), MRP2, BCR-ABL, STI-571 resistance-associated protein, cyclooxygenase-2, nuclear factor κ, XRCC1, ERCC1, GSTP1, mutant-β-tubulin, and growth factors.

[0123] Furthermore, useful targets include global regulatory elements exemplified by cytoplasmic polyadenylated element-binding proteins (CEPBs). For example, CEPB4 is overexpressed in glioblastoma and pancreatic cancer, where this protein activates hundreds of genes associated with tumor growth and is undetectable in healthy cells (Oritz-Zapater et al., 2011). Therefore, according to this specification, treatment of glioblastoma can be achieved by administering a composition containing intact, bacterial-derived mini-cells containing an agent that counteracts CEPB4 overexpression (e.g., siRNA or other functional nucleic acid molecules that disrupt CEPB4 expression by tumor cells).

[0124] Further examples of useful targets for functional nucleic acids include the replication protein A (RPA) trimer complex, composed of 70kDa (RPA1), 32kDa (RPA2), and 14kDa (RPA3) subunits, which is essential for DNA replication in all organisms. Iftode et al., 1999.

[0125] Other useful targets are those important for mitosis and maintaining genomic stability. An example is polo-like kinase (PLK1), which has been found to be overexpressed in a wide range of cancer cells. See Example 3, Figure 12. The inventors of this disclosure have also found that siRNA inhibiting Plk1 (siPlk1) expression inhibits the proliferation of mesothelioma and adrenocortical cancer cells. See Example 10. Therefore, in some embodiments, the mini-cells of this disclosure contain Plk1.

[0126] Other useful targets are those involved in DNA replication and repair. For example, ribonucleotide reductase (RR), which catalyzes the conversion of ribonucleoside 5'-bisphosphate to the corresponding 2'-deoxyribonucleoside 5'-triphosphate necessary for DNA replication and repair, can be a therapeutic target for cancer. See D'Angiolella et al., 2012. Human RR comprises two subunits, RRM1 and RRM2, and functional nucleic acids targeting both subunits are useful in the present invention. The inventors of this disclosure have shown that siRNA targeting RRM1 (siRRM1) potently inhibits mesothelioma and adrenocortical cancer cell proliferation when delivered to mini-cells. See Example 10. Therefore, in some embodiments, the mini-cells include siRNA that inhibits ribonucleotide reductase M1 (RRM1) expression.

[0127] B. Type I interferon agonist The compositions of the present invention may include type I interferon agonists, i.e., agents that increase the level (e.g., activity or expression level) of type I interferon. Human type I interferons (IFNs) are a large subgroup of interferon proteins that help regulate the activity of the immune system. Interferons bind to interferon receptors. All type I IFNs bind to a specific cell surface receptor complex known as IFN-α (IFNAR), which consists of IFNAR1 and IFNAR2 chains. IFNARs are mammalian type I IFNs called IFN-α (alpha), IFN-β (beta), IFN-κ (kappa), IFN-δ (delta), IFN-ε (epsilon), IFN-τ (tau), IFN-ω (omega), and IFN-ζ (zeta, also known as limitin).

[0128] i. Oligonucleotides Figure 2 shows a graphical depiction of an exemplary embodiment of minicells containing immunomodulatory 60-mer double-stranded DNA. We discovered that delivery of type I interferon agonists, such as double-stranded DNA, to EGFR-targeted minicells acts as an immunostimulant (i.e., enhances the antitumor effect) of cytotoxic drug-loaded minicells. See Example 11. Therefore, combining minicells packaged with the highly toxic drug PNU-159682 enhanced the antitumor effect, and this treatment was well-tolerated by patients with advanced pancreatic cancer. See Example 12.

[0129] Type I interferon (IFN) expression can be induced by delivering double-stranded DNA to target cells. Specifically, innate immune activation by cytosolic DNA derived from microbial pathogens is a potent inducer of type I IFN and pro-inflammatory cytokines mediated by cytosolic DNA sensors such as cGAMP, cyclic GMP-AMP synthase (cGAS), and IFNγ-inducible factor 16 (IFI16). See, for example, Hansen et al., 2014; and Unterholzner et al., 2013. cGAS has the enzymatic ability to post-bind to double-stranded DNA and to produce second messenger cyclic GMP-AMP that docks on endoplasmic reticulum-binding protein-stimulating factor (STING) of the IFN gene. Barber et al., 2011. This leads to the homodimerization of STING, migration from the ER (Dobbs et al., 2015), and recruitment of TANK-binding kinase 1 to phosphorylate STING, thereby generating the transcription factor IFN regulator 3, which initiates IFN expression. See Dobbs et al., 2015; Wang et al., 2014; and Liu et al., 2015. Thus, type I IFN expression can be induced by delivering double-stranded DNA to target cells that can be recognized by cytoplasmic DNA sensors, as described above and in the cited references.

[0130] In some embodiments, the compositions disclosed herein include intact mini-cells containing a type I IFN agonist. In some embodiments, the type I IFN agonist is an oligonucleotide suitable for DNA sensor-mediated induction of type I IFN, as described herein. In some embodiments, the oligonucleotide comprises a sequence of at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, or at least about 200 nucleotides. In other embodiments, the oligonucleotide comprises a sequence of nucleotides from about 10 to about 200, or any amount between these two values. In some embodiments, the oligonucleotide comprises a sequence of at least about 40 nucleotides, at least about 50 nucleotides, or at least about 60 nucleotides.

[0131] In other embodiments, polynucleotide products of the enzyme polynucleotide phosphorylase (PNPase1) can be used as synthetic inducers of IFN activity. Field et al., 1967. Similarly, dsRNA-mimicking polyinosine:polycytidylic acid (poly(I:C)) has been shown to function as an agonist for both TLR3 and MDA5. Alexopoulou et al., 2001; and Gitlin et al., 2006. Thus, in some embodiments, the oligonucleotide is a polynucleotide product of PNPase1, poly(I:C), polyICLC, imiquimod, imidazochyolinesquimod, or CpG oligodeoxynucleotide.

[0132] Synthetic oligonucleotides can also be designed and used as agonists for nucleic acid sensors. For example, TLR9-activating synthetic CpG oligodeoxynucleic acid (CpG-ODN) was designed based on the immunostimulatory properties of bacterial DNA, which are rich in non-metallated CpG motifs in contrast to human DNA. Krieg et al., 1995. Optimization of sequence features and skeletal modifications resulted in CpGODN subtypes that preferentially activate either B cells or pDCs. Thus, as is considered herein, CpG-ODN may be methylated, unmethylated, or a combination of both.

[0133] Many molecules are known to be stimulants of type I IFN secretion, and these molecules, along with their agonists, are suitable for delivery via minicells to induce type I IFN secretion. These molecules include, but are not limited to, double-stranded RNA (dsRNA), poly(dA:dT)DNA, double-stranded Z-DNA and B-DNA, DNA longer than 36 bp and DNA-RNA hybrids (dsDNA), the bacterial second messenger cyclic diGMP, TLR3, TLR4, TLR7, TLR8 and TLR9 agonists, and STING agonists, which are well described below.

[0134] ii. Double-stranded RNA (dsRNA) Double-stranded RNA is an inducer of type I IFN. The RNA helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are cytoplasmic receptors that induce type I IFN secretion. These receptors (RIG-I-like receptors) transmit signals via the mitochondrial localization adapter molecules IPS-1 or MAVS and the kinases TBK1 and IKKi, activating IRF3 and inducing the transcription of readily available type I IFN genes (Kawai & Akiyoshi, 2010). RIG-I and MDA5 respond to viral RNA triphosphated at their 5' ends (Leung and Amarasinghe, 2016; Lu et al., 2010; Marq et al., 2011; Wang et al., 2010).

[0135] iii. Poly(dA:dT)DNA RNA polymerase III is a cytosolic DNA sensor for poly(dA:dT)DNA (Ablasser et al., 2009). In the cytosol, RNA polymerase III converts poly(dA:dT) to RNA with 5' triphosphorylation. The converted 5'-pppRNA then initiates the RIG-I-MAVS pathway and NFκB activation, inducing type I IFN secretion.

[0136] iv. Double-stranded Z-DNA and B-DNA Cytosolic DNA sensors, IRF DNA-dependent activators (DAIs) or Z-DNA binding protein 1 are known to induce type I IFNs in response to right-handed dsDNA higher-order structures (B-DNA) via a TBK1 and IRF3-mediated mechanism (Kawai and Akira, 2010). RNA polymerase III also transcribes B-DNA to 5'-pppRNA and subsequently activates type I IFN transcription via RIG-I (Chiu et al., 2009). Phosphorylation of these transcription factors helps promote the expression of all genes in the type I IFN family, thereby amplifying type I IFN production. Many cytosolic DNA sensors have been reported to recognize intracellular pathogenic DNA. See, for example, Figure 26, excerpted from Xia et al., "DNA sensor cGAS-mediated immune recognition," Protein Cell, 7(11): 777-791 (2016).

[0137] For example, DDX41 (Zhang et al., 2011b), IFI16 (Orzalli et al., 2012; Unterholzner et al., 2010), and DAI (Takaoka et al., 2007) detect double-stranded DNA (dsDNA) and activate the STINGTBK1IRF3 pathway. LRRFIP1 binds to dsDNA and triggers IRF3 activation via β-catenin (Yang et al., 2010). DHX9 and DHX36 associate with dsDNA and lead to NFκB activation via MyD88 (Kim et al., 2010). Ku70 binds to dsDNA and induces type I interferon (IFN) through the activation of IRF1 and ASCRF7 (Zhang et al., 2011a). AIM2 interacts with dsDNA and activates the inflammasome by recruiting ASC and pro-caspase-1 (Burckstummer et al., 2009; Fernandes-Alnemri et al., 2009; Hornung et al., 2009). Notably, Sox2 is expressed in the neutrophil cytosol and, upon binding to dsDNA, activates the Tab2 / TAK1 complex in a sequence-dependent manner (Xia et al., 2015).

[0138] v. DNA longer than 36 bp (dsDNA) and DNA-RNA hybrids cGAS is a DNA sensor that recognizes cytoplasmic DNA (Ablasser et al., 2013a; Ablasser et al., 2013b; Gao et al., 2013a; Li et al., 2013b; Schoggins et al., 2014; Sun et al., 2013; Wu et al., 2013). Double-stranded DNA (dsDNA) longer than 36 bp is optimal for cGAS activation (Gao et al., 2013b). After DNA binding, cGAS undergoes a conformational change that allows ATP and GTP to enter the catalytic pocket, leading to the synthesis of CGAMP, a strong activator of the STING-TBK1 axis (Civril et al., 2013; Gao et al., 2013b; Kranzusch et al., 2013; Wu et al., 2013; Zhang et al., 2014). cGAS can be activated by dsDNA and DNA-RNA hybrids (Mankan et al., 2014).

[0139] vi. Cyclic-diGMP, a recent second messenger The bacterial second messenger cyclic-diGMP is independent of DAI or other known cytoplasmic receptors, but potently induces type I IFNs via a mechanism requiring TBK1 and IRF3 (McWhirter et al., 2009).

[0140] vii. TLR3, TLR4, TLR7, TLR8, and TLR9 agonists In some cell types, such as macrophages and dendritic cells, type I intercellular neurons (IFNs) are produced in response to triggers from dsRNA and lipopolysaccharides on the transmembrane receptors Toll-like receptor 3 (TLR3) and TLR4, respectively. TLR3 and TLR4 transmit signals via the adapter molecule TRIF, which then associate with TBK1 to activate IRF3 (Kawai & Akira, 2010).

[0141] Plasma cell-like DCs (pDCs), which are naturally occurring IFN-producing cells (Colonna et al., 2004), preferentially express the intracellular endosomal receptors TLR7 and TLR9, and induce signaling via the adapter protein MyD88, enabling them to respond to single-stranded RNA and DNA viruses, respectively (Colonna et al., 2004). These receptors undergo spatiotemporal regulation upon TLR ligation so that these cells constitutively express IRF7 and IRF8, and the MyD88-IRF7 complex is retained in the endosomal compartment, thereby inducing type I IFN production. As a result, pDCs alone are highly efficient at inducing type I IFN (Colonna et al., 2004).

[0142] The TLR4 agonist glucopyranosyllipid immunostimulant (GLA) has been tested alone or in combination with an anti-PDmAb [Immune Design 2016] (J. Meulen and S. Brady, "Immune Design", Hum. Vaccin. Immunother., 13(1):15(2017)). The TLR3 agonist Polyiltonol® and the TLR7 / 8 agonist MEDI9197 have also been tested in patients with advanced, reachable solid tumors (MedImmune 2016; Oncovir 201). ("Activating natural host defense; Hiltonol (poly-ICLC) and malignant tumors, ASalzar, Oncovir, Inc., www.oncovir.com / id2 (accessed July 11, 2018); and Gupta et al., "Abstract CT091: Safety and pharmacodynamic activity of MEDI9197, TLR 7 / 8 agonists managed in-subject with solid tumors," Cancer Research, AACR Annual Meeting 2017; April 1-5, 2017 (published July 2017)). Intratumoral injection of TLR agonists such as CpG-enriched oligodeoxyribonucleotides (CpGODN, PF-3512676) in conjunction with low-dose radiotherapy has shown a clinical response in patients with advanced non-Hodgkin lymphoma in phase I / II clinical trials [Dynavax 2016] (Adamus et al., 2018).

[0143] viii. STING Agonist Cyclic dinucleotides (CDNs) [cyclic di(guanosine 5'-monophosphate), cyclic di(adenosine 5'-monophosphate), and cyclic GMP] are a class of pathogen-associated molecular pattern molecules (PAMPs) that activate the TBK1 / IRF3 / 1 interferon signaling axis via cytoplasmic pattern recognition receptor stimulants (STINGs) of interferon genes.

[0144] Novel STING agonists are being developed to induce type I interferon responses. One major approach involves rational modification of CDNs to improve efficiency, which has led to the development of synthetic dithio mixed-binding CDNs (Corrales et al., 2015). One compound (ML RRS2CDA or ADUS100) bound to both human and mouse STING and showed potent antitumor effects in multiple animal models (Corrales et al., 2015). Phase 1 clinical trials of ADUS100 are underway in patients with cutaneously accessible solid tumors and lymphomas (Aduro Biotech, 2016).

[0145] Analysis of the 1000 Genomes Project database (http: / / www.1000genomes.org / ) identified five human STING variants, including the WT allele, reference (REF) allele (R232H), HAQ allele (R71H, G230A, R293Q), AQ allele (G230A, R293Q), and Q allele (R293Q) (Yi et al., 2013).

[0146] A rationally designed synthetic CDN agonist, ML RRS2 CDA, has been developed, exhibiting enhanced stability, human STING activation, cellular uptake, and antitumor efficacy, as well as lower reactogenicity, compared to natural STING ligands produced by bacterial or host cell cGAS (Corrales et al., 2015; Fu et al., 2015).

[0147] Rp,Rp(R,R) dithio-substituted diastereomer CDNs are resistant to phosphodiesterase digestion, stimulate higher IFN-β expression in cultured human cells, and induce more potent antitumor immunity compared to CDNs without dithio modification (Corrales et al., 2015; Fu et al., 2015). To increase affinity for human STING, MLRRS2 CDA contains a non-canonical structure defined by a phosphate crosslink with one 2'5' and one 3'5' mixed phosphodiester bond (2',3'CDN). The 2',3' mixed bond structure confers increased STING binding affinity (Gao et al., 2013b) and is also found in endogenous cGAMP produced by eukaryotic cGAS. MLRR-S2CDA was shown to activate a wide range of known human STING alleles in a HEK293T cell STING signaling assay of IFN-β in human perivascular hematopoietic cells (PBMCs) isolated from multiple donors with different STING types, including donor homodigoses against the REF allele known to be resistant to signaling induced by bacterial 3',3'CDN (Corrales et al., 2015; Fu et al., 2015), and in the induction dose-dependent expression of induced IFN-β.

