Cancer immunotherapy with enveloped self-amplifying mRNA CARG-2020 that modulates IL-12, IL-17 and PD-L1 pathways to prevent tumor recurrence.

Targeting multiple immune mechanisms may overcome therapy resistance and further improve cancer immunotherapy for humans. Here, we describe the application of virus-like vesicles (VLV) for delivery of three immunomodulators alone and in combination, as a promising approach for cancer immunotherapy. VLV vectors were designed to deliver single chain interleukin (IL)-12, short-hairpin RNA (shRNA) targeting programmed death ligand 1 (PD-L1), and a dominant-negative form of IL-17 receptor A (dn-IL17RA) as a single payload or as a combination payload. Intralesional delivery of the VLV vector expressing IL-12 alone, as well as the trivalent vector (designated CARG-2020) eradicated large established tumors. However, only CARG-2020 prevented tumor recurrence and provided long-term survival benefit to the tumor-bearing mice, indicating a benefit of the combined immunomodulation. The abscopal effects of CARG-2020 on the non-injected contralateral tumors, as well as protection from the tumor cell re-challenge, suggest immune-mediated mechanism of protection and establishment of immunological memory. Mechanistically, CARG-2020 potently activates Th1 immune mechanisms and inhibits expression of genes related to T cell exhaustion and cancer-promoting inflammation. The ability of CARG-2020 to prevent tumor recurrence and to provide survival benefit makes it a promising candidate for its development for human cancer immunotherapy.

Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory.

Current immunotherapies have proven effective in strengthening antitumor immune responses, but constant opposing signals from tumor cells and the surrounding microenvironment eventually lead to immune escape. We hypothesized that in situ release of antigens and regulation of both the innate and adaptive arms of the immune system would provide a robust and long-term antitumor effect by creating immunologic memory against tumors. To achieve this, we developed CARG-2020, a genetically modified virus-like vesicle (VLV) that is a self-amplifying RNA with oncolytic capacity and encodes immune regulatory genes. CARG-2020 carries three immune modulators: (i) the pleiotropic antitumor cytokine IL12, in which the subunits (p35 and p40) are tethered together; (ii) the extracellular domain (ECD) of the protumor IL17RA, which serves as a dominant-negative antagonist; and (iii) a shRNA targeting PD-L1. Using a mouse model of ovarian cancer, we demonstrated the oncolytic effect and immune-modulatory capacities of CARG-2020. By enhancing IL12 and blocking IL17 and PD-L1, CARG-2020 successfully reactivated immune surveillance by promoting M1, instead of M2, macrophage differentiation, inhibiting MDSC expansion and establishing a potent CD8+ T cell-mediated antitumoral response. Furthermore, we demonstrated that this therapeutic approach provided tumor-specific and long-term protection against the establishment of new tumors. Our results provide a rationale for the further development of this platform as a therapeutic modality for ovarian cancer patients to enhance antitumor responses and prevent a recurrence.

Immune modulation of innate and adaptive responses restores immune surveillance and establishes anti-tumor immunological memory.

Current immunotherapies have proven effective in strengthening anti-tumor immune responses but constant opposing signals from tumor cells and surrounding microenvironment eventually lead to immune escape. We hypothesize that in situ release of antigens and regulation of both the innate and adaptive arms of the immune system will provide a robust and long-term anti-tumor effect by creating immunological memory against the tumor. To achieve this, we developed CARG-2020, a virus-like-vesicle (VLV). It is a genetically modified and self-amplifying RNA with oncolytic capacity and encodes immune regulatory genes. CARG-2020 carries three transgenes: 1 ) the pleiotropic antitumor cytokine IL-12 in which the subunits (p35 and p40) are tethered together; 2) the extracellular domain (ECD) of the pro- tumor IL-17RA, which can serve as a dominant negative antagonist; and 3) shRNA for PD-L1. Using a mouse model of ovarian cancer, we demonstrate the oncolytic effect and immune modulatory capacities of CARG-2020. By enhancing IL-12 and blocking IL-17 and PD-L1, CARG-2020 successfully reactivates immune surveillance by promoting M1 instead of M2 macrophage differentiation, inhibiting MDSC expansion, and establishing a potent CD8+ T cell mediated anti-tumoral response. Furthermore, we demonstrate that this therapeutic approach provides tumor-specific and long-term protection preventing the establishment of new tumors. Our results provide rationale for the further development of this platform as a therapeutic modality for ovarian cancer patients to enhance the anti-tumor response and to prevent recurrence.

