408 oncolytic vaccines in combination with pd l1 blockade for the treatment of melanoma

The Immunological escape of tumors represents one of the main obstacles to the treatment of malignancies. The approval of drugs able to disrupt the immune suppressive pathways through anti-CTLA-4 monoclonal antibodies represented a milestone in the history of immunotherapy. However, treatment with these immune checkpoint inhibitors (ICIs) seems to be effective only in small cohorts of patients. It has been proposed that the efficacy of ICIs relies on the presence of an undergoing immunological response. For this reason, we hypothesized that oncolytic vaccines, able to elicit a tumor specific response, would synergize with anti-PD-L1 therapy. B16 murine melanomas were established in immunocompetent C57 mice. Then mice were treated with anti-PD-L1 monotherapy, PeptiCRAd (oncolytic vaccine) monotherapy or a combination of the two. The growth of the tumors was analyzed. At the end of the experiment, all the mice mice were euthanized and organs collected for immunological analysis. We investigated antigen-specific T-cell responses and immune suppressive background by flow cytometry and ELISPOT assays

Physical PEGylation to Prevent Insulin Fibrillation

Insulin is one of the most marketed therapeutic proteins worldwide. However, its formulation suffers from fibrillation, which affects the long-term storage limiting the development of novel devices for sustained delivery including portable infusion devices. We have investigated the effect of physical PEGylation on structural and colloidal stability of insulin by using 2 PEGylating agents terminating with polycyclic hydrophobic moieties, cholane and cholesterol: mPEG5kDa-cholane and mPEG5kDa-cholesterol, respectively. Microcalorimetric analyses showed that mPEG5kDa-cholane and mPEG5kDa-cholesterol efficiently bind insulin with binding constants (Ka) of 3.98 104 and 1.14 105 M-1, respectively. At room temperature, the 2 PEGylating agents yielded comparable structural stabilization of \u3b1-helix conformation and decreased dimerization of insulin. However, melting studies showed that mPEG5kDa-cholesterol has superior stabilizing effect of the protein conformation than mPEG5kDa-cholane. Furthermore, the fibrillation study showed that at a 1:1 and 1:5 insulin/polymer molar ratios, mPEG5kDa-cholesterol delays insulin fibrillation 40% and 26% more efficiently, respectively, as compared to mPEG5kDa-cholane which was confirmed by transmission electron microscopy imaging. Insulin was released from the mPEG5kDa-cholane and mPEG5kDa-cholesterol assemblies with comparable kinetic profiles. The physical PEGylation has a beneficial effect on the stabilization and shielding of the insulin structure into the monomeric form, which is not prone to fibrillation and aggregation

Isoflavones in aglycone solution enhance ultraviolet B-induced DNA damage repair efficiency.

The isoflavones daidzein and genistein are natural compounds which have anti-inflammatory and photoprotective activities, and may be effective in the repair of ultraviolet (UV)-induced photodamage. In this study, an alcoholic solution of aglycone isoflavones with a genistein:daidzein ratio of 1:4 [Rottapharm (RPH)-aglycone] was examined for its effects on the repair of DNA damage induced by a single dose of UVB irradiation (20 mJ/cm2). For this purpose, human skin cells were first UVB-irradiated and then treated with RPH-aglycone. Comet assay analysis was used to estimate the UVB-induced DNA damage at different time points after treatment by measuring the tail moment parameter. We found that treatment with 10 μmol/L RPH-aglycone solution resulted in a significantly reduced tail moment at 1 h after treatment, and 34-35% enhancement of damage repair at 4 h after treatment. These results suggest that isoflavone aglycones are protective against UVB-induced DNA damage. © 2014 British Association of Dermatologists

Quetiapine as add-on treatment for bipolar I disorder: efficacy in preventing relapse of depressive episodes