[0148] C. Type II interferon agonist The compositions and methods of the present invention may include type II IFN agonists, i.e., agents that increase the level (e.g., activity or expression level) of type II interferon. The class of type II interferon (IFN) now includes a member called IFN-γ (gamma). Mature IFN-γ is an antiparallel homodimer that binds to the IFN-γ receptor (IFNGR) complex and induces signaling in target cells. IFNGR consists of two subunits of each molecule, named IFNGR1 and IFNGR2. IFN-γ is involved in regulating immune and inflammatory responses; in humans, there is only one type of interferon-γ. It is produced in activated T cells and natural killer cells. IFN-γ enhances the action of type I IFN. IFN-γ released from Th1 cells recruits leukocytes to the site of infection, promoting inflammation. It also stimulates macrophages to kill ingested bacteria. IFN-γ released from Th1 cells is also important for regulating the Th2 response. IFN-γ plays a crucial role in regulating the immune response, and its production can lead to autoimmune diseases.

[0149] Therefore, one embodiment of the present invention encompasses a composition comprising minicells containing a type II IFN agonist. Although the minicells are derived from bacteria, the minicells themselves do not activate the type II interferon response in human patients. See Example 15. The inventors discovered that the addition of IFNγ increased the antitumor efficacy of doxorubicin-loaded EGFR-targeted EDV and induced tumor regression in a xenograft model. See Example 13. Furthermore, compositions comprising (i) EFFR-targeted minicells loaded with the highly toxic chemotherapeutic agent PNU159682, (ii) untargeted minicells loaded with double-stranded DNA containing 20 nucleotides, and (iii) minicells containing the IFNγ product Imukin were well-tolerated and induced anticancer effects in dogs with late-stage endogenous tumors. See Example 14.

[0150] Type II interferons (IFNs) play a crucial role in antitumor immunity by activating cytotoxic T cells. See, for example, Chikuma et al., 2017. While IFNγ cytokines are released from natural killer cells upon binding to innate antigens, sphingoglycolipid compounds can function as potent activators of both innate and adaptive immune responses. We have discovered that exposure to sphingoglycolipids induces a potent cytokine response by innate natural killer T (iNKT) cells containing type II interferons, IFN-γ, and numerous interleukins (Th1-, Th2-, and / or Th17-type cytokines). See, for example, Carreno et al., 2016. Subsequently, iNKT cells induce DC maturation and exhibit T cell helper-like functions that lead to the development of a cytotoxic T cell response.

[0151] Examples of glycosphingolips useful for inducing IFN type II responses are described herein, including α-galactosylceramide (α-C-GalCer) in C-glycoside form and α-galactatosylceramide. (α-GalCer), galactosylceramide in 12-carbon acyl form (β-GalCer), β-D-glucopyranosylceramide (β-GlcCer), 1,2-diacyl-3-0-galactosyl-sn-glycerol (BbGL-II), diacylglycerol-containing glycolipid (Glc-DAG-s2), ganglioside (GD3), gangliotriceramide (Gg3Cer), glycosylphosphatidylinositol (GPI), α-glucuronosylceramide (GSL-1 or GSL-4), isoglobotrihexylceramide (iGb3), lipophosphoglycan (LPG), α-galactosylceramide analog (OCH), and treitolceramide. In certain embodiments, the mini-cells disclosed herein contain α-galactosylceramide (α-GalCer) as a type II IFN agonist.

[0152] The INFII agonist α-GC is known to stimulate the immune system through the activation of a type of leukocyte known as natural killer T cells (NKT cells) (Birkholz et al., 2015). Knowing that mini-cells can facilitate the presentation of α-GC to target cells, and as will be further discussed in Example 17, the applicant has developed mini-cells α-GC We transitioned to research on mini-cell-enhanced immune activation using this technology, which allowed us to complement treatments consisting of mini-cell-enhanced delivery of chemotherapy drugs.

[0153] As shown in Example 18, the applicant used the chemotherapy doxorubicin ( Ep Miniature cells Dox Tumor-containing mice administered mini-cells containing α-GC (mini-cells) α-GC ) and, Ep Miniature cells Dox We found that mice treated with this method showed a significant cessation of tumor progression compared to mice treated with only this method. These observations indicate that mini-cell compositions incorporating an INFII agonist instead of an INFI agonist are effective in treating tumors in mice.

[0154] Minicells can directly deliver type II IFN agonists to immune system cells in terms of enhancing iNKT cell activation and type II interferon IFN-γ production in vivo. Alternatively, untargeted EDV is taken up by phagocytic cells of the immune system, where it is degraded in endosomes, and αGC is presented to iNKT cells for immune activation. Thus, in some embodiments, minicells provide targeted delivery of type II interferon agonists. In other embodiments, the composition disclosed herein comprises untargeted minicells containing a type II interferon agonist.

[0155] IFN-γ production is regulated by cytokines secreted by antigen-presenting cells (APCs), most notably interleukin (IL)-12 and IL-18. These cytokines act as bridges linking IFN-γ production and infection in the innate immune response. Macrophage recognition of many pathogens induces the secretion of IL-12 and chemokines. These chemokines attract NK cells to the site of inflammation, and IL-12 promotes IFN-γ synthesis in these cells. In macrophages, natural killer cells, and T cells, combined stimulation of IL-12 and IL-18 further increases IFN-γ production. Therefore, any one or a combination of these proteins are suitable agents for the purposes of this disclosure.

[0156] Negative regulators of IFN-γ production include IL-4, IL-10, transforming growth factor β, and glucocorticoids. Proteins and nucleic acids that inhibit these factors can stimulate IFN-γ production.

[0157] Additionally, polynucleotides encoding IFN-γ, or genes that activate the production and / or secretion of IFN-γ, are also suitable for use in this context.

[0158] Drugs that increase IFN-γ levels can also be considered viral vaccines. Many viral vaccines are available that can induce IFN-γ production without causing infection or other types of adverse effects. A typical example of this type of viral vaccine is the influenza vaccine.

[0159] The data indicate that the serum concentration of IFN-γ necessary to effectively activate the host immune response against tumor cells is low when the patient also receives drug loading, bispecific antibody-targeted mini-cells, or dead bacterial cells. Thus, in one aspect, the method of the present invention results in an increase in serum IFN-γ concentration to approximately 30,000 pg / mL or less. In another aspect, the serum IFN-γ concentration increases to no higher than approximately 5,000 pg / mL, 1,000 pg / mL, 900 pg / mL, 800 pg / mL, 700 pg / mL, 600 pg / mL, 500 pg / mL, 400 pg / mL, 300 pg / mL, 200 pg / mL, or 100 pg / mL. In further stages, the resulting serum IFNγ concentration is at least about 10 pg / mL, or at least about 20 pg / mL, 30 pg / mL, 40 pg / mL, 50 pg / mL, 60 pg / mL, 70 pg / mL, 80 pg / mL, 90 pg / mL, 100 pg / mL, 150 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, or 500 pg / mL.

[0160] In some embodiments, the drug is IFNγ protein, or a modified protein or analog. In some embodiments, administration achieves approximately 0.02 ng to 1 microgram of IFN-γ per ml of host blood. In one embodiment, the IFNγ concentrations achieved in host blood are approximately 0.1 ng / ml to 500 ng / ml, approximately 0.2 ng / ml to 200 ng / ml, approximately 0.5 ng / ml to 100 ng / ml, approximately 1 ng / ml to 50 ng / ml, or approximately 2 ng / ml to 20 ng / ml.

[0161] III. Intact Bacterial Mini-Cells The term “minicell” is used here to mean a derivative of a bacterial cell that lacks chromosomes (“achromosome”) and arises from a disruption in the coordination of cell division and DNA segregation during binary fission. Minicells differ from other small vesicles such as so-called “membrane blebs” (less than approximately 0.2 μm in size). These vesicles are spontaneously generated and released under certain circumstances, but not by specific gene rearrangements or episomal gene expression. By the same token, intact minicells are distinguished from similar ghosts. Similar ghosts are not generated due to specific gene rearrangements or episomal gene expression. The bacterial-derived minicells used in this disclosure are completely intact and therefore distinguished from other achromosome-free bacterial cell derivatives characterized by an outer membrane or delimiting membrane that is destroyed or degraded, and even removed. See U.S. Patent No. 7,183,105, column 111, line 54 et seq. The intact membranes characterizing the minicells of this disclosure allow for the retention of a therapeutic payload within the minicell until the payload is released after uptake, within the tumor invention.

[0162] Minicells, or EDVs, are anucleated, non-viable nanoparticles produced as a result of derepressing the polarity sites of cells by inactivating genes that control normal bacterial cell division. (Ma et al., 2004) Derepression means that bacteria divide centrally and polarly, and the polar division resulting from the minicells demonstrated by the inventors of this disclosure can function as leak-resistant microreservoir carriers, enabling efficient packaging of various chemotherapeutic drugs. Furthermore, in contrast to current stealth liposome drug carriers (liposomal doxorubicin) that can only package about 14,000 molecules per particle (Park et al., Breast Cancer Res, 4(3):95-99 (2002)), or "armed antibodies" that can carry fewer than five drug molecules, EDVs can readily accommodate payloads of up to one million drug molecules. Additionally, EDVs can target overexpressed receptors on the surface of cancer cells using bispecific antibodies (see Part D below), which enables highly significant tumor growth inhibition and / or regression both in vitro and in vivo.

[0163] The mini-cells used in this invention can be prepared from bacterial cells such as Ecoli and Styphymurium. Chromosome replication in prokaryotes is linked to normal binary fission, including mesocellular septum formation. For example, in E. coli, mutations in the min gene, such as minCD, can eliminate the inhibition of septum formation at the cell pole during cell division, resulting in the production of normal daughter cells and chromosome-less mini-cells. See de Boer et al., J. Bacteriol., 174:63-70 (1992); Raskin & de Boer, J. Bacteriol., 181:6419-s24 (1999); Hu & Lutkenhaus, Mol. Microbio., 34:82-90 (1999); Harry, Mol. Microbiol., 40:795 (2001).

[0164] In addition to min operon mutations, chromosome-less mini-cells can also be generated following a series of other gene rearrangements or mutations that affect septum formation, for example in Bacillus subtilis divIVB1. See Reeve and Cornett, J Virol., 15:1308 (1975). Mini-cells can also be formed following disturbances in the gene expression levels of proteins involved in cell division / chromosome segregation. For example, overexpression of minE leads to polar division and mini-cell production. Similarly, chromosome-less minicells can result from deletions in chromosome segregation, such as the smc mutation in Bacilus subtilis (Britton et al., Genes Dev., 12: 1254 (1998)), the spoOJ deletion in Bsubtilis (Ireton et al., JBacteriol., 176: 532029 (1994)), the mukB mutation in Ecoli (Hiraga et al., JBacteriol., 171:1496 (1989)), and the parC mutation in Ecoli (Stewart and D'Ari, JBacteriol., 174:4513 (1992)). Furthermore, CafA can enhance the rate of cell division and / or inhibit post-replication chromosome segregation (Okada et al., JBacteriol., 176:917 (1994)), leading to the formation of linked cells and chromosome-less minicells.

[0165] Therefore, minicells for this disclosure can be prepared from any bacterial cell, which may be of Gram-positive or Gram-negative origin due to the conserved nature of bacterial cell division in these bacteria. Furthermore, the minicells used in this disclosure should, as described above, have intact cell walls (i.e., “intact minicells”) and should be distinguished from and isolated from other vesicles (e.g., membrane blebs) that are not due to specific genetic rearrangements or episomal gene expression.

[0166] In a given embodiment, the parent (source) bacteria of the minicells may be Gram-positive or Gram-negative. In one embodiment, the parent bacteria are selected from one or more BV4, which includes Terra- / Glidobacteria (BV1), Proteobacteria (BV2), Spirochaetes, Sphingobacteria, and Plantobacteria. In another aspect, the bacteria are selected from one or more Firmicutes (BV3), such as Bacilli, Clostridia, or Tenericutes / Mollicutes, or Actinobacteria (BV5), such as Actinomycetales or Bifidobacteriales.

[0167] According to the present invention, dead bacterial cells are non-viable prokaryotic cells of bacteria, cyanobatelia, eubacteria, and archaea, as defined in the second edition of Bergey's Manual of Systematic Biology. Such cells are considered “intact” if they have intact cell walls and / or cell membranes and contain genetic material (nucleic acids) endogenous to the bacterial species. A method for preparing dead bacterial cells is described, for example, in U.S. Patent Application Publication No. 2008 / 0038296, which is incorporated herein by reference.

[0168] In yet another context, these bacteria include Eobacteria (Chloroflexia, Deinococcus sarmus), cyanobacteria, Thermofulbacteria, thermophiles (Achifica, Thermotogae), Alpha, Beta, Gamma (Enterobacteriaceae) Delta or Epsilon Proteobacteria, Spirobacteria, Spirochetes, Fibrobacter, Chlorobacteria / Bacteroides, Chlamydia / Berchomecrobia, Plantomyces, Acidobacteria, Chrysiogenes, Deferibacteria, Fusobacteria, Gematimona One or more species are selected from Detes, Nitrospira, Synergistes, Dictyoglomus, Lentisphaera Bacillus, Bacillus, Listeriaceae, Staphylococcus, Enterococci, Enterococcus, Lactobacillus, Leuconostoc, Clostridium, Thermoanaerobacter, Mycoplasmales, Anaeroplasmales, Acholeplasmales, Actiomycyne, Actinomyces, Corynebacterium, Corynebacterium, furan kinase, Microcossine, Brevibacterium, and Bifidobacteria.

[0169] For pharmaceutical use, the compositions of this disclosure should comprise minicells or dead bacterial cells isolated as completely as possible from immunogenic components and other toxic contaminants. Methodologies for purifying bacterial minicells to remove free endotoxins and parental bacterial cells are described, for example, in WO 2004 / 113507, which is incorporated herein by reference in its entirety. Briefly, the removal of (a) smaller vesicles, such as membrane blebs, generally smaller than 0.2 μm, (b) free endotoxins released from the cell membrane, and (c) parental bacteria, whether alive or dead, and their debris (which is also a source of free endotoxins) is achieved. Such removal can be carried out, among other things, using 0.2 μm filtration to remove smaller vesicles and cellular debris, 0.45 μm filtration to induce parental cells to form filaments and then remove the parental cells, antibiotics to kill living bacterial cells, and antibodies against free endotoxins.

[0170] The basis of the purification procedure is our discovery that, despite differences in their bacterial sources, all intact mini-cells are approximately 400 nm in size, i.e., larger than membrane vesicles and other smaller vesicles, but smaller than the parent bacteria. The size determination of mini-cells can be achieved by using solid-state methods such as electron microscopy, or by liquid-based techniques, such as dynamic light scattering. The size values ​​obtained by each such technique may have an error range, and these values ​​may differ somewhat between techniques. Thus, the size of dry mini-cells can be measured as approximately 400 nm ± 50 nm by electron microscopy. Dynamic light scattering can measure the same mini-cells as approximately 500 nm ± 50 nm in size. Furthermore, drug-packaged ligand-targeted mini-cells can also be measured as approximately 400 nm to 600 nm ± 50 nm using dynamic light scattering.

