PROJECT SUMMARY – PROJECT 1 The effective treatment of glioblastoma (GBM) using oncolytic Herpes Simplex Virus (oHSV) will almost certainly require both broad intratumoral virus spread and tumor destruction and virus-induced anti-tumor immunity. The tumor microenvironment (TME) must be supportive of adaptive immune responses through the recruitment of active tumor antigen presenting cells and responsive CD8+ T cells. However, GBM tumors are notoriously “cold” tumors that are metabolically adept at preventing the development of a pro-inflammatory TME. This immune- suppressive GBM TME exhibits upregulated expression of (1) the checkpoint proteins TIGIT and PD-1, and their respective inhibitory ligands, and (2) the ectoenzymes CD39 and CD73 that increase local production of adenosine (ADO); both pathways confer immunosuppression and tumor aggression. Here we propose to improve the anti-tumor activity of rQNestin34.5v.1 an F strain oHSV-1. This virus has been engineered to allow tumor-specific expression of natural viral genes that thwart innate anti-viral cellular responses (e.g. ICP34.5). rQNestin34.5v.2, a modified version of rQNestin34.5v.1 with GFP removed, was shown to be safe in a patient trial with evidence of intratumoral viral antigen expression for extended time periods (NCT03152318). Three aims are designed to evaluate rQNestin34.5v.1 enhancement in patient derived xenograft (PDX) models and in syngeneic GBM mouse models. We propose in Aim 1, to test the hypothesis that syncytial mutant variants of rQNestin34.5v.1 will exhibit enhanced oncolytic activity, augmenting viral spread and persistence within the tumor. In Aim 2, we propose to test the hypothesis that the therapeutic efficacy of rQNestin34.5v.1 can be improved by arming the vector with a combination of IL-12, the checkpoint inhibitors (CPI) anti-PD-1 and anti- TIGIT (Aim 2A), or with the adenosine deaminase (ADA) gene (Aim 2B). The efficacy of ADA-armed oHSV will be tested in combination with oHSV expressing PTEN and anti-CD73 (oHSV-P10-CD73; Project 3) and depending on the outcome, both arming genes will be