<p>Abstract</p> <p>Objective</p> <p>To assess the long-term response to add-on quetiapine therapy in patients with bipolar I disorder who were not adequately responding to standard medications.</p> <p>Methods</p> <p>Outpatients with bipolar I disorder (DSM-IV-TR) responding inadequately to standard treatment were observed before and after the addition of quetiapine. Symptom severity was evaluated using the Clinical Global Impressions scale for Bipolar Disorder (CGI-BP) each month. Relapses included hospitalization, treatment in a day hospital or clinic, scores ≥ 1 point higher than previous CGI-BP scores and/or upward titration of quetiapine or other medications.</p> <p>Results</p> <p>Sixty-one patients (age range of 18–68 years) were observed prospectively for an average of 7.5 months (range 3–18 months) prior to addition of quetiapine and subsequently followed for an average of 15.7 months (range 6–42 months). The final mean quetiapine dose was 537.1 ± 91.7 mg/d. Prior to quetiapine addition, an annual relapse rate of 2.09 episodes was recorded, relating to 0.94 depressive and 1.15 manic or mixed episodes. Following quetiapine addition, annual relapse rates were reduced to 0.61 episodes, representing 0.14 depressive and 0.46 manic or mixed episodes. Compared with the period of add-on quetiapine treatment, the relative risk of relapse <it>prior </it>to quetiapine therapy was 3.4 for all episodes (χ²= 24.8, <it>P </it>< 0.001), 6.7 for depressive episodes (χ²= 24.7, <it>P </it>< 0.001), and 2.5 for manic or mixed episodes (χ²= 9.0, <it>P </it>< 0.05).</p> <p>Conclusion</p> <p>This naturalistic follow-up study provides preliminary evidence for the efficacy of long-term add-on quetiapine treatment in the prevention of relapses of manic or mixed and depressive episodes of bipolar I disorder, and particularly in the prevention of depressive episodes.</p

Optimization of Early Steps in Oncolytic Adenovirus ONCOS-401 Production in T-175 and HYPERFlasks

Oncolytic adenoviruses can trigger lysis of tumor cells, induce an antitumor immune response, bypass classical chemotherapeutic resistance strategies of tumors, and provide opportunities for combination strategies. A major challenge is the development of scalable production methods for viral seed stocks and sufficient quantities of clinical grade viruses. Because of promising clinical signals in a compassionate use program (Advanced Therapy Access Program) which supported further development, we chose the oncolytic adenovirus ONCOS-401 as a testbed for a new approach to scale up. We found that the best viral production conditions in both T-175 flasks and HYPERFlasks included A549 cells grown to 220,000 cells/cm2 (80% confluency), with ONCOS-401 infection at 30 multiplicity of infection (MOI), and an incubation period of 66 h. The Lysis A harvesting method with benzonase provided the highest viral yield from both T-175 and HYPERFlasks (10,887 ± 100 and 14,559 ± 802 infectious viral particles/cell, respectively). T-175 flasks and HYPERFlasks produced up to 2.1 × 109 ± 0.2 and 1.75 × 109 ± 0.08 infectious particles of ONCOS-401 per cm2 of surface area, respectively. Our findings suggest a suitable stepwise process that can be applied to optimizing the initial production of other oncolytic viruses

Optimization of Early Steps in Oncolytic Adenovirus ONCOS-401 Production in T-175 and HYPERFlasks

Oncolytic adenoviruses can trigger lysis of tumor cells, induce an antitumor immune response, bypass classical chemotherapeutic resistance strategies of tumors, and provide opportunities for combination strategies. A major challenge is the development of scalable production methods for viral seed stocks and sufficient quantities of clinical grade viruses. Because of promising clinical signals in a compassionate use program (Advanced Therapy Access Program) which supported further development, we chose the oncolytic adenovirus ONCOS-401 as a testbed for a new approach to scale up. We found that the best viral production conditions in both T-175 flasks and HYPERFlasks included A549 cells grown to 220,000 cells/cm2 (80% confluency), with ONCOS-401 infection at 30 multiplicity of infection (MOI), and an incubation period of 66 h. The Lysis A harvesting method with benzonase provided the highest viral yield from both T-175 and HYPERFlasks (10,887 ± 100 and 14,559 ± 802 infectious viral particles/cell, respectively). T-175 flasks and HYPERFlasks produced up to 2.1 × 109 ± 0.2 and 1.75 × 109 ± 0.08 infectious particles of ONCOS-401 per cm2 of surface area, respectively. Our findings suggest a suitable stepwise process that can be applied to optimizing the initial production of other oncolytic viruses