[0171] This scattering of size values ​​is readily applicable, for example, to the purpose of isolating minicells from immunogenic components and other toxic contaminants, as described above. That is, intact bacterial minicells are characterized by cytoplasm surrounded by a rigid membrane, which gives the minicell a rigid spherical structure. This structure is evident in transmission electron microscopy, where the diameter of the minicell is measured across the minicell between the outer boundaries of the rigid membrane. This measurement yielded the aforementioned size value of 400 nm ± 50 nm.

[0172] Another structural element of minicells derived from dead bacterial cells or Gram-negative bacteria is the O-polysaccharide component of lipopolysaccharide (LPS), which is embedded in the outer membrane via lipid A anchors. This component is a chain of repeating carbohydrate residue units, with as many as 70 to 100 repeating units, each containing 4 to 5 sugars. Since these chains are not rigid in a liquid environment as they are in vivo, they can adopt a wavy, flexible structure that gives seaweed its typical appearance in coral sea environments; that is, the chains move with the liquid while remaining fixed to the minicell membrane.

[0173] Influenced by the O-polysaccharide component, dynamic light scattering can provide mini-cell size values ​​of approximately 500 nm to 600 nm, as described above. Nevertheless, mini-cells of both Gram-negative and Gram-positive bacteria readily pass through 0.45 μm filtration, demonstrating an effective mini-cell size of 400 nm ± 50 nm. The above-mentioned size variation is encompassed within the present invention and is particularly indicated by the modifying phrase "approximately" in terms such as "size of approximately 400 nm".

[0174] With respect to toxic contaminants, the compositions of this disclosure preferably contain less than about 350 EU of free endotoxins. In relation to this, approximately 250 EU or less, approximately 200 EU or less, approximately 150 EU or less, approximately 100 EU or less, approximately 90 EU or less, approximately 80 EU or less, approximately 70 EU or less, approximately 60 EU or less, approximately 50 EU or less, approximately 40 EU or less, approximately 30 EU or less, approximately 20 EU or less, approximately 15 EU or less, approximately 10 EU or less, approximately 9 EU or less, approximately 8 EU or less, approximately 7 EU or less, approximately 6 EU or less, approximately 5 EU or less, approximately 4 EU or less, approximately 3 EU or less, approximately 2 EU or less, approximately 1 EU or less, approximately 0.9 EU or less, approximately 0.8 EU or less, approximately 0.7 EU or less, approximately 0.6 EU or less, approximately 0.5 EU or less, approximately 0.4 EU or less, approximately 0.4 EU or less, approximately 0.3 EU or less, approximately 0.2 EU or less, approximately 0.1 EU or less, approximately 0.05 EU or less, or approximately 0.01 EU or less.

[0175] The compositions of the present invention also include at least about 10 9 Miniature cells or dead bacterial cells, e.g., at least about 1 × 10⁻¹⁶ 9 , at least about 2 × 10 9 , at least about 5 × 10 9 , or at least 8 × 10 9 It may include. In some embodiments, the composition is about 10 11 Miniature cells or dead bacterial cells, such as a few cells or fewer, e.g., about 1 x 10⁶ 11 The following, or approximately 9 x 10 10 Below, or approximately 8 x 10 10 Includes the following:

[0176] IV. Loading of active substances into mini-cells or sterilized bacterial cells Activators or antitumor agents (e.g., small molecule drugs, proteins, and functional nucleic acids) can be directly packaged into mini-cells by co-incubating multiple intact mini-cells with the activator in a buffer. The buffer composition can be varied according to conditions well known in this field to optimize the loading of the activator in intact mini-cells. The buffer can also be varied depending on the drug (e.g., depending on the nucleotide sequence or length of the nucleic acid loaded into the mini-cells in the case of nucleic acid payloads). Exemplary buffers suitable for loading include, but are not limited to, phosphate-buffered saline (PBS). Once packaged, the activator remains in the mini-cells and is protected from degradation. Long-term incubation studies using siRNA-packaged mini-cells incubated in sterile saline have shown, for example, no siRNA leakage.

[0177] Functional nucleic acids or proteins, which can be encoded by nucleic acids, can be introduced into minicells by transforming parent bacterial cells with a plasmid-like vector encoding the activator. When minicells are formed from parent bacterial cells, they hold a specific copy of the plasmid and / or expression product, the antitumor agent. Further details of packaging into minicells and the expression product are provided in International Publication No. 03 / 033519, the contents of which are incorporated in whole by reference into this invention.

[0178] The data presented in WO 03 / 033519 demonstrates, for example, that recombinant minicells carrying mammalian gene expression plasmids can be delivered to phagocytic and non-phagocytic cells. International Publication No. 03 / 033519 also describes the genetic transformation of minicell-producing parent bacterial strains using heterologous nucleic acids supported on episomal replication plasmid DNA. Upon separation of the parent organism and minicells, a portion of the episomal DNA is separated into the minicells. The resulting recombinant minicells are readily engulfed by mammalian phagocytic cells and degraded within intracellular phagolysosomes. Furthermore, a portion of the recombinant DNA escapes the phagolysosome membrane and is transported to the mammalian cell nucleus, where the recombinant gene is expressed.

[0179] In other embodiments, multiple nucleic acids directed to different mRNA targets can be packaged into the same minicell. Such approaches can be used to combat drug resistance and apoptosis resistance. For example, cancer patients routinely exhibit resistance to chemotherapy drugs. Such resistance can be mediated, among other things, by the overexpression of genes such as multidrug resistance (MDR) pumps and anti-apoptotic genes. To combat this resistance, minicells can be packaged with functional nucleic acids at therapeutically significant concentrations against MDR-linked genes and administered to patients before chemotherapy. Furthermore, packaging multiple functional nucleic acids into the same minicell directed to different mRNA targets can enhance therapeutic success, as most molecular targets undergo mutation and possess multiple alleles. Further details of direct packaging of nucleic acids into minicells are provided in International Publication No. 2009 / 027830, the contents of which are incorporated in whole by reference herein.

[0180] Small molecule drugs, whether hydrophilic or hydrophobic, can be packaged within minicells by creating a concentration gradient between the extracellular medium containing the minicells and the minicell cytoplasm. If the extracellular medium contains a higher concentration of the drug than the minicell cytoplasm, the drug will naturally move down this concentration gradient into the minicell cytoplasm. However, if the concentration gradient is reversed, the drug will not move out of the minicells. Further details of the drug loading process and its remarkable properties can be found, for example, in U.S. Patent Application Publication 2008 / 0051469 (the contents of which are specifically incorporated by reference).

[0181] To load minicells with drugs that are not normally water-soluble, the drug can first be dissolved in a suitable solvent. For example, paclitaxel can be dissolved in a 1:1 blend of ethanol and Cremophore EL (polyethoxylated castor oil), and then diluted in PBS to obtain a paclitaxel solution that is partially diluted in an aqueous medium and supports a minimal amount of organic solvent to ensure that the drug remains in the solution. Minicells can then be incubated in this final medium for drug loading. Thus, we have found that even hydrophobic drugs can diffuse into the cytoplasm or membrane of minicells to achieve high and therapeutically significant cytoplasmic drug loading. This is unexpected because the minicell membrane is composed of a hydrophobic phospholipid bilayer, which is expected to prevent the diffusion of hydrophobic molecules into the cytoplasm. The loading of a diverse range of representative small molecule drugs into mini-cells has been demonstrated, with various sizes and chemical properties shown: doxorubicin, paclitaxel, fluoro-paclitaxel, cisplatin, vinblastine, monsatrol, thymidylate synthase (TS) inhibitor OSI-7904, irinotecan, 5-fluorouracil, gemcitabine, and carboplatin. Furthermore, across the board, the resulting small molecule drug-packaged mini-cells exhibit significant antitumor efficacy in vitro and in vivo.

[0182] V. Targeting specific mammalian cells and tumors with minicells The inventors discovered that blood vessels surrounding tumor cells exhibit a loss of integrity. That is, even within the blood-brain barrier (BBB) ​​environment, the vessels have large fenestrations and are "leaky." Once cancer cells colonize, they secrete substances that promote the formation of new blood vessels—a process called angiogenesis. These vessels grow rapidly and, unlike normal vessels, are leaky through "holes" (fenestrations) ranging from 50 nm to 1.2 μm (highly permeable vascular system). Drug delivery particles such as liposomes are currently thought to achieve tumor targeting through passive methods, including extravasation from the leaky vascular system supporting the tumor microenvironment (Hobbs et al., 1998). While the abnormal tumor microenvironment is characterized by interstitial hypertension, and this phenomenon has been shown to limit access to anti-cancer antibody therapies, it does not appear to be an absolute barrier, as exemplified by immunoliposomes (Nielsen et al., 2002) and antibodies conjugated to Quantum Dots (Gao et al., 2004). This phenomenon also applies to EDV, which has the additional advantage of possessing specifically targeted tumor antibodies. After intravenous injection, EDV leaks into the tumor microenvironment, and then active targeting occurs via binding to cancer cell surface receptors and endocytosis. Therefore, contrary to conventional understanding, particles the same size as minicells, i.e., much larger than the aforementioned consensus pore size limit of BBB, are nevertheless smaller than the fenestrations of the leaky vessel walls and thus can passively spill into the tumor microenvironment through these fenestrations.

[0183] Upon entering the tumor microenvironment, minicells can induce receptor-mediated internalization by host tumor cells and be taken up by them. Thus, minicells packaged with antitumor agents release the drug into the cytoplasm of tumor cells, killing them.

[0184] According to further aspects of this disclosure, mini-cells or killed bacterial cells of the above composition are directed to target mammalian tumor cells via ligands. In some embodiments, the ligand is “dual-specific,” i.e., the ligand exhibits specificity to both mini-cell and mammalian (tumor) cell components, resulting in a given vesicle binding to the target cell, thereby causing the latter to engulf the former. The use of dual-specific ligands for targeting mini-cells to tumor cells is further described in International Publication No. 05 / 056749 and International Publication No. 05 / 079854, and the use of dual-specific ligands for targeting killed bacterial cells to tumor cells is further described in U.S. Patent No. 8,591,862, the contents of which are incorporated herein by reference in their entirety. Once such a ligand attaches to a vesicle, the ligand’s non-occupying specificity (“single-specificity”) remains relevant until it interacts with the target (tumor) mammalian cell. Numerous tumor-targeting ligands are known in this field (Hong et al., 2011; Hoelder et al., 2012; Galluzzi et al., 2013). Several peptides, such as somatostatin (SST) peptide, vasoactive intestinal peptide (VIP), Arg (RGD) peptide, and bombesin / gastrin-releasing peptide (BBN / GRP), have been successfully characterized for tumor receptor imaging (De Jong et al., 2009; Tweedle, 2009; Schottelius and Wester, 2009; Igarashi et al., 2011; Laverman et al., 2012).

[0185] Tumor-targeting peptide sequences can be selected primarily by three different methods: (1) derivatization from native proteins (Nagpal et al., 2011); (2) chemosynthesis and structure-based rational engineering (Andersson et al., 2000; Merrifield, 2006); and (3) screening of peptide libraries (Gray and Brown, 2013). Of these methods, phage display technology is a conventional method but the most widely used, offering many advantages (e.g., ease of handling, and the ability to effectively screen a large number of different peptides (Deutscher, 2010)). Receptors overexpressed in tumor cells, rather than normal cells, are excellent candidates for in vivo tumor imaging. To date, many tumor-targeting peptides and their analogues have been identified, as described below.

[0186] The Arg-Gly-Asp(RGD) peptide RGD specifically binds to integrin receptors (Ruoslahti, 1996). Integrins consist of two subunits (α and β subunits). The integrin family, particularly αvβ3, is associated with tumor angiogenesis and metastasis. These are overexpressed on endothelial cells during angiogenesis but are barely detectable in most normal organs. Therefore, they are widely used in diagnostic imaging.

[0187] Bombesin (BBN) / digestible-release peptide (GRP) - Amphibian BN and related peptides constitute a family of neuropeptides that exhibit various physiological effects such as exocrine regulation, endocrine division, thermoregulation, sucrose regulation, and cell growth (Ohki-Hamazaki et al., 2005). There are four subtypes of bombesin-like peptide receptors: neuromedin B receptor, bombesin 3 receptor, GRP receptor, and bombesin 4 receptor. These receptors are overexpressed in many tumors, including breast cancer, ovarian cancer, and gastrointestinal stromal tumors.

[0188] Cholecystokinin (CCK) / gastrin peptides: CCK and gastrin are structurally and functionally similar peptides that exert various physiological effects in the gastrointestinal tract and central nervous system (Matsuno et al., 1997). Three receptors for CCK (CCK1, CCK2, and CCK2i4sv) have been identified, all belonging to the GPCR superfamily. Among these, the CCK2 / gastrin receptor is frequently found in human cancers such as stromal ovarian cancer and astrocytoma.

[0189] α-Melanocyte-stimulating hormone (α-MSH)-α-MSH is a linear tridecapeptide primarily responsible for regulating skin pigmentation (Singh and Mukhopadhyay, 2014). α-MSH and its analogs exhibit binding affinity to the melanocortin-1 receptor (MC-1r), which is expressed in over 80% of human melanoma metastases, and are widely used as a medium for melanoma-targeted imaging and radiotherapy.

[0190] Neuropeptide Y (NPY) is a 36-amino acid peptide belonging to the pancreatic polypeptide family (Tatemoto, 2004). The NPY receptor is overexpressed in various tumors, including neuroblastoma, sarcoma, and breast cancer.

[0191] Neutrotensin (NT)-NT is a 13-amino acid peptide that targets the NT receptor, which has been identified in various tumors, including pancreatic ductal adenocarcinoma, small cell lung cancer, and medullary thyroid carcinoma (Tyler-McMahon et al., 2000). Therefore, it is an attractive candidate for cancer imaging.

[0192] Prostate-specific membrane antigen (PSMA): Prostate cancer cells overexpress PSMA on their cell surface (Silver et al., 2007; Ghosh and Heston, 2004; Mhawech-Fauceglia et al., 2007; Santoni et al., 2014). 68 Ga]GaPSMA-HPEDCC([ 68Ga]GaPSMA-11[PET] (also known as GaPSMA-11), monoclonal antibody (mAb) [ 177 Lu]Lu / [ 90 Y]y-J591 (therapy), [ 123 I]I-MIP-1072 (plane / SPECT), [ 131 I]I-MIP-1095 (therapy), and for PET 68 Ga or for treatment 177 There are several available radiopharmaceuticals that target PSMA, including the Lu-labeled theranostic agents PSMA-I&T and DKFZ-PSMA-617 (PSMA-617).

[0193] Somatostatin (SST) peptide is a naturally occurring cyclopeptide hormone having either 14 or 28 amino acids (Weckbecker et al., 2003). It can inhibit the secretion of insulin, glucagon, and other hormones. Somatostatin receptors (SSTRs; five subtypes SSTR1-SSTR5) are overexpressed in many tumors, including gliomas, neuroendocrine tumors, and mammary tumors. GEP-type neuroendocrine neoplasms (NENs) most commonly originate from the pancreas, jejunum, ileum, cecum, rectum, appendix, and colon. A common feature of all GEP-NENs is the combined characteristics of endocrine and neuronal cells. Well-differentiated NENs overexpress somatostatin receptors (SSTRs), particularly the SSTR-2 subtype.

[0194] Substance P is an undecapeptide belonging to the neuropeptide family known as tachykinins (Strand, 1999). Substance P is a specific endogenous ligand known to the neurokinin 1 receptor (NK1R) and is known to be expressed in various cancer cells.

[0195] T140 is a 14-amino acid peptide with one disulfide crosslink and is an inverse agonist of chemokine receptor type 4 (CXCR4) (Burger et al., 2005). Its derivatives are widely used as CXCR4 contrast agents.