Inhibition of GSK_3ß by Iridoid Glycosides of Snowberry (Symphoricarpos albus) Effective in the Treatment of Alzheimer's Disease Using Computational Drug Design Methods.

The inhibition of glycogen synthase kinase-3ß (GSK-3ß) activity prevents tau hyperphosphorylation and binds it to the microtubule network. Therefore, a GSK-3ß inhibitor may be a recommended drug for Alzheimer's treatment. In silico methods are currently considered as one of the fastest and most cost-effective available alternatives for drug/design discovery in the field of treatment. In this study, computational drug design was conducted to introduce compounds that play an effective role in inhibiting the GSK-3ß enzyme by molecular docking and molecular dynamics simulation. The iridoid glycosides of the common snowberry (Symphoricarpos albus), including loganin, secologanin, and loganetin, are compounds that have an effect on improving memory and cognitive impairment and the results of which on Alzheimer's have been studied as well. In this study, in the molecular docking phase, loganin was considered a more potent inhibitor of this protein by establishing a hydrogen bond with the ATP-binding site of GSK-3ß protein and the most negative binding energy to secologanin and loganetin. Moreover, by molecular dynamics simulation of these ligands and GSK-3ß protein, all structures were found to be stable during the simulation. In addition, the protein structure represented no change and remained stable by binding ligands to GSK-3ß protein. Furthermore, loganin and loganetin have higher binding free energy than secologanin; thus, these compounds could effectively bind to the active site of GSK-3ß protein. Hence, loganin and loganetin as iridoid glycosides can be effective in Alzheimer's prevention and treatment, and thus, further in vitro and in vivo studies can focus on these iridoid glycosides as an alternative treatment.

Modified Alphavirus-Vesiculovirus Hybrid Vaccine Vectors for Homologous Prime-Boost Immunotherapy of Chronic Hepatitis B.

Abstract： Virus-like vesicles (VLV) are hybrid vectors based on an evolved Semliki Forest virus (SFV) RNA replicon and the envelope glycoprotein (G) from vesicular stomatitis virus (VSV) [...].

Virus-like Vesicles Expressing Multiple Antigens for Immunotherapy of Chronic Hepatitis B.

Infections with hepatitis B virus (HBV) can initiate chronic hepatitis and liver injury, causing more than 600,000 deaths each year worldwide. Current treatments for chronic hepatitis B are inadequate and leave an unmet need for immunotherapeutic approaches. We designed virus-like vesicles (VLV) as self-amplifying RNA replicons expressing three HBV antigens (polymerase, core, and middle surface) from a single vector (HBV-VLV) to break immune exhaustion despite persistent HBV replication. The HBV-VLV induces HBV-specific T cells in naive mice and renders them resistant to acute challenge with HBV. Using a chronic model of HBV infection, we demonstrate efficacy of HBV-VLV priming in combination with DNA booster immunization, as 40% of treated mice showed a decline of serum HBV surface antigen below the detection limit and marked reduction in liver HBV RNA accompanied by induction of HBsAg-specific CD8 T cells. These results warrant further evaluation of HBV-VLV for immunotherapy of chronic hepatitis B.

A multifunctional cytoprotective agent that reduces neurodegeneration after ischemia.

Cellular and molecular pathways underlying ischemic neurotoxicity are multifaceted and complex. Although many potentially neuroprotective agents have been investigated, the simplicity of their protective mechanisms has often resulted in insufficient clinical utility. We describe a previously uncharacterized class of potent neuroprotective compounds, represented by PAN-811, that effectively block both ischemic and hypoxic neurotoxicity. PAN-811 disrupts neurotoxic pathways by at least two modes of action. It causes a reduction of intracellular-free calcium as well as free radical scavenging resulting in a significant decrease in necrotic and apoptotic cell death. In a rat model of ischemic stroke, administration of PAN-811 i.c.v. 1 h after middle cerebral artery occlusion resulted in a 59% reduction in the volume of infarction. Human trials of PAN-811 for an unrelated indication have established a favorable safety and pharmacodynamic profile within the dose range required for neuroprotection warranting its clinical trial as a neuroprotective drug.

Phase I and pharmacokinetic study of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) using a single intravenous dose schedule.