Heterologous and cross-species tropism of cancer-derived extracellular vesicles

Extracellular vesicles (EVs) are naturally occurring cargo delivery vesicles that have recently received considerable attention for their roles in intercellular communication in many physiological and pathological processes, including tumourigenesis. EVs generated by different tissues demonstrated specific homing: in particular, cancer-derived EVs showed a selective tropism for the tumor tissue from which the vesicles originated. For this property, EVs have been proposed as drug delivery tools for anti-cancer therapies, although the limited knowledge about their in vivo tropism hinders their therapeutic applications. The current study aimed to characterize the targeting properties of cancer- derived EVs in vitro and their biodistribution in vivo, by using an imaging approach. Methods. EVs were generated from: i) murine lung (LL/2) and colon (MC-38) cancer lines, ii) human lung cancer cell line (A549) and iii) human liver biopsy samples from healthy individuals. EVs were loaded with fluorescent dyes alone or in combination with a biopharmaceutical agent, the oncolytic adenovirus (OV), characterized for charge and size and tested for their activity in cancer cell lines. Finally, optical imaging was extensively applied to study in vivo and ex vivo the biodistribution of EVs originated from different sources in different mouse models of cancer, including xenograft, syngeneic graft and the MMTV-NeuT genetically modified animal. Results. We initially demonstrated that even loading EVs even with a large biopharmaceutical oncolytic viruses (OVs) did not significantly change their charge and dimension properties, while increasing their anti-neoplastic activity compared to the virus or EVs alone. Interestingly, this activity was observed even if the EVs derived from lung cancer were applied to colon carcinoma cell lines and vice versa, suggesting that the EV uptake occurred in vitro without any specificity for the cancer cells from which the vesicles originated. When administered i.v (intravenously) to the mouse models of cancer, the tumour-derived EVs, but not the EVs derived from a healthy tissue, demonstrated a selective accumulation of the fluorescence at the tumour site 24 h after injection; adding OVs to the formulation also did not change the tumour-specific tropism of the EVs also in vivo. Most interestingly, the in vivo experiments confirmed the in vitro observation of the generalized tropism of tumour-derived EVs for any neoplastic tissue, independent of the tumour type or even the species originating the vesicles. Conclusions. Taken together, our in vitro and in vivo data demonstrate for the first time a heterologous, cross-species tumour-tropism for cancer-derived EVs. This finding challenges our current view on the homing properties of EVs and opens new avenues for the selective delivery of diagnostic/therapeutic agents to solid tumours

622. Oncolytic Adenoviruses Loaded With Active Drugs as a Novel Drug Delivery System for Cancer Therapy

L-carnosine (β-Ala-His) is a naturally occurring histidine dipeptide, normally found in brain, kidney and in large amounts in muscle. L-carnosine has biological functions, including antioxidant activity, ability to chelate metal ions, as well as anti-inflammatory and anti-senescence properties. Recent studies have demonstrated that 50-100 mM of L-carnosine decreases cell proliferation in a colon cancer cell line HCT116, bearing a mutation in codon 13 of the RAS proto-oncogene. In addition, pre-treatment with L-carnosine decreases the intracellular concentration of Adenosine Triphosphate (ATP) and Reactive Oxygen Species (ROS) and inhibits the cell cycle progression in the G1 phase. The proto-oncogene KRAS is mutated in a wide array of human cancers and is important both in tumour progression and resistance to anticancer drugs. To overcome treatment limitations due to the high intracellular concentration required we have hypothesized that L-carnosine can be conjugated on the capsid of oncolytic viruses. Oncolytic viruses are viruses that are able to replicate specifically in and destroy tumor cells and this property is either inherent or genetically-engineered. The association of viruses with specific drugs, would increase the efficacy of the treatment of human neoplasia due to the synergistic action of virus and drug. First we have developed a strategy to conjugate peptides on viral capsid, based on electrostatic interaction. Then, using different cancer cell lines we found that oncolytic virus coated with L-carnosine with a tail of positively charged polylysine was able to enhance a positive anticancer synergistic effect. Finally, in order to investigate the molecular mechanisms underlying the effect of tumor reduction by oncolytic virus coated with modified L-carnosine, we have used three different approaches. First, we have examined, in samples with virus alone, or in combination with L-carnosine, the oncolytic replication by evaluating the E1A expression, second the apoptotic mechanism by expression of specific genes and at end the autophagy regulation via the amount of LC3-II. In conclusion, we have developed a model to use oncolytic adenovirus as a scaffold to deliver active drugs. Once validated the proposed model could be used as a novel drug delivery system for cancer therapy

659 oncolytic adenovirus loaded with bioactive modified peptide as a novel approach to treat cancer

Cancer is still a leading cause of death worldwide. Although many kinds of treatment have been developed during the past decades, there is still a lack of effective therapy for advanced cancer. Currently treatments such as surgery, chemotherapy and radiotherapy can help to improve patient prognosis and increase patient life expectancy. Therefore new treatment strategies against cancer are in high demand. Efficient anticancer agent and its targeted delivery into the tumor mass is a key prerequisite for the successful cancer therapy. Oncolytic virotherapy is emerging as a potential approach to treat cancer, using viruses, which are specifically engineered to selectively infect, replicate in and kill cancer cells without causing damage to normal cells. Their combination with chemotherapeutic agents have shown promising results due to the synergistic effect of viruses and drugs; therefore the combinatorial therapy is considered a beneficial approach for cancer treatment. Taken into account these considerations we optimized a strategy to conjugate peptides on the viral capsid, based on electrostatic interaction and used this strategy to deliver an active anti-tumor dipeptide. We used L-carnosine, a naturally occurring histidine dipeptide with anti-proliferative activity. A modified L-carnosine, positively charged was absorbed onto the viral capsid of an oncolytic adenovirus to generate a virus-carnosine complex. The complex showed enhanced anti tumor efficacy in vitro and in vivo and higher infectious titer compared to a naked oncolytic adenovirus in colorectal and lung cancer cells. The in vivo efficacy of the complex was analyzed in lung and colon cancer xenograft models, displaying a significant reduction in tumor growth and synergistic effect between virus and dipeptide. Moreover, we studied the molecular mechanisms underlying the effects of complex on tumor growth reduction. Complex can induce apoptosis in both cells lines, by using two different mechanisms, enhancing viral replication and affecting the expression of Hsp27. Our system could be used in further studies also for specific delivery of other active drugs

Oncolytic adenovirus loaded with L-carnosine as novel strategy to enhance the antitumor activity

Oncolytic viruses are able to specifically replicate, infect, and kill only cancer cells. Their combination with chemotherapeutic drugs has shown promising results due to the synergistic action of virus and drugs; the combinatorial therapy is considered a potential clinically relevant approach for cancer. In this study, we optimized a strategy to absorb peptides on the viral capsid, based on electrostatic interaction, and used this strategy to deliver an active antitumor drug. We used L-carnosine, a naturally occurring histidine dipeptide with a significant antiproliferative activity. An ad hoc modified, positively charged L-carnosine was combined with the capsid of an oncolytic adenovirus to generate an electrostatic virus-carnosine complex. This complex showed enhanced antitumor efficacy in vitro and in vivo in different tumor models. In HCT-116 colorectal and A549 lung cancer cell lines, the complex showed higher transduction ratio and infectious titer compared with an uncoated oncolytic adenovirus. The in vivo efficacy of the complex was tested in lung and colon cancer xenograft models, showing a significant reduction in tumor growth. Importantly, we investigated the molecular mechanisms underlying the effects of complex on tumor growth reduction. We found that complex induces apoptosis in both cell lines, by using two different mechanisms, enhancing viral replication and affecting the expression of Hsp27. Our system could be used in future studies also for delivery of other bioactive drugs. Mol Cancer Ther; 15(4); 651-60. ©2016 AACR