[0196] Tumor molecular target peptide 1 (TMTP1) is a 5-amino acid peptide that has been found to specifically bind to highly metastatic cancer cells, particularly cancer cells derived from typical liver micrometastases (Yang et al., 2008).

[0197] Vasoactive intestinal peptide (VIP) is a neuropeptide consisting of 28 amino acids (Igarashi et al., 2011). It promotes vasodilation and cell proliferation. Its effects are mainly regulated by two receptor subtypes (VPAC1 and VPAC2). VIP receptors are expressed in large quantities in many tumors, including pancreatic adenocarcinoma and neuroendocrine tumors.

[0198] Ligands can attach to the cell membrane of vesicles through interactions between the ligand and components on the cell membrane, such as polysaccharides, glycoproteins, or polypeptides. Expressed ligands are immobilized on the surface of vesicles so that the tumor surface component binding portion of the ligand is exposed, and as a result, when the vesicle comes into contact with mammalian tumor cells, that portion can bind to target mammalian cell surface receptors.

[0199] Alternatively, the ligand may be expressed and presented by the living counterpart of a bacterial vesicle, such as the parent cell of a minicell, or by the bacterial cell before it becomes a killed cell. In this case, the ligand does not need to be specific to the vesicle, but only to components characteristic of mammalian cells. That is, such components do not need to be specific to the tumor cell itself, or even to the specific type of tumor cell being treated, as long as the tumor cell is present on its surface. There isn't one.

[0200] When administered intravenously, the vesicles rapidly accumulate in the tumor microenvironment. This accumulation, occurring as a function of the leaky tumor vascular system described above, leads to the delivery of the vesicle-packaged therapeutic payload to tumor cells, which then internalize the packaged vesicles.

[0201] The inventors have found that this delivery approach is applicable to a range of mammalian tumor cells, including cells that are typically resistant to specific adhesion and endocytosis of minicells. For example, ligands containing antibodies directed towards anti-HER2 receptors or anti-EGF receptors can bind minicells to their respective receptors on a range of target non-phagocytic cells, such as lung, ovarian, brain, breast, prostate, and skin cancer cells.

[0202] The binding thus achieved precedes the uptake of the vesicle by each type of non-phagocytic cell. In other words, in the context of the present invention, a suitable target cell presents a cell surface receptor, and its binding, via a ligand on the vesicle, induces endocytosis of that vesicle.

[0203] More specifically, the inventors discovered that the interaction between (a) a ligand on minicells or dead bacterial cells and (b) a mammalian cell surface receptor can activate an uptake pathway, which we call the "receptor-mediated endocytosis" (rME) pathway, into the late endosomal / lysosomal compartment of target host cells such as tumor cells. Through this rME pathway, the inventors found that bacterial vesicles are processed through early endosomes, late endosomes, and lysosomes, resulting in the release of their payloads into the cytoplasm of mammalian host cells. Furthermore, the nucleic acid payloads not only escape complete degradation in the late endosomal / lysosomal compartment but are also expressed by the host cells.

[0204] For this delivery approach, the tumor-targeting ligand can be "dual-specific" as described above, since it binds to a surface component on the payload-carrying vesicle and a surface component on the target cell, respectively, and its interaction with the latter component leads to the uptake of the vesicle into the rME pathway. In any case, according to the present invention, a given target cell surface receptor can be a candidate for ligand binding if the interaction with the component substantially accesses an endocytosis pathway involving cytoplasmic internalization from the target cell surface. Such candidates can be readily evaluated for suitability in the present invention by co-incubating in vitro a cell type that presents the candidate component on its surface with mini-cells carrying the ligand to which the candidate binds, and also by an assay that binds to a fluorescent dye or other marker according to detection, for example, visually via a confocal microscope. (This type of in vitro assay is described by MacDiarmid et al., 2007b, in the legend on page 436 of Figure 3) Thus, the internal migration of the observed marker constitutes a positive indicator by the assay, such as that the tested target cell surface receptor is suitable for the present invention.

[0205] According to the present invention, the ligand can be any polypeptide or polysaccharide exhibiting the desired specificity or specificity. A preferred ligand is an antibody. In this use, the term “antibody” encompasses immunoglobulin molecules obtained by in vitro or in vivo generation of an immunogenic response, and therefore the “antibody” category encompasses monoclonal antibodies and humanized antibodies (e.g., single-chain antibody fragments (scFv), bispecific antibodies, etc.). A large number of different bispecific protein and antibody-based ligands are known, as demonstrated by the review by Caravella and Lugovskoy (Curr. Opin. Chem. Biol., 14:520-28 (2010)) (which is incorporated here in its entirety for reference). Antibodies useful in accordance with this disclosure can be obtained by known recombinant DNA techniques.

[0206] Therefore, as a non-limiting example, mini-cells can be targeted to cells in the tumor being treated using antibodies that have specificity for surface components such as tumor antigens. Exemplary cell surface receptors in this regard include any of the RTKs, epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and insulin-like growth factor receptor (IGFR), each of which is highly expressed in some solid tumors, including brain tumors, and the folate receptor is overexpressed in some pituitary adenomas. Such bispecific ligands can be targeted to mutant or modified receptors (e.g., the IL13Rα2 receptor expressed in 50%–80% of human glioblastoma pleomorphic tumors) (see Wykosky et al., 2008; Jarboe et al., 2007; Devinski et al., 2000; and Okada et al., 1994), but different from its physiological counterpart IL4R / IL13R expressed in normal tissues. See Hershey, 2003. Therefore, IL13Rα2 is virtually absent in normal brain cells. See Debinski and Gibo, 2000. Furthermore, tumors that metastasize to the brain may overexpress certain receptors, which could also be suitable targets. For example, Da Silva et al., 2010, showed that brain metastases from breast cancer express all members of the HER family of RTKs. HER2 was amplified and overexpressed in 20% of brain metastases, EGFR was overexpressed in 21%, HER3 was overexpressed in 60%, and HER4 was overexpressed in 22%. Interestingly, HER3 expression was increased in breast cancer cells present in the brain.

[0207] Examples of candidate target cell surface receptors are receptor-type tyrosine kinases or members of the "RKT" family. RKTs are a family of transmembrane proteins that undergo constitutive endocytosis at a similar rate to other endometrial proteins. See Goh and Sorkin, 2013. The RKT family was described by Lemmon and Schlessinger in Cell, 141(7):1117-134 (2010). Exemplary RTKs are ErbB EGFR, ErbB2, ErbB3, ErbB4 Ins InsR, IGF1R, InsRR PDGF PDGFRα, PDGFRβ, CSF1R / Fms, Kit / SCFR, Fit3 / Flk2 VEGF VEGFR1 / Fit1, VEGFR2 / KDR, VEGFR3 / Fit4 FGF FGFR1, FGFR2, FGFR3, FGFR4 PTK7 PTK7 / Cck4 Trk TrkA, TrkB, TrkC Ror Ror1, Ror2 MuSK MET, Ron AXL, Mer, Tyro3 Tie Tie1, Tie2 Eph EphA1-8, EPHA10, EphB1-4, EphB6 Ret Ryk DDR DDR1, DDR2 Ros LMR LMR1, LMR2, LMR3 ALK, LTK This is STYK1 SuRTK106 / STYK1.

[0208] Other suitable candidates for appropriate target cell surface receptors include the family of membrane-bound high-affinity folate-binding proteins (folate receptors), which bind to folate and reduced folate derivatives and mediate the intracellular delivery of tetrahydrofolate; the family of membrane-bound cytokine receptors, which play a role in the internal translocation of congeneral cytokines such as IL-13; surface antigens such as CD20, CD33, mesothelin, and HM1.24, which are expressed on certain cancer cells and mediate the internal translocation of congeneral monoclonal antibodies, e.g., rituximab in the case of CD20; and the family of adhesion receptors (integrins), which are transported through the endosomal pathway and are major bridgers in cancer cell adhesion. In one embodiment of the present invention, tumor cell surface receptors include integrin, neuromedin B receptor, bombesin 3 receptor, GRP receptor, bombesin 4 receptor, CCK2 / gastrin, melanocortin-1 receptor (MC-1r), neuropeptide Y (NPY) receptor, neurotensin (NT) receptor, prostate-specific membrane antigen (PSMA), somatostatin (SST) receptor, neurokinin 1 receptor (NK1R), chemokine receptor type 4 (CXCR4), vasoactive intestinal peptide (VIP), epidermal growth factor receptor ( These include EGFR, vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), insulin-like growth factor receptor (IGFR), and combinations thereof.

[0209] According to another embodiment of the present invention, the cell surface receptor is an antigen that is uniquely expressed on target cells in a disease state but remains unexpressed, expressed at low levels, or is not present in a healthy state. Examples of such target antigens that can be specifically bound by the targeted ligand of the present invention may be advantageously selected from EpCAM, CCR5, CD19, HER-2neu, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5, MUC7, BhcG, and Lewis-Y. CD20, CD33, CD30, ganglioside GD3, 9-O-acetyl GD3, GM1, fucosyl SA, GD2, carboanhydrase IX (MN / CA IX), CD44v6, sonic hedgehog (Shh), Wue-1, plasma cell antigen, (membrane-bound) IgE, chondroitin sulfate proteoglycan (MCSP), CCR8, TNF-α precursor, STEAP, prostate stem cell antigen (PSCA), Ly-6, desmoglein 4, E-cadherin neoepitope, fetal acetylcholine receptor, CA19-9 marker and Müllerian duct inhibitor (MIS) receptor type II sTn (sialylated Tn antigen; TAG-72), FAP (fibroblast-activating antigen), endosialin, EGFRVIIILG, SAS, and CD63.

[0210] VI. Formulations The present invention includes, within its scope, compositions or formulations comprising minicells having a payload of one or more combinations of (1) an antitumor agent, (2) a type I IFN agonist, and / or (3) a type II IFN agonist. In a composition comprising all three components, the antitumor agent, type I IFN agonist, and type II IFN agonist may be contained in one or more minicells. For example: (a) an antitumor agent, a type I IFN agonist, and a type II IFN agonist may be contained within the same mini-cell; (b) an antitumor agent and a type I IFN agonist may be contained within a first mini-cell, and a type II IFN agonist may be contained within a second mini-cell; (c) an antitumor agent and a type II IFN agonist may be contained within a first mini-cell, and a type I IFN agonist may be contained within a second mini-cell; or (d) an antitumor agent may be contained within a first mini-cell, and a type II IFN agonist may be contained within a second mini-cell, and a type II IFN agonist may be contained within a second mini-cell; or (e) an antitumor agent may be contained within a first mini-cell, a type I IFN agonist may be contained within a second mini-cell, and a type II IFN agonist may be contained within a third mini-cell.

[0211] The present invention includes, within its scope, compositions or formulations comprising mini-cells having a payload of (1) an antitumor agent and (2) a combination of a type I IFN agonist or a type II IFN agonist. In some embodiments, the antitumor agent and the type I IFN agonist or INFII agonist may be contained in one or more mini-cells. For example, (a) the antitumor agent and the type I IFN agonist may be contained in the same mini-cell; (b) the antitumor agent may be contained in a first mini-cell and the type I IFN agonist in a second mini-cell; (c) the antitumor agent and the type II IFN agonist may be contained in the same mini-cell; or (d) the antitumor agent may be contained in a first mini-cell and the type II IFN agonist in a second mini-cell.

[0212] In exemplary embodiments, the composition disclosed herein comprises the antitumor agent siPlk1, the interferon type I agonist 60-mer double-stranded DNA, and / or the interferon type II agonist α-galactosylceramide, wherein siPlk1, 60-mer double-stranded DNA, and α-galactosylceramide are contained within one or more minicellular cells.

[0213] In another exemplary embodiment, the composition disclosed herein comprises the antitumor agent siRRM1, the interferon type I agonist 60-mer double-stranded DNA, and / or the interferon type II agonist α-galactosylceramide, wherein siRRM1, 60-mer double-stranded DNA, and α-galactosylceramide are contained within one or more minicellular cells.

[0214] In another exemplary embodiment, the composition disclosed herein comprises the antitumor agent PNU159682, the interferon type I agonist 60-mer double-stranded DNA, and / or the interferon type II agonist α-galactosylceramide, wherein PNU159682, 60-mer double-stranded DNA, and / or α-galactosylceramide are contained within one or more minicellular cells.

[0215] The formulation also optionally includes a bispecific ligand for targeting minicells to target cells. The minicells and ligands may be any of those described herein. Thus, the bispecific ligands of the present invention can bind to surface components of minicells and surface components of target mammalian cells.

[0216] The formulations of the present invention comprising mini-cells, drugs, and optionally bispecific ligands (i.e., formulations comprising such mini-cells, drugs, and ligands having other components that do not excessively interfere with the drug or drug delivery quality of the composition) can be formulated in a conventional manner using one or more pharmaceutically acceptable carriers or excipients.

[0217] The formulations or compositions of this disclosure may be presented in unit dosage form, for example, in ampoules or vials, or in multi-dose containers, with or without added preservatives. The formulations may be solutions, suspensions, or emulsions in an oily or aqueous vehicle and may comprise the formulation agent (e.g., suspenders, stabilizers, and / or dispersants). Suitable solutions are isotonic with the recipient's blood and are exemplified by saline, Ringer's solution, and dextrose solution. Alternatively, the formulation may be in lyophilized powder form for reconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water or saline. The formulations may also be in depot formulation form. Such long-acting formulations may be administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. In some embodiments, administration includes enteral or parenteral administration. In some embodiments, administration includes administration selected from oral, buccal, sublingual, intranasal, rectal, vaginal, intravenous, intramuscular, and subcutaneous injection.

[0218] In some embodiments, a mini-cell-containing composition is provided that contains a therapeutically effective amount of an antitumor agent. The “therapeutically effective” amount of the antitumor agent is, in accordance with this disclosure, the dose of the agent in question, such as siRNA or a hypercytotoxic agent, that elicits a pharmacological response when administered to a subject.

[0219] Accordingly, in the context of this disclosure, the therapeutically effective dose may be measured by the prevention or improvement of a tumor or tumor symptoms in either an animal model or a human subject when mini-cells containing the therapeutic payload are administered, as further described below. The amount that demonstrates a “therapeutically effective dose” in a given example (e.g., a particular subject) may not be effective for 100% of subjects treated similarly for a tumor, even if such a dosage is considered a “therapeuticly effective dose” by a skilled practitioner. The appropriate dose in this regard also varies, for example, as a function of the type, stage, and severity of the tumor.

[0220] When "therapeutically effective" is used to refer to the number of minicells in a pharmaceutical composition, that number can be determined based on which antitumor agent is packaged in the minicells and the potency of that agent in treating the tumor. In this regard, the therapeutic effect can be measured using clinical or pathological parameters such as tumor mass. Thus, a decrease in tumor mass or a decreased increase can be used to measure the therapeutic effect.

[0221] A. Route of Administration The formulations of the present invention can be administered either locally or systemically, via various routes and to various sites within a mammalian body to achieve the desired therapeutic effect. Delivery can be achieved, for example, by oral administration, by application of the formulation to a body cavity, by inhalation or ventilation, or by parenteral, intramuscular, intravenous, intraportal, intrahepatic, intraperitoneal, subcutaneous, intratumoral, or intradermal administration. The mode and site of administration depend on the location of the target cells. For example, tumor metastases can be treated more efficiently via intravenous delivery of the target minicells. Primary ovarian cancer can be treated via intraperitoneal delivery of the target minicells. Combinations of routes can also be used. For example, in metastatic bladder cancer, the cytotoxic drug-loaded and receptor-targeted minicells can be administered intravesically and intravenously, and the immunostimulant-packaged (receptor-targeted or non-targeted) minicells can be administered intravenously together with the targeted drug-packaged minicells. In situ administration of the targeted, drug-packaged minicells can target bladder surface-exposed tumors, while a complete combination of intravenously administered minicells can target tissue-localized tumors and can also induce an antitumor immune response.

