Project Summary: Oncolytic virotherapy (OV) represents a novel method to treat a variety of solid tumors by inducing anti-tumor immune responses. While this therapy has been extremely efficacious in a wide variety of preclinical models, translating these successes into human patients has proven challenging. It has recently become clear that one of the major reasons for these failures is the existence of immune-regulatory mechanisms which dampen the efficacy of virally induced anti-tumor immunity. However, the exact nature of these regulatory pathways remains unclear. Our lab has previously developed a novel oncolytic MYXV which expresses a soluble PD1 inhibitor and an IL12 fusion protein (vPD1/IL12). This construct is highly effective at treating disseminated disease, however, ~50% of tumor models remain at least partially non-responsive and viral treatment is associated with the development of immune-related adverse events. In our follow-up studies into the mechanisms mediating the efficacy of vPD1/IL12 we have observed that the virus induces high levels of TNF during therapy. Strikingly, both genetic elimination or antibody-based blockade of this TNF results in a significant INCREASE in therapeutic efficacy as well as a significant REDUCTION in toxicity. To our knowledge, no other studies have demonstrated a positive impact of TNF blockade on OV and therefore both the mechanism(s) involved as well as how to leverage this finding into improved clinical outcomes remains unclear. We therefore put forth the current proposal which contains three specific aims designed to build off of our exciting preliminary data by: identifying the mechanism through which TNF restricts OV efficacy, understanding how TNF mediates OV- induced toxicities, and determining clinically applicable methods to apply TNF-blockade during OV. This work will advance not only the clinical use of our existing vPD1/IL12 virus, but also improve our understanding of the basic mechanisms involved in successful OV.