PURPOSE: To perform a phase I and pharmacokinetics study of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) a new ribonucleotide reductase inhibitor using a single intravenous (2-h) schedule every 4 weeks. 3-AP was given at a starting dose of 5 mg/m(2) with escalation based on a modified Fibonacci scheme. PATIENTS AND METHODS: A total of 27 patients with advanced cancer were entered into the study. Doses of 3-AP ranged from 5 mg/m(2) to 105 mg/m(2). Blood and urine samples were collected and 3-AP was measured by HPLC. RESULTS: A total of 46 courses were evaluable. One patient developed grade 4 thrombocytopenia at the lowest dose level, and one patient had grade 3 anemia. Two patients developed grade 3 coagulation abnormalities. The only other toxicities of more than grade l occurring in more than 10% of patients were fever and asthenia. No toxicities were observed at the highest dose level. Peak serum concentration of 3-AP increased linearly with dose. No tumor responses were observed in this heavily pretreated population, although eight patients had stabilization of their disease. CONCLUSIONS: Relevant tumor inhibitory concentrations were achieved without significant toxicity using doses up to 105 mg/m(2) on this single intravenous dose schedule. Prolonged administration schedules and combinations with other cytotoxic agents, strategies predicted to have greater antitumor efficacy according to preclinical studies, are under investigation.

Toxicological evaluation of 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylaminocarbonyl)hydrazine (VNP40101M), a novel alkylating agent with potential antitumor activity, with intravenous administration in rats and dogs.

These studies investigated the toxicological effects of 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylaminocarbonyl) hydrazine, VNP40101M, a novel alkylating antitumor agent, in animals. Sprague-Dawley rats (2-10/sex/time point at each dose) and Beagle dogs (1-3/sex/time point at each dose) were treated with VNP40101M (0 [vehicle], 1, 3, 10, and 20 mg/kg in rats and 0, 0.3, 1, and 3 mg/kg in dogs), given intravenously (IV, bolus via the tail or slow push via the cephalic or saphenous vein, respectively) once daily for 5 consecutive days. Clinical signs, mortality, body weight, clinical pathology, gross necropsy, organ weights, and histopathology were evaluated for as long as 43 days in rats and 50 days in dogs. In rats, the toxic doses were found to be at 10 and 20 mg/kg, which induced mainly pulmonary toxicity and mortality. The pulmonary toxicity was reflected by an increase in lung weight; clear, pink or red fluid within the thoracic cavity observed at necropsy; and histopathological evidence of alveolar edema, vascular congestion, alveolar histiocytosis, and vascular thrombi. Although some of these effects were observed in rats treated with 3 mg/kg, the incidence was low (approximately 7%-30%) and may be reversible (based on the time-dependent reduction in the magnitude of lung weight increases). Therefore, the maximum tolerated dose (MTD, or the maximum dose that did not induce significant toxicity or induced reversible toxicity) was > or = 3 mg/kg. VNP40101M at 1 mg/kg did not induce any toxicity, other than low incidence of alveolar edema (2/30 rats), and increased incidences of capillary ectasis/congestion and alveolar histocytosis (2-6/30 rats vs. 1/30-36 in control rats). Therefore, the low effect level (LOEL) is considered to be 1 mg/kg in rats when given IV for 5 days. In dogs, LOEL, MTD, and toxic dose levels were comparable (based on a body weight/surface area conversion) to those in rats, except for some gastrointestinal (GI) effects (i.e., red lesion in the ileum) observed at 0.3 mg/kg (equivalent to 1 mg/kg, or similar to the LOEL in rats) and the associated effects (slight body weight loss and inappetence). For dogs treated with 1 mg/kg (equivalent to approximately 3 mg/kg, or MTD, in rats), VNP40101M induced the same GI effects seen in dogs treated with 0.3 mg/kg of VNP40101M. Additionally, a transient reduction in white blood cell counts was also observed. Three mg/kg (equivalent to approximately 10 mg/kg, or toxic dose level, in rats) was toxic to dogs, as reflected by the poor clinical condition of these dogs, which subsequently required euthanasia. In conclusion, VNP40101M, when given IV once daily for 5 consecutive days, has a LOEL of 1 mg/kg, a MTD of 3 mg/kg, and toxic doses at > or = 10 mg/kg in rats. The primary toxicity of VNP40101M was pulmonary toxicity and mortality. Based on an interspecies body weight/surface area conversion, VNP40101M had comparable LOEL (0.3 mg/kg), MTD (1 mg/kg), and toxic doses (> or = 3 mg/kg) in dogs, except that dogs appeared to be more sensitive to the GI effects of VNP40101M.