[0222] B. Purity The minicells of the present invention are substantially free of contaminating parental bacterial cells. Thus, minicell-containing formulations preferably contain less than about 1 contaminating parental bacterial cell per 10 7 minicells, less than about 1 contaminating parental bacterial cell per 10 8 minicells, less than about 1 contaminating parental bacterial cell per 10 9 minicells, less than about 1 contaminating parental bacterial cell per 10 10 minicells, or less than about 1 contaminating parental bacterial cell per 10 11Each minicell contains less than approximately one contaminating parental bacterial cell.

[0223] Methods for purifying minicells are well known in the art and are described in PCT / IB02 / 04632. One such method combines cross-flow filtration (feed flow rate parallel to the membrane surface; Forbes, 1987) and dead-end filtration (feed flow rate perpendicular to the membrane surface). If necessary, differential centrifugation at low centrifugal force can be performed before the filtration combination to remove some portion of the bacterial cells and thereby concentrate the supernatant for the minicells.

[0224] Another purification method involves density gradient centrifugation in a biocompatible medium. After centrifugation, the mini-cell band is collected from the gradient, and optionally, the mini-cells are subjected to further rounds of density gradient centrifugation to maximize purity. This method may further include a preliminary step of performing differential centrifugation on the mini-cell-containing sample. When performed at low centrifugal force, differential centrifugation removes some of the parent bacterial cells, thereby concentrating the supernatant for the mini-cells.

[0225] A particularly effective purification method utilizes bacterial filamentation to increase the purity of minicells. Therefore, a minicell purification method may include (a) subjecting a sample containing minicells to conditions that induce the parent bacterial cells to take on a filamentous morphology, and (b) filtering the sample to obtain a purified minicell preparation.

[0226] It is also possible to combine known mini-cell purification methods. One very effective combination of methods is as follows: Step A: Differentiation centrifugation of mini-cell-producing bacterial cell cultures. This step can be performed at 2,000g for approximately 20 minutes, removing most of the parent bacterial cells while leaving the mini-cells in the supernatant; Step B: Density gradient centrifugation using an isotonic and non-toxic density gradient medium. This step separates minicells from many contaminants, including parent bacterial cells, while minimizing minicell loss. Preferably, this step is repeated within the purification method; Step C: Cross-flow filtration through a 0.45 μm filter to further reduce contamination by parent bacterial cells; Step D: Stress-induced filament formation in the remaining parent bacterial cells. This can be achieved by exposing the mini-cell suspension to one of several stress-inducing environmental conditions; Process E: Antibiotic treatment to kill parent bacterial cells; Step F: Cross-flow filtration to remove small contaminants such as membrane blebs, membrane fragments, bacterial debris, nucleic acids, and culture medium components, and to concentrate the mini-cells. A 0.2 μm filtration can be used to separate the mini-cells from small contaminants, and a 0.1 μm filtration can be used to concentrate the mini-cells; Process G: Dead-end filtration to remove filamentous dead bacterial cells. A 0.45 μm filtration can be used for the process; Step H: Removal of endotoxins from mini-cell preparations. Anti-lipid A coated magnetic beads can be used in this step.

[0227] In general, the formulations disclosed herein may be used in appropriate doses determined by routine testing to obtain optimal physiological effects while minimizing any potential toxicity. The method of administration may be selected depending on various factors, including the patient's age, weight, sex, medical condition; the severity of the condition being treated; the route of administration; and the patient's renal and hepatic function.

[0228] Achieving optimal precision in mini-cell and drug concentrations within the range that produces maximum efficacy with minimal side effects may require regimens based on mini-cell dynamics and drug availability to target sites and target cells. When determining the optimal concentration of a therapeutic regimen, the distribution, equilibrium, and excretion of mini-cells or drugs may be considered. When used in combination, the doses of mini-cells and drugs may be adjusted to achieve the desired effect.

[0229] Furthermore, the dosage of the formulation can be optimized using a pharmacokinetic / pharmacodynamic modeling system. For example, one or more drug regimens may be selected, and a pharmacokinetic / pharmacodynamic model may be used to determine the pharmacokinetic / pharmacodynamic profiles of one or more drug regimens. Then, based on the specific pharmacokinetic / pharmacodynamic profile, one of the drug regimens may be selected for administration that achieves the desired pharmacokinetic / pharmacodynamic response. See, for example, WO 00 / 67776.

[0230] Specifically, the formulation can be administered at least once a week over several weeks. In one embodiment, the formulation is administered at least once a week over several weeks to several months.

[0231] More specifically, the formulation can be administered at least once a day for approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. Alternatively, the formulation may be administered approximately once a day, or every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days or more.

[0232] Alternatively, the formulation may be administered approximately once a week, or approximately once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks or longer. Alternatively, the formulation may be administered at least once a week for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks or longer.

[0233] Alternatively, the formulation may be administered approximately twice a week for approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks or longer. Alternatively, the formulation may be administered at least once a week for approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks or longer.

[0234] Alternatively, the formulation can be administered approximately once a month, or approximately once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer.

[0235] The drug may be administered once a day, or the total daily dose may be divided and administered two, three, or four times a day.

[0236] In the method of administering mini-cells before the drug, the drug can be administered at any point in time, from minutes to hours after the mini-cell administration. Alternatively, the drug can be administered at any point in time, from hours to days, or possibly weeks to months, after the mini-cells.

[0237] More specifically, mini-cells may be administered at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours before drug administration. Furthermore, mini-cells may be administered at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days before drug administration. In yet another embodiment, mini-cells may be administered at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 weeks or more before the drug. In yet another embodiment, mini-cells may be administered at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 months before the drug.

[0238] In another embodiment, the mini-cells are administered after the drug. The administration of the mini-cells may occur at any point from a few minutes to a few hours after the administration of the drug. Alternatively, the mini-cells may be administered at any point from a few hours to a few days, or possibly from a few weeks to a few months, after the drug.

[0239] VII. Methods of treating cancer The compositions described herein may be used to treat subjects suffering from cancer. The methods disclosed herein involve administering an effective amount of the composition according to the present invention to a subject, comprising at least one antitumor agent, an interferon type I agonist, an interferon type II agonist, or a combination of an interferon type I agonist and an interferon type II agonist. The antineoplastic agent, interferon type I agonist, interferon type II agonist, or a combination of an interferon type I agonist and an interferon type II agonist consists of one or more mini-cells.

[0240] In another context, a composition used to treat a subject suffering from cancer further comprises a pharmaceutically acceptable carrier.

[0241] In another context, the methods disclosed herein are useful for treating subjects suffering from cancer, where the subjects are humans, non-human primates, dogs, cats, cattle, sheep, horses, rabbits, mice, or rats.

[0242] In another context, the methods disclosed herein are useful for treating cancerous diseases. In some embodiments, cancer includes lung cancer, breast cancer, brain cancer, liver cancer, colon cancer, pancreatic cancer, or bladder cancer.

[0243] In some embodiments, cancers include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphomas, anal cancer, appendiceal cancer; astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bladder cancer, brainstem glioma, brain tumors (brainstem glioma, atypical teratoid / rhabdoid tumor of the central nervous system, germ cell tumor of the central nervous system, astrocytoma, craniopharyngioma, ependymocyte, ependymocyte, medulloblastoma, medullary epithelioma, pineal parenchymal tumor, supratentorial primitive neuroectodermal tumor and pinealoblastoma, breast cancer, bronchial tumor, Burkitt Trimphoma, cancer of unknown primary site, carcinoid tumor; cancer of unknown major site, atypical teratoma / rhabdoid tumor of the central nervous system, cervical cancer, childhood cancer, chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic spinal proliferative disorder, colorectal cancer, colon cancer, craniopharyngioma, cutaneous T-cell lymphoma, endocrine islet cell tumor, endometrial cancer, ependymoma, esophageal cancer, sensory neuroblastoma, Ewing's sarcoma; extracranial embryo, extragonadal germ cell tumor, Extrahepatic germ cell tumors, gastric (stomach) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gliomas, hairy cell leukemia, head and neck cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors; Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer; lip cancer, liver cancer, malignant fibrous histiocytoma, bone cancer, medulloblastoma, medullary epithelioma; melanoma, Merkel cell carcinoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of unknown primary origin for the neck, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasmacytic neoplasm, mycosis fungoides, myelodysplastic syndrome, spinal proliferative neoplasms; nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-melanoma, skin cancer, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma, other brain and spinal cord tumors, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell carcinoma Low-grade ovarian tumors, pancreatic cancer, papillomatosis; sinus cancer;This includes parathyroid cancer, pelvic cancer, penile cancer, pharyngeal cancer, pineal gland tumor, pineoblastoma, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuroblastoma, primary central nervous system (CNS) lymphoma, primary hepatocellular carcinoma, prostate cancer, rectal cancer, renal cell carcinoma, renal cell carcinoma, airway cancer, rhabdomyosarcoma, salivary gland cancer, Sézary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell neck cancer, gastric cancer, supratentorial primitive neuroectodermal tumor, T-cell carcinoma, testicular cancer, pharyngeal cancer, thymic carcinoma, thymoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, trophoblast tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, or Wilms' tumor.

[0244] In some embodiments, brain cancer or brain tumor is selected from the group consisting of brainstem glioma, atypical teratomatoid / rhabdoid tumor of the central nervous system, germ cell tumor of the central nervous system, astrocytoma, craniopharyngioma, ependymoblastoma, ependymodium, medulloblastoma, medullary epithelioma, pineal parenchymal tumor of intermediate differentiation, supratentorial undifferentiated primitive neuroectodermal tumor, and pineoblastoma.

[0245] VIII.Definitions Unless otherwise defined, the technical and scientific terms used herein have meanings that are generally understood by those skilled in the art. The materials, reagents, etc., referenced in the following description and examples are available from commercial sources unless otherwise noted.

[0246] For convenience, the meanings of certain terms and phrases used in this specification, the examples, and the appended claims are provided below. Other terms and phrases are defined throughout this specification.

[0247] The singular forms "a," "an," and "the" include multiple quotations unless the context clearly indicates otherwise.

[0248] The term “about” means that the number understood is not limited to the exact number set forth herein, and is intended to refer to a number substantially around the enumerated number without departing from the scope of the invention. Where used herein, “about” will be understood by those skilled in the art and will vary to some extent depending on the context in which it is used. Where there is a use of a term that is not obvious to those skilled in the art given the context in which it is used, “about” will mean up to ±10% of the particular term.

[0249] As used interchangeably herein, “individual,” “subject,” “host,” and “patient” refer to any mammalian subject to which diagnosis, treatment, or therapy is desired. In one preferred embodiment, the individual, subject, host, or patient is a human. Other subjects include, but are not limited to, cattle, horses, dogs, cats, guinea pigs, rabbits, rats, primates, and mice.

[0250] In this specification, the terms “cancer,” “neoplasm,” “tumor,” “malignant tumor,” and “carcinoma” as used interchangeably refer to cells or tissues exhibiting an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. There are several main types of cancer. Cancer is cancer that originates in the tissues covering the skin or internal organs. Sarcoma is cancer that originates in the bone, cartilage, fat, muscle, blood vessels, and other connective or supporting tissues. Leukemia is cancer that originates in hematopoietic tissues such as bone marrow, in which a large number of abnormal blood cells are produced and enter the bloodstream. Lymphoma and multiple myeloma are cancers that originate in the cells of the immune system. Central nervous system cancers are cancers that originate in the tissues of the brain and spinal cord. The methods and compositions of the present invention are particularly applicable to precancerous, malignant, premetastatic, metastatic, and nonmetastatic cells.

[0251] The terms “treatment,” “to treat,” and “to cure” refer to obtaining desired pharmacological and / or physiological effects in a patient with a tumor. These effects may be prophylactic, in that they completely or partially prevent the tumor or its symptoms, and / or therapeutic, in that they partially or completely stabilize or cure the tumor and / or adverse effects caused by the tumor. Treatment encompasses any treatment of tumors in mammals, particularly humans. The desired effect is a tumor response, which can be measured in particular as a reduction in tumor mass or inhibition of tumor mass growth. In addition to the tumor response, other desired effects include increased overall survival, progression-free survival, or time to tumor recurrence, or The reduction of adverse effects can also be used clinically as a desired therapeutic effect.

[0252] As used herein, the term “administer” includes administering directly to another, self-administering, and prescribing or directing the administration of any drug as disclosed herein.

[0253] Where used herein, the terms “effective dose” and “therapeutic effective dose” mean, respectively, the amount or plasma concentration of the activator administered to a subject that provides the specific pharmacological effect of the activator in a subject requiring such treatment. It is emphasized that an effective dose of an activator is not always effective in treating the conditions / diseases described herein, even if such a dose is considered effective by those skilled in the art.

[0254] As used herein, the term “activator” refers to any small molecule drug, protein, functional nucleic acid, or polynucleotide that is useful for treating a subject and encodes a functional nucleic acid. An activator may be any of the antitumor agents, functional acids, interferon type I agonists, or type II agonists described herein.

[0255] The term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions, and / or dosage forms that are within the bounds of sound medical judgment, suitable for in vivo use without excessive toxicity, irritation, allergic reactions, or other problems or complications, commensurate with an appropriate benefit-risk ratio.

[0256] The term "endocytosis" encompasses categories including (1) phagocytosis and (2) pinocytosis itself, (2a) macropinocytosis that does not require receptor binding, as well as (2b) clathrin-mediated endocytosis, (2c) caveolae-mediated endocytosis, and (2d) clathrin / caveolae-independent endocytosis, all of which tend to access late endosomal / lysosomal pathways. The inventors discovered that interaction between ligands on minicells and mammalian cell surface receptors activates specific endocytic pathways, involving receptor-mediated endocytosis (rME) to late endosomal / lysosomal compartments. Thanks to such endocytic pathways, the inventors further discovered that minicells can release their payloads into the cytoplasm of target mammalian cells. If the payload is an encoding nucleic acid, the nucleic acid is not only completely degraded in the late endosomal / lysosomal compartment but is also expressed in the target mammalian cells.

[0257] The following examples are not limiting and are merely illustrative, providing a more complete understanding of the present invention. The examples demonstrate that drug-resistant tumor cells can be effectively treated in vivo by (1) administration of targeted recombinant mini-cells carrying RNAi sequences designed to reduce or eliminate the expression of genes encoding drug resistance, and (2) administration of targeted, drug-packaged mini-cells carrying drugs to which cancer cells are made sensitive.

[0258] The following embodiments are provided to illustrate the present invention. However, it should be understood that the present invention should not be limited to the specific conditions or details described in these embodiments. Throughout this specification, any references to publicly available documents, including U.S. patents, are incorporated by reference. [Examples]

[0259] Example 1: Preclinical trial using mice This example demonstrates that mini-cells (EDVs) provide efficient delivery of chemotherapeutic drugs and inhibit tumor growth in a mouse xenograft model. EDV targeting technology has been tested in mouse xenograft models of various cancers, including colon cancer, breast cancer, ovarian cancer, leukemia, lung cancer, mesothelioma, and uterine cancer. Furthermore, various targeting moieties were utilized as EDVs were targeted to tumor cell surface receptors, including EGFR, human epidermal growth factor receptor 2 (HER2), mesothelin (MSLN), and CD33. Finally, the targeted EDVs tested included a wide variety of cytotoxic agents, including doxorubicin, paclitaxel, monastrole, irinotecan, hypercytotoxic agents such as PNU-159682, and novel thymidylate synthase inhibitors (OSIs). PNU-159682 is an anthracycline analog that is thousands of hours more cytotoxic than doxorubicin. OSI7904 is a benzoquinazoline folate analog with antitumor activity. As a thymidylate synthase inhibitor, OSI-7904 non-competitively binds to thymidylate synthase, thereby inhibiting thymine nucleotide synthesis and DNA replication.

[0260] In all cases, tumor stabilization or regression was observed when using specifically targeted EDVs and drug-packaged EDVs, even in large tumors (>1000 mm). 3 Tumors were also observed (see Table 5). Control mice treated with the free drug (not packaged in an EDV) showed expected toxicity of phlebitis, ultimately leading to weight loss and death. These toxicities were not observed with repeated administration of the EDV-packaged drug.

[0261] Surprisingly, the concentration of cytotoxic drugs delivered via EDV was 8,000 times lower than that delivered systemically, yet the concentration of cytotoxic drugs delivered by EDV was sufficient to maximize antitumor efficacy.

[0262] [Table 5-1]

[0263] [Table 5-2] Example 2: Preclinical trial using dogs This example demonstrated that drug-loaded mini-cells (EDVs) can be safely administered to dogs.

[0264] In a canine toxicity study using a series of dogs with endogenous tumors (n=41), targeted EDV and drug-packaged EDV could be safely administered to a single animal up to 98 times over a period of more than two years. Some dogs experienced mild temperature increases (up to 1) after administration, accompanied by elevated interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor α (TNF-α), but these were not associated with serious adverse events.

[0265] Furthermore, preclinical studies in dogs have shown that CD3-targeted doxorubicin-packaged EDV can inhibit tumor growth. Two dogs with advanced non-Hodgkin lymphoma were treated with CD3-targeted, doxorubicin-packaged EDV, and both showed significant tumor regression, as evidenced by a very significant reduction in lymph node size. MacDiarmid et al., 2007b. More than 60% of dogs with angiosarcoma showed tumor stabilization or regression when treated with CD33-targeted EDV and doxorubicin-packaged EDV.

[0266] In another study of dogs with late-stage brain tumors (n = 17), animals were treated with EGFR-targeted EDV loaded with doxorubicin. MacDiarmid et al., 2016. One dog (11 dogs received more than 20 doses) received up to 98 repeated doses of 1×10 10 of EGFR mini-cells Dox administered, and no signs of toxicity were observed. The objective response rate was 23.53% (4 out of 17 dogs; 95% confidence interval, 6.8 - 49.8%). Of the 15 dogs evaluated for tumor response, 2 showed a complete response (CR) to treatment, 2 showed a partial response (PR) to treatment (a 90 - 98.95% reduction in tumor volume), 10 showed stable disease (SD), and 1 showed progressive disease (PD).

[0267] In two dogs with brain cancer 123 In the biodistribution test using iodine radiolabeled EGFR-targeted EDV, it was shown that the targeted EDV was localized in the brain tumor, suggesting that the EDV could enter the peritumoral area by bypassing the blood-brain barrier. Localization in the gastrointestinal tract suggests excretion via feces.

[0268] Example 3: Preclinical trial using monkeys This example demonstrated that the mini-cell (EDV) technology was well tolerated by monkeys. Three rhesus monkey trials were conducted to evaluate the toxicity of empty EDV (up to 2×10 10 per dose), EGFR-targeted doxorubicin-loaded EDV (up to 2×10 10 per dose), and EGFR-targeted paclitaxel-loaded EDV (up to 1×10 11 per dose). EDV was administered to monkeys once a week for 5 weeks (a 35-day repeated-dose study).

[0269] As observed in dogs, there was a transient temperature spike (up to 1), accompanied by a post-administration increase in IL-6. The inflammatory marker C-reactive protein also increased during these periods, but no significant toxicity or adverse events were observed. A mild increase in TNF-α was observed during treatment with EDV alone, which was loaded with EGFR-targeted doxorubicin. The EDV technology was safely administered to a total of 72 monkeys.

[0270] Example 4: Inflammatory and immune responses in preclinical trials These examples demonstrate that only minor inflammatory responses were observed in preclinical mouse studies (Example 1), preclinical dog studies (Example 2), and preclinical monkey studies (Example 3). These responses resolved rapidly, as did the response 4 hours after administration. No significant changes were observed in other hematological and biochemical parameters. Appearance and behavior remained healthy throughout the administration period.

[0271] Antibody formation was evaluated in canine and monkey studies. The immune responses considered in response to targeted EDV administration were as follows: • The serum antibody response to the EDV surface-exposed immunodominant antigen is the O-polysaccharide component of LPS (IgG or IgM reaction). The anti-O-polysaccharide antibody response is T cell-independent and does not show a memory response. • Serum antibody response to mouse IgG monoclonal antibodies used in the construction of BsAbs to target EDV to tumor cell surface receptors (e.g., EGFR).

[0272] In dogs with angiosarcoma or brain tumors, serum anti-LPS IgG titers increased to an average of approximately 10,000 by 3-4 doses of targeted EDV and doxorubicin-packaged EDV. No further increases in titer were observed with subsequent doses. In three studies using monkeys (healthy animals), anti-LPS IgG titers generally showed a mild increase in the first 2-3 doses before reaching a plateau. Since the expected anti-O-polysaccharide antibody titers for vaccines against Gram-negative bacteria are generally in the millions, the response is primarily dose-dependent, and the highest dose level... EGFR EDVDox Then it rose to its highest potency, just over 100.

[0273] The anti-LPSIgM titer response in the monkey study was also mild. EGFR EDV Dox With treatment using [specific method], the titer rose slightly above 100, while with treatment using non-targeted EDV, it rose to 1,000. After doses 3-4, the titer did not increase further.

[0274] The immunogenic response to the monoclonal antibody used to construct the BsAb was also measured in studies using monkeys, and a mild titer increase in response to the EGFR antibody was observed in monkeys administered with EGFR-targeted EDV (mouse IgG). These results suggest that administration of drug-packaged EDV targeting BsAb may not induce a significant anti-LPS immune response that could interfere with the efficacy of subsequent administrations. This suggests that repeated administration is likely to be a viable treatment option, and is therefore particularly relevant to cancer patients who are likely to have impaired immune systems.

[0275] Example 5: Erbitux target in advanced solid tumors, paclitaxel package EDV ( EGFR(Erb) EDV Pac Phase 1 clinical trial to evaluate ) This example demonstrates promising results using mini-cells (EDVs) to deliver paclitaxel (Taxol®) to advanced solid tumors.

[0276] This trial demonstrated that elbitux (cetuximab)-targeted paclitaxel-packaged EDV was well-tolerated in human patients. However, a significant number of patients had to discontinue the trial due to adverse events or dose-limiting toxicities. Furthermore, while this treatment strategy achieved disease stabilization, none of the patients in this study demonstrated a partial or complete response to the treatment. The results of this trial were published by Solomon et al. in 2015.

[0277] The first human trial involved EGFR-targeted paclitaxel loading EDV ( EGFR(Erb) EDV Pac Designed as a dose-escalation study to determine the safety, tolerability, and maximum tolerated dose or recommended Phase 2 dose of EDV. Note that the antibody used to target EGFR with EDV is based on the Erbitux sequence. Other purposes include intravenous administration. EGFR(Erb) EDV Pac The objectives were to evaluate the immune and inflammatory responses to the disease, and to assess the response to treatment according to RECIST criteria.

[0278] This trial was conducted at three oncology clinics in Melbourne, Australia, and was registered with the Australia and New Zealand Clinical Trials Registry (number ACTRN12609000672257). The final trial report is available, and the trial was published in Solomon et al. in 2015.

[0279] The patients were adults aged 18 or older with advanced epithelial malignancies for whom standard curative treatment was unavailable.

[0280] EGFR(Erb) EDV Pac The drug was administered weekly as a 20-minute intravenous infusion in a 5-week treatment cycle. A treatment-free week was then provided during which patients underwent radiological evaluation of the tumor by MRI, CT, or FDG-PET. Patients could continue with further cycles of treatment if the tumor was stable, responding to treatment, or providing clinical benefit, and no dose-limiting toxicities (DLTs) or other adverse events (AEs) requiring discontinuation of treatment were experienced.

[0281] The single dose containing paclitaxel is 1 × 10⁻⁶ 8 , 1 x 10 9 , 3 x 10 9 , 1 x 10 10 , 1.5×10 10 , 2×10 10 , and 5×10 10A total of 236 doses were provided across seven stages of EGFR (Erb)-targeted EDV. Of the 28 patients, 22 completed at least one cycle of treatment (5 doses per week), and one patient received 45 doses over 9 cycles (approximately 14 months). No treatment-related deaths were reported. The maximum tolerated dose was 1 × 10⁶. 10 EGFR(Erb) EDV Pac It was identified that significant toxicity was observed at doses above this level, particularly in the form of prolonged fever and transient elevation of liver function tests (LFT). The treatment was generally well-tolerated, and acceptable safety findings were observed in the indicated population.

[0282] A summary of the clinical trials and findings is shown in Table 6 below.

[0283] [Table 6] The main adverse events that were deemed "probably related" to the investigational drug were low-grade fever and chills, which occurred in up to 60% of patients (severity grade 1-2). In most patients, a mild, transient increase in cytokines IL-6, IL-8, and IL-10 was observed 4 hours after administration. These levels returned to pre-administration levels within 24 hours after administration. This is consistent with a mild inflammatory response to the treatment.

[0284] Serious treatment-related adverse events were observed in 5 cases, and dose-limiting toxicities or adverse events requiring dose reduction were observed in 8 cases. These events are summarized in Table 7 and described below.

[0285] [Table 7] 1 x 10 8In the treatment population, elevated LFT levels were observed, meeting the DLT criteria. This event was deemed not serious, and the definition of DLT was revised for subsequent dose levels. Four patients at dose levels above the MTD showed elevated LFT levels that did not meet the revised DLT criteria; however, these patients also developed serious treatment-related clinical symptoms (fever, chills, nausea, vomiting), and the safety committee decided to reduce the dose.

[0286] 1 x 10 8 EGFR(Erb) EDV Pac Three patients were enrolled in the initial dose, and in one patient, a grade 3 decrease in phosphate concentration was observed after 3 out of 5 doses. In both cases, levels returned to normal within 24 hours post-administration, and no clinical symptoms were observed. In another patient, asymptomatic grade 3 elevations of the liver enzymes alanine transaminase (ALT) and aspartate transaminase (AST) were observed 4 hours after the 3rd dose, but these levels returned to baseline by the next dose. These events met the original dose-limiting toxicity (DLT) criteria in the clinical trial protocol, and the ongoing patient dose was 5 × 10⁶ 7 EGFR(Erb) EDV Pac The number of doses decreased, and one patient received a total of 45 doses during the study period. In addition, three patients received 1 × 10 8 EGFR(Erb) EDV Pac The subjects were recruited to participate in the study, and no drug-related adverse events were reported in these three individuals. The definition of DLT in the clinical trial protocol was revised to exclude biochemical abnormalities that resolved within 7 days of administration.

[0287] Two days after the second dose, 1 x 10 9 EGFR(Erb) EDV PacThe dose was increased, but one patient experienced severe joint pain. This was accompanied by a significant increase in the cytokine interferon-alpha, suggesting a viral infection. The patient was hospitalized for observation and later diagnosed with reactive arthritis, which was classified as a serious adverse event (SAE). After some consideration, the safety committee decided to proceed cautiously and defined this event as a DLT. Thus, this group was expanded to six patients. No other patients in this trial experienced a similar event. At the completion of the first cycle, safety data supported further dose increases.

[0288] The group of 3 patients was divided into the following doses: 3 × 10 9 EGFR(Erb) EDV Pac One patient discontinued treatment after receiving only one dose due to a rapidly progressing disease and subsequently died as a result of the disease. A fourth patient was recruited at the same dose level. All patients tolerated this dose without major concerns, and safety data supported further dose increases. One patient in this group achieved disease stabilization after the first cycle and completed two cycles with a total of 10 doses. One patient received only 4 out of 5 doses due to disease infiltration into the bone marrow.

[0289] A group of three patients, the next dose level, 1 × 10 10 EGFR(Erb) EDV Pac Supplementation: Patients tolerated this dose level without any significant concerns, and at the completion of cycle 1, safety data supported further dose increases. Two out of three patients achieved disease stabilization and completed 3 and 5 cycles of 15 and 25 doses, respectively.

[0290] The two patients were mobilized to the following dose levels: 5 × 10 10 EGFR(Erb) EDV PacBoth patients received a single dose at this level and experienced an elevation of grade 3–4 in liver enzymes ALT and AST. Although these changes were transient and did not meet the revised DLT requirements of the clinical trial protocol, these patients experienced other adverse events such as fever, malaise, and nausea (one patient was hospitalized due to a serious adverse event), so these patients were not given 1 × 10⁶ doses. 10 EGFR(Erb) EDV Pac The dosage was reduced. These patients also experienced significant elevations in the inflammatory markers IL-6, IL-8, IL-10, and TNF-α. One of these two patients continued treatment to achieve disease stabilization and completed two full cycles after the dose reduction.

[0291] To identify the maximum tolerated dose (MTD), one patient was given 2 × 10⁶ doses. 10 EGFR(Erb) EDV Pac The patient was supplemented to an intermediate dose level, receiving a single dose at this level, and similarly experienced transient grade 3-4 elevations in ALT and AST, accompanied by fever, malaise, nausea and vomiting, as well as elevated inflammatory markers. Clinically significant elevations of lactate dehydrogenase (LDH) and gamma-glutamyltransferase (GGT) were also observed. Again, although these parameters did not meet the revised DLT criteria in the protocol, the elevation of liver enzymes was considered a serious adverse event, 1 × 10⁶ 10 EGFR(Erb) EDV Pac It was determined that reducing the dosage was clinically appropriate. This subject achieved stable disease and completed 4 cycles of 19 doses.

[0292] In a further attempt to identify MTD, three patients were divided into 1.5 × 10⁶ 10 EGFR(Erb) EDV Pac The intermediate dose was supplemented, but one of these patients received the initial dose without any adverse reactions but did not continue treatment due to a rapidly progressing illness. Another patient developed grade 3 hypotension, which was considered a dose limiting factor for serious adverse events, so the dose was reduced to 5 × 109 EGFR(Erb) EDV Pac The dose was reduced to 1 × 10⁶. The last patient in this group experienced a grade 3 increase in AST with treatment-related symptoms (fever, rigidity, vomiting) and elevated inflammatory markers after the initial dose, and the subsequent dose was reduced to 1 × 10⁶. 10 EGFR(Erb) EDV Pac I lost weight.

[0293] Therefore, EGFR(Erb) EDV Pac The MTD for this is 1 × 10 10 This was the dose level, and it was concluded that three more patients were mobilized to this dose level. One patient was discontinued after one dose due to suspected cytokine release syndrome. The patient had a pre-existing cough and occasionally experienced episodes of syncope due to a supraclavicular mass compressing the brachiocephalic vein. Between 2 and 4 hours after administration, the patient developed a fever, began coughing, and experienced 3 to 4 episodes of syncope witnessed by staff. The patient was hospitalized for observation, and although no increase in IFNγ was detected, they were diagnosed with cytokine release syndrome. The patient experienced elevated IL-6, IL-8, and IL-10 levels, similar to those observed in other patients at this dose level and above, but this is more likely to represent an inflammatory response to the bacterial component of the formulation rather than typical cytokine release syndrome. Although this event was not a serious adverse event, the safety committee determined that it did not meet the DLT criteria due to the involvement of a pre-existing condition. The remaining two patients completed the first course of treatment without major concern.

[0294] No deaths due to adverse events occurred during the study treatment. Overall, this treatment was well-tolerated, and there are no particular safety concerns for the intended target population.

[0295] Antibodies against Salmonella typhimurium (anti-LPS) and Erbitux were negative in all cases at screening. All patients except one were EGFR(Erb) EDV PacPositive Salmonella antibody titers were developed after treatment with (27 / 28 = 96%). Anti-LPS antibody titers peaked by administration 3 (Day 15) and were maintained at that level despite repeated administration. No patients showed positive Erbitux antibody titers.

[0296] Tumor response was evaluated in 22 patients who completed Cycle 1. The best response observed was stable disease (SD) (no patients achieved partial or complete response according to RECIST criteria). At the end of Cycle 1, 10 out of 22 patients (45.5%) were stable, and 12 out of 22 patients (55.5%) showed disease progression (PD). One patient at dose level 1 completed 9 cycles of complete treatment, and from the end of the 4th cycle, the disease alternated between stable and progressive states. She had the longest time to the onset of PD, at 197 days.

[0297] In conclusion, the initial trial in men demonstrated good tolerability of EDV packaged with paclitaxel, with 45.5% of patients showing disease stabilization. This example also demonstrates the desirability of improving cancer treatment strategies to improve survival and disease response.

[0298] Example 6: EGFR-targeted doxorubicin package EDV in recurrent glioblastoma EGFR(V) EDV Dox Phase 1 clinical trial to evaluate ) This example demonstrates that treatment with EGFR-targeted doxorubicin-packaged mini-cells (EDVs) was well-tolerated in patients with relapsed glioblastoma. While 50% of patients showed disease stabilization, no patients experienced partial or complete responses. Whittle et al., J. Clin. Neurosci., 22(12):1889-1894(2015).

[0299] The relapsed glioblastoma trial involved EGFR-targeted doxorubicin loading EDV ( V EDV DoxDesigned as a dose-escalation study to determine the safety, tolerability, and maximum tolerated dose or recommended Phase 2 dose of EDV. Note that the antibody used to target EGFR with EDV is the same antibody (Vectivix-based sequence) as for the current protocol. Other purposes include intravenous administration. V EDV Dox The objectives were to evaluate the immune and inflammatory responses to the disease, and to assess the response to treatment according to the response assessment criteria in neuro-oncology (RANO).

[0300] This study commenced on February 5, 2013, and concluded on June 26, 2014. It was conducted at four oncology clinics in Sydney and Melbourne, Australia, and was registered with the Australia-New Zealand Clinical Trials Registry (number ACTRN12613000297729). The final clinical trial report is available. This study is based on a draft list by Whittle et al., JClin. Neurosci., 22(12):1889-1894 (2015).

[0301] The patients were adults aged 18 years or older who had a pathologically confirmed, recurrent World Health Organization (WHO) Grade IV glioblastoma and had experienced disease recurrence or progression after receiving standard treatment (including maximally safe surgical resection, standard adjuvant radiotherapy / temozolomide, and maintenance temozolomide therapy).

[0302] V EDV Dox The drug was administered weekly as a 20-minute intravenous infusion in an 8-week treatment cycle. At the end of each cycle, patients underwent radiological evaluation of the tumor by magnetic resonance imaging (MRI). If the tumor was stable, responding to treatment, or providing clinical benefit from treatment, patients could continue with further cycles of treatment and did not experience any DLTs or other adverse events requiring discontinuation of treatment.

[0303] 2 x 10 per round 9 , 5×10 9, 8×10 9 V EDV Dox A total of 197 doses were administered in three stages. Of the 14 patients, 8 completed at least one cycle of treatment (8 doses at one week each), and one patient received 47 doses over approximately 6 cycles (about 12 months). No administration-related deaths were observed, and no patients experienced dose-limiting toxicity or other adverse events requiring discontinuation of treatment. A summary of the clinical trial and findings is shown in Table 8 below.

[0304] [Table 8] The main adverse events that were "probably related" to the investigational drug were mild fever, nausea, and rigidity (chills), observed in up to 50% of patients (generally grade 1-2 severity). In most patients, a mild, transient increase in cytokines IL-6, IL-8, IL-10, and TNF-14 was observed 3 hours after administration. These levels returned to pre-administration levels within 24 hours post-administration. This is consistent with a mild inflammatory response to the treatment.

[0305] Five patients experienced treatment-related adverse events of grade 3 or higher according to the National Cancer Institute's Common Terminology Criteria for Adverse Events (NCI-CTCAE). These events are summarized in Table 9 below and described in the following explanation.

[0306] [Table 9] Two cases of grade 3 or higher adverse events occurred and were judged to be serious. 5×10 9 One patient was hospitalized in the evening after the second dose of cycle 1 due to a serious adverse event: grade 3 malaise accompanied by grade 1 fever. This was not considered dose-limiting toxicity because the patient had tested positive for anti-product antibodies (antibodies against the Salmonella LPS component of EDV) at trial enrollment. The patient received two further treatments, and no recurrence of the event occurred.

[0307] 8×10 9 In another patient in the treatment group, a serious adverse event of grade 3 symptomatic hypotension occurred 4 hours after the first cycle of administration, requiring hospitalization for intravenous fluid replacement. This was not considered a DLT because it was likely due to an EColi urinary tract infection that the patient had been treated for. The patient subsequently received three additional doses of the investigational drug.

[0308] Grade 3 or higher adverse events occurred in three cases, but none were considered serious. In one case, grade 3 elevated liver enzymes were observed after partial administration. These were asymptomatic and transient, and returned to baseline between doses, so they were not considered dose-limiting toxicities or serious adverse events. Grade 3 hypophosphatemia was observed in two cases, and grade 4 lymphopenia in one case. These were also transient and did not require therapeutic intervention, so they were not considered dose-limiting toxicities or serious adverse events.

[0309] Overall, this treatment is well-tolerated, and there are no particular safety concerns for the intended target population.

[0310] To evaluate the immune response to EDV treatment, antibodies against Salmonella were assessed at screening. At screening, 13 out of 14 patients (93%) were negative for Salmonella antibodies. One patient, assigned to dose level 2, was positive at screening. An initial increase in antibody titer was observed in all patients by the third dose. Despite one patient receiving a total of 47 doses, titer was maintained without exceeding subsequent dose levels. No patients developed antibodies against Vectibix.

[0311] To evaluate efficacy, tumor reduction was assessed in eight patients who completed the first cycle. While no patients experienced complete or partial remission, 50% showed disease stability, and no patients experienced disease progression. One patient reported stable disease throughout the entire trial period and received nearly six cycles (approximately 12 months) of treatment.

[0312] Eight patients who completed at least one cycle of treatment were followed up for survival. All eight patients survived beyond the median background survival of 5–7 months, with a median overall survival (OS) of 15.1 months (range 9.1–>18.4). Four subjects were alive at the time of last contact and were censored at that point. Of these, two (14.3%) who completed at least one cycle of treatment survived for >18 months.

[0313] In conclusion, treatment of recurrent glioblastoma with EGFR-targeted doxorubicin packaged EDV showed promising results with few adverse events and disease stabilization in 50% of patients. However, improved treatment strategies are needed.

[0314] Example 7: MicroRNA-16 mimic in mesothelioma ( EGFR(V) EDV miRNA16a A Phase 1 clinical trial evaluating EGFR-targeted EDV, a package of EGFR-targeted EDV. In this example, EGFR-targeted EDV packaged with microRNA-16 showed a partial response in one of 16 mesothelioma patients whose efficacy was tested, while disease stabilization occurred in 62.5% of patients and disease progression in 31.3%. The trial data are published in van Zandwijk et al., Lancet Oncol., 18(10):1386-1396 (2017) and Kao et al., Am.J. Respir. Crit. Care Med., 191(12): 1467-1469 (2015). This treatment strategy was generally favorable.

[0315] In an open-label, multicenter, exploratory phase 1 trial involving patients with relapsed malignant pleural mesothelioma... V EDV miRNA16a The primary endpoint of the trial was evaluated. V EDV miRNA16a To establish the maximum tolerated dose and DLT, and to evaluate the effects of repeated administration, V EDV miRNA16aThe objective was to detect early signs of efficacy. Note that the antibody used to target EGFR with EDV was the same as the antibody in the current protocol (Vectivix-based sequence). The second endpoint of the trial was V EDV miRNA16a The objectives were to assess the quality of life of patients treated with the drug and to monitor changes in the overall condition and pulmonary function parameters of the Eastern Collaborative Oncology Group (ECOG) during treatment. Exploratory endpoints included evaluating changes in immune and cytokine markers during treatment.

[0316] To be eligible, patients must have a histological or cytological record of MPM with evidence of EGFR expression in tumor tissue. Patients were men and women aged 18 years or older with an ECOG performance status of 0 or 1 and a life expectancy of at least 3 months. Patients had to show disease progression during or after administration of a standard first or second-line treatment regimen and had to have adequate bone marrow, liver, and kidney function.

[0317] V EDV miRNA16a The drug was administered once or twice a week as a 20-minute intravenous infusion in an 8-week treatment cycle. At the end of each cycle, patients underwent radiographic evaluation of the tumor. Tumor reduction was assessed according to the RECIST (Revised Response Evaluation Criteria for Solid Tumors) standards. Disease progression was assessed using spirometry, FDG-PET scans, and CT scans.

[0318] This trial commenced on October 2, 2014, at three oncology clinics in Sydney, Australia, and concluded on November 24, 2016. The trial was registered with the Australia & New Zealand Clinical Trials Registry (number ACTRN 12614001248651) and Clinical Trials.gov (number NCT02369198). A total of 27 patients were recruited across five groups, with 26 patients receiving a total of 316 doses. V EDV miRNA16a(One subject died before receiving any treatment and was excluded from further analysis.) This study was published by Kao et al., 2015 and van Zandwijk et al., 2017.

[0319] The evaluated dosage was 5 × 10 9 2.5 x 10 once or twice a week 9 The drug was administered twice a week. To avoid an increased cytokine response, the dose was reduced to 1 × 10⁶. 9 The indication for gradually increasing the dose to the phase 1 equivalent dose was also evaluated. The indication for dexamethasone (dex) was also evaluated, and dex was gradually reduced in preparation for subsequent dose premedication. MTD was 5 × 10 once weekly. 9 V EDV miR16a It was identified as [the specified condition]. This treatment was generally successful, and acceptable safety findings were observed in the indicated population.

[0320] A summary of the clinical trials and findings is shown in Table 10 below.

[0321] [Table 10] Of the 24 patients, 16 completed at least one cycle of treatment, and 2 received a total of 40 or more doses (more than 5 full cycles of treatment). The best observed response was a partial response in one patient. This case is described below. V EDV miR16a The patients showed near-complete remission in response to treatment. Ten patients (62.5%) showed disease stabilization, while five patients (31.3%) showed disease progression. The median survival time was 36.5 weeks, or 8.4 months (range 9.3 to >119.6 weeks), with nine patients (60.0%) surviving for six months, of which five were well-survived more than 12 months after the start of treatment. See Figure 4.

[0322] Of particular note is one patient (patient #5 from group 1) who showed a dramatic clinical response at the end of the first cycle (see Kao et al., Am. J. Respir. Crit. Care Med., 191(12):467-1469 (2015)). At the end of the 8-week period, a “complete” metabolic response was revealed on PET-CT scan, and a partial response was observed on chest CT scan, confirmed at 4 weeks. The objective imaging response was accompanied by significant improvement in respiratory function parameters.

[0323] The most common adverse events associated with administration were infusion-related reactions (96.2%), such as chills, rigidity, fever, tachycardia, or hypertension (including night sweats). Most of these were mild to moderate in severity. Non-cardiac chest pain at the tumor site was also experienced by 14 patients (53.8%) after infusion. These reactions were addressed by revisions to the clinical trial protocol. The revised protocol included a modified dose escalation schedule for all subjects in the first cycle, resulting in fewer and less severe infusion-related reactions. Clinical laboratory tests showed transient increases in inflammatory cytokines and neutrophils, as well as in the majority of patients. V EDV miRNA16a A transient decrease in lymphocytes immediately after injection was observed, consistent with a mild inflammatory response.

[0324] Eight patients reported nine serious adverse events, which were considered to be at least potentially related to the treatment. Non-cardiac chest pain and infusion-related reactions occurred in two patients each. Dose-limiting toxicity occurred in three patients, and two more patients experienced toxicity that was considered to be dose-limiting toxicity, but these did not meet the criteria for dose-limiting toxicity because they occurred outside the scope of dose-limiting toxicity. No administration-related deaths were reported. These events are summarized in Table 11 below and described in the following explanation.

[0325] [Table 11] Three patients were added to group 1 (5 x 10 weeks). 9In one of these cases, dose-limiting toxicity was observed in the form of noncardiac chest pain around the tumor site. This subject progressed to a reduced dose and then gradually returned to full strength. This population was expanded to enroll a total of six subjects, but no further DLTs were observed.

[0326] Two out of two subjects in group 2 (5x10 twice a week) 9 Toxicity leading to dose reduction or discontinuation of the trial was observed in the following subjects. The first was an infusion-related reaction classified as a dose-limiting toxicity. This subject progressed to a reduced dose and then gradually returned to full strength. The second event was a persistent electrocardiogram change complicated by coronary ischemia, which occurred during the fourth week of administration and was therefore not classified as a dose-limiting toxicity (discussed further below). This subject was excluded from the trial, and no further subjects were enrolled in this population (maximum dose).

[0327] Six subjects were added to an additional group (Group 3, 5 x 10 once a week). 9 A group was enrolled in a dose-escalation study where all subjects began with dose reduction and then gradually increased to a sufficient strength to minimize infusion reactions to the investigational drug. No subjects in this group experienced dose-limiting toxicity.

[0328] Two subjects were enrolled in group 4 (2.5 × 10 9 (Twice a week + dose escalation), no dose-limiting toxicity was observed. However, due to the significant clinical burden associated with twice-weekly administration, recruitment for this group was discontinued.

[0329] An additional group of 9 subjects (group 5, 5 days a week x 10 days) 9Participants were enrolled in the dose escalation (+dose escalation + dex-limiting indication) and the medication plan, including dose escalation and tapering of dexamethasone premedication, was evaluated. See Figure 4. In this population, one subject received no treatment. Of the remaining eight subjects, two experienced toxicity leading to dose reduction or discontinuation of the study. The first was takotsubo (stress-related) cardiomyopathy, which was classified as a dose-limiting toxicity (discussed further below). This subject discontinued the study. The second was an anaphylactic-like reaction, which occurred during the 7th week administration and was therefore not classified as a dose-limiting toxicity. However, this subject was excluded from the study, and recruitment of this population was discontinued. The maximum tolerable dose was 5 × 10 once weekly. 9 V EDV miRNA16a It was decided that...

[0330] Numerous cardiac events were observed in this trial, which had not been previously observed in trials of different EDV products for other indications. Two patients experienced serious adverse events (ischemia and takotsubo cardiomyopathy) leading to dose reduction or discontinuation. These events occurred 7 days after administration, and given the patient's history of coronary artery disease, the ischemia was deemed unlikely to be attributable to the investigational drug. This patient also experienced electrocardiogram changes earlier in the administration period. An acute fluid response preceded the cardiomyopathy event, which was likely a result of dex tapering. This patient also had a history of coronary artery disease. Three additional patients experienced transient electrocardiogram changes (T-wave abnormalities) after administration, but these were not classified as serious. These were not associated with elevated troponin levels, ischemia, or changes in ejection fraction. All events resolved, and the patients received additional doses without further electrocardiogram abnormalities. Nevertheless, given the advanced age of patients with malignant pleural mesothelioma and the associated morbidity, these observations led the Safety Committee to recommend stricter cardiac exclusion criteria and additional cardiac monitoring for the remainder of the trial and any future trials.

[0331] V EDV miRNA16a Comprehensive treatment by means of 5 × 10 9The EDV was well tolerated up to the maximum tolerated dose, and this particular patient population was given adequate monitoring and an adapted dose-escalation plan, without any significant concerns.

[0332] Thus, this example demonstrated promising results in treating mesothelioma by using EGFR-targeted EDV to deliver miRNA16a to cancer cells. However, the results of this example also demonstrate the need for improved therapeutic strategies for mesothelioma.

[0333] Example 8: In vitro cytotoxicity assays revealed that the highly toxic drug PNU-159682 inhibits tumor cell proliferation more effectively than other chemotherapeutic agents. This example demonstrates that PNU-159682, a highly toxic cytotoxic chemotherapeutic agent, is a more potent cancer cell proliferation inhibitor than a wide range of other chemotherapeutic agents.

[0334] PNU-159682 is a potent metabolite of the anthracycline nemorubicin (MMDX), and possesses cytotoxicity more than 3,000 times greater than its parent compounds (MMDX and doxorubicin). Therefore, PNU-159682 is considered a "supertoxic" chemotherapeutic drug. The use of such a drug is generally not possible in typical chemotherapy treatments because the toxicity levels of supertoxic drugs can lead to severe adverse events, including death.

[0335] PNU-159682 and other indicated chemotherapeutic agents were added to tumor cell lines at the concentrations shown in Figures 5-10.

[0336] All cells were incubated for a further 72 hours, followed by CellTiter 96 AQu eous One Solution Cell Proliferation Assay (Promega Corp, Madison, W IUSA) was used according to the manufacturer's instructions to perform colorimetric MTS cell proliferation analysis (Cory et al., Cancer Commun., 3(7):207-12 (1991)). Colorimetric measurements were read at 490 nm.

[0337] Greater Cytotoxicity: The results of these in vitro cytotoxicity analyses showed that PNU-159682 exhibited greater cytotoxicity against the human lung cancer cell line A549 compared to the cytotoxicity observed with a range of known chemotherapeutic agents, as shown in Figure 5A. In particular, PNU159682 inhibited A549 cells far more significantly than doxorubicin. Figure 5B.

[0338] Efficacy against cancer cells known to be resistant to conventional chemotherapy agents: Furthermore, Figure 6 shows that PNU-159682 and duocalmycin showed potent cytotoxic effects against two adrenocortical cancer cell lines derived from stage IV patients that were highly resistant to doxorubicin, mitotane, paclitaxel, oxaliplatin, and mitoxantrone. Furthermore, Figure 7 shows that PNU-159682 inhibited the proliferation of the human breast cancer cell line MDA-MB-468, which was resistant to doxorubicin, paclitaxel, and docetaxel. Figure 8 shows that PNU-159682 inhibited the proliferation of the human colorectal cancer cell lines Caco-2 (Figure 8A) and HCT116 (Figure 8B), which were resistant to doxorubicin and cisplatin. Figure 9 shows that PNU-159682 can inhibit the proliferation of the glioblastoma cell line U87-MG, which was resistant to doxorubicin and paclitaxel IVAX. Figure 10A shows that PNU-159682 can inhibit the proliferation of a gemicitabine-sensitive human pancreatic cell line (MiaPaca-2 cells, Figure 10A), even though these cells were resistant to doxorubicin, gemzar, matoltoxate, oxaliplatin, irinotecan, and 5-fluorouracil (5-FU). Figure 10B shows that PNU-159682 can inhibit the proliferation of a gemicitabine-resistant human pancreatic cell line (MiaPaca-2 GemR cells, Figure 10B), even if these cells were resistant to gemzar.

[0339] These data suggest that the use of highly toxic chemotherapeutic agents such as PNU-159682 in cancer treatment is highly desirable because these agents may be useful in treating cancers that have developed resistance to conventional non-highly toxic chemotherapeutic agents.

[0340] Example 9: PNU159682 delivered with EGFR-targeted EDV can overcome drug resistance in human lung cancer cells in a mouse xenograft model. This example demonstrates that using mini-cells (EDVs) to deliver highly toxic chemotherapeutic agents such as PNU-159682 effectively inhibits tumor growth in a lung cancer xenograft model.

[0341] A549 (lung cancer) cells were cultured in the presence of doxorubicin (Dox), and doxorubicin-resistant clones were selected to induce doxorubicin resistance. These cells were then transplanted as xenografts into Balb / c nu / nu mice. The tumor volume was approximately 150 mm². 3 When this was reached, four different groups of mice (n=7 per group) were divided into (i) physiological saline and (ii) doxorubicin ( EGFR EDV TM Dox (iii) PNU-159682( EGFR EDV TM 682 (iv) EGFR targeting EDV loaded with ) and PNU-159682 (EDV) at the time indicated by the solid arrow in Figure 11. TM 682 Intravenous (IV) administration was performed using a non-targeted EDV loaded with EGFR. The composition of EDVs targeted at EGFR and loaded with PNU-159682 is shown graphically in Figure 1.

[0342] The results shown in Figure 11 are: EGFR DV TM Dox This indicates that it did not have an antitumor effect, and therefore the tumor showed resistance to DOX. In contrast, EGFR EDV TM 682Mice treated with this method showed complete recovery. Tumors treated with physiological saline were 500 mm 3 ~700mm 3 When the tumor volume reaches this point, the treatment is performed at the point indicated by the asterisk (*) in Figure 11. EGFR EDV TM 682 I changed it to this. Surprisingly, the result was 500mm 3 ~700mm 3 It showed very significant antitumor efficacy even in tumors that had reached a certain volume.

[0343] Example 10: Delivery of functional DNA agents via EGFR-targeted EDV effectively inhibits the proliferation of mesothelioma (MSTO) and adrenocortic carcinoma (ACC) cancer cells. This example demonstrates that using mini-cells (EDVs) to deliver siRNA targeting polo-like kinase 1 (Plk1), ribonucleotide reductase enzyme 1 (RRM1), or miRNA16a can effectively inhibit the proliferation of cancer cells.

[0344] Kinase 1 (Plk1) and ribonucleotide reduction enzyme 1 (RM1) were shown to be overexpressed in several non-small cell lung cancer (NSCLC) cell lines, including A549, A549MDR (Dox-resistant A549 cell line, multidrug-resistant membrane pump, overexpressing MDR), H2122, H358, and H441. Figure 12 shows the expression of GAPDH(g), KSP(K), Plk1(P), and RRM1(R), which are shown in comparison to GAPDH expression in the NSCLC cell lines shown.

[0345] To test whether Plk1 or RRM1 are useful targets for cancer treatment, inhibitory non-coding small interfering RNAs (siRNAs) targeting RRM1 (siRRM1) and Plk1 (siPlk1) were synthesized and packaged in EDV for delivery to cancer cell lines.

[0346] siRNA targeting RRM1 was found to inhibit the proliferation of mesothelioma and adrenocortical carcinoma cells. EGFR-targeted, siRRM1-packaged EDV was transfected into MSTO (mesoheloma cell line) or H295R (adrenocortical cell line). Cell proliferation was measured 5 days after transfection, and the results are shown in Figure 13. Compared to control transfections using untargeted siRRM1-packaged EDV or EGFR-targeted siNonsense-packaged EDV, a very significant inhibition of cell proliferation was observed.

[0347] In a mesothelioma (MSTO) xenograft study in Balb / c nu / nu mice, intravenous (IV) treatment with EGFR-targeted siRRM1-packaged EDV demonstrated significantly greater antitumor efficacy compared to saline or EGFR-targeted siScrambled-packaged EDV, as shown in Figure 14. Tumors isolated from mice treated with siRRM1-packaged EDV were significantly smaller than those from mice treated with negative controls, as shown in Figure 15.

[0348] MiRNA16a was found to inhibit the proliferation of mesothelioma cancer cells. In a mesothelioma (MSTO) xenograft study in Balb / c nu / nu mice, intravenous (IV) treatment with EGFR-targeted miRNA16a-packaged EDV showed significantly greater antitumor efficacy compared to saline or EGFR-targeted si-scrambled packaged EDV, as shown in Figure ...

Claims

1. (a) At least one antitumor agent comprising PNU-159682, doxorubicin, or mitoxantrone, in a therapeutically effective amount to purified intact bacterial minicells, and (b) An anticancer drug composition comprising an interferon type II agonist selected from the group consisting of C-glycoside type α-galactosylceramide (α-C-GalCer), α-galactosylceramide (α-GalCer), and combinations thereof.

2. Element (b) of the composition is The anticancer agent composition according to claim 1, comprising a therapeutically effective amount of purified, intact bacterial mini-cells containing an interferon type II agonist.

3. (a) The anticancer agent composition according to claim 1 or 2, wherein the antitumor agent and the interferon type II agonist are packaged in two or more purified intact bacterial minicells.

4. The aforementioned antitumor agent and an interferon type II agonist are included, where, (a) The antitumor agent and the interferon type II agonist are contained within the same minicell, or (b) The antitumor agent is contained within the first mini-cell, and the interferon type II agonist is contained within the second mini-cell. The anticancer agent composition according to any one of claims 1 to 3.

5. The anticancer agent composition according to any one of claims 1 to 4, wherein the composition does not contain an interferon type I agonist.

6. The anticancer agent composition according to any one of claims 1 to 5, wherein at least one antitumor agent is selected from the group consisting of PNU-159682 and doxorubicin.

7. An anticancer agent composition according to any one of claims 1 to 6, wherein at least one antitumor agent is PNU-159682.

8. The anticancer drug composition according to any one of claims 1 to 7, wherein the interferon type II agonist is α-galactosylceramide (α-GalCer).

9. (a) a bispecific ligand bound to minicells containing an antitumor agent, and / or (b) A bispecific ligand bound to a minicell containing an interferon type I agonist, and / or (c) The anticancer drug composition according to any one of claims 1 to 8, further comprising a bispecific ligand bound to minicells containing an interferon type II agonist.

10. The bispecific ligand is (a) comprising a first arm having specificity for minicell surface structures and a second arm having specificity for nonphagocytic mammalian cell surface receptors, and / or (b) comprising a first arm having specificity for minicell surface structures and a second arm having specificity for nonphagocytic mammalian cell surface receptors, wherein the minicell surface structure is the O-polysaccharide component of lipopolysaccharide on the minicell surface, and / or (c) comprising a first arm having specificity for minicell surface structures and a second arm having specificity for nonphagous mammalian cell surface receptors, wherein the nonphagous mammalian cell surface receptors can activate receptor-mediated endocytosis of minicells, and / or (d) comprising a bispecific antibody or antibody fragment, and / or (e) The anticancer composition according to claim 9, wherein the antibody or antibody fragment comprises a first multivalent arm having specificity for a bacterial minicell surface structure and a second multivalent arm having specificity for a cancer cell surface receptor, the cancer cell surface receptor being able to activate receptor-mediated endocytosis of minicells.

11. The composition is 10 ７ Less than 1 contaminated parent bacterial cell per minicell, 10 ８ Less than 1 contaminated parent bacterial cell per minicell, 10 ９ Less than 1 contaminated parent bacterial cell per minicell, 10 １０ Approximately less than 1 contaminated parental bacterial cell per minicell, or 10 １１ An anticancer agent composition according to any one of claims 1 to 10, comprising less than approximately one contaminated parental bacterial cell per minicell.

12. The anticancer agent composition according to any one of claims 1 to 11, further comprising a pharmaceutically acceptable carrier.

13. The anticancer drug composition according to any one of claims 1 to 12, wherein the mini-cells have a diameter of approximately 400 nm.

14. The anticancer agent composition according to any one of claims 1 to 13, wherein the composition does not contain parental bacterial cell contamination that can be removed by 200 nm filtration.

15. The composition is (a) at least 10 ９ , (b) at least 1 × 10 ９ , (c) at least 2 × 10 ９ , (d) At least 5 × 10 ９ , (e) at least 8 × 10 ９ , (f)10 １１ the following, (g) 1×10 １１ the following, (h) 9 x 10 １０ The following, or (i) 8 x 10 １０ The anticancer agent composition according to any one of claims 1 to 14, comprising the following mini-cells or dead bacterial cells.

16. An anticancer agent composition according to any one of claims 1 to 15, for use in a method of treating a subject as needed, comprising administering an effective amount of the composition to the subject.

17. The anticancer drug composition according to claim 16, wherein the subject is a human, a non-human primate, a dog, a cat, a cow, a sheep, a horse, a rabbit, a mouse, or a rat.

18. (a) The subject has cancer, and / or (b) The subject has cancer, and the cancer is selected from the group consisting of lung cancer, breast cancer, brain cancer, liver cancer, colon cancer, pancreatic cancer, and bladder cancer, and / or (c) The subject has cancer, and the cancer is acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, appendiceal cancer, astrocytoma, atypical teratoma / rhabdoid tumor, basal cell carcinoma, bladder cancer, brainstem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, cancer of unknown primary site, carcinoid tumor, cancer of unknown major site, atypical teratoma-like / rhabdoid tumor of the central nervous system, germ cell tumor of the central nervous system, cervical cancer, childhood cancer, chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, colorectal cancer, colorectal cancer. Craniopharyngioma, cutaneous T-cell lymphoma, endocrine pancreatic islet cell tumor, endometrial cancer, ependymoblastoma, ependymomas, esophageal cancer, sensory neurosarcoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, bile duct cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal cell tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastoma, glioma, hairy cell leukemia, cardiac tumor, head and neck cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip cancer, liver cancer, malignant fibrous tissue Bone cancer, liposarcoma, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of unknown primary origin, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasmacytic neoplasm, mycosis fungoides, myelodysplastic syndrome, nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma, other brain and spinal cord tumors, ovarian germ cell tumors, low-grade ovarian tumors, pancreatic cancer, papillomatosis, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, pharyngeal cancer, pineal parenchymal cell tumors showing intermediate differentiation Cystic tumors, pineal blastomas, pituitary tumors, plasma cell neoplasms / multiple myeloma, pleuropneumonoma, primary central nervous system (CNS) lymphoma, primary hepatocellular carcinoma, pleuropneumonoma, primary hepatocellular carcinoma, prostate cancer, renal cell carcinoma, renal cell carcinoma, renal cell carcinoma, retinoblastoma, retinoblastoma, rhabdomyosarcoma, Sézary syndrome, salivary gland cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, cervical cancer, gastric (stomach) cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, testicular cancer, pharyngeal cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, chorioblastoma,Ureteral cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms tumor and / or, (d) The anticancer composition according to claim 16 or 17, wherein the subject is suffering from a brain tumor or brain tumor selected from the group consisting of brainstem glioma, atypical teratomatoid / rhabdoid tumor of the central nervous system, germ cell tumor of the central nervous system, astrocytoma, craniopharyngioma, ependymoblastoma, ependymodium, medulloblastoma, medullary epithelioma, pineal parenchymal tumor of intermediate differentiation, supratentorial primitive neuroectodermal tumor, and pinealoblastoma.

19. The composition is (a) at least once a week for several weeks, and / or (b) at least once a week for several weeks to several months, and / or (c) at least once a week for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks or longer, and / or (d) twice a week, and / or (e) The anticancer drug composition according to any one of claims 16 to 18, administered twice a week for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks or longer.

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