Title: Development of recombinant VSV-based oncolytics for cancer immunotherapy
Abstract:
Oncolytic viruses (OV) are a relatively novel class of anti-tumor therapies. Their selective replication in tumor cells and activation of the host immune response lead to the death of only malignant cells setting OV-based therapies apart from the non-specific conventional chemo-/ radiotherapies and even target-specific antibody-based therapies.
Vesicular stomatitis virus (VSV) offers a promising platform for OV development, especially in combination with immunotherapeutic approaches. In this study, the optimized viral rescue protocol allowed the amplification of the virus in suspension cultures and did not rely on helper vaccinia virus. The oncolytic potency of rVSV-delM51-GFP was tested in syngeneic in vivo model with fifty-two male C57BL/6 mice injected subcutaneously with 1x106 murine melanoma (an aggressive tumor model) B16-F10 cells. Eight days post inoculation, the mice were injected intratumorally with 1x106 TCID of VSV-GFP per mouse (group 1), 1x108 TCID of VSV-GFP per mouse (group 2) or placebo control (group 3) at days 0 and 3. Tumor sizes were measured every 2 days for 31 days. Partially inhibited tumor growth was observed in group 1 and 2 mice as compared to group 3 mice (max 52,5% and 41,8%, respectively, on day 11). The TGI% index in group 1 remained positive at 20-30% until the end of the study, but fell to zero in group 2 by day 25. Statistically significant differences in tumor volumes between groups 1 and 3 remained for 12 days of the study (Mann-Whitney U-test, p<0.05). Overall, the morbidity of mice in group 3 exceeded that of mice in groups 1 and 2. The increase in the median survival of mice in group 1 was 6.1 days compared to group 3 (Kaplan-Meier survival analysis). Virus persisted in melanoma cells for at least 21 day as demonstrated by immunohistochemistry analysis of tumor tissue. OV-induced tumor immunity can be enhanced by delivery of anti-tumor cytokines, such as IL-12 (interleukin -12) and GM-CSF (granulocyte-macrophage colony-stimulating factor). IL-12 induces T-helper 1 (Th1) differentiation and activates interferon (IFN)-γ production in natural killer (NK) cells, CD4+ and CD8+ T lymphocytes. GM-CSF induces the activation of antigen-presenting cells (APCs) and promotes dendritic cell (DC) differentiation. OV-mediated targeted cytokine delivery to the tumors avoids significant toxicity associated with systemic delivery while also boosting the immune response. To achieve this synergistic effect, a novel recombinant VSV (rVSV)-mIL12-mGMCSF, co-expressing mouse IL-12 and GM-CSF, was produced. The cytopathic effect was assessed in BHK21 cells with titer of 2 x 106 TCID50 / ml. Expression of the chimeric mIL12-mGM-CSF protein was detected by Western Blotting. Functional activity of IL-12 produced in viral supernatants of BHK21 cells was assessed by both ELISA and in HEK-Blue™ IL-12 reporter cell line stably expressing IL-12 receptor and signaling pathway genes, as well as the STAT4-inducible SEAP reporter gene. The levels of SEAP reporter expression induced by the rVSV-produced IL-12 were 1.3 times higher than those induced by the commercial IL-12 control used at 10ng/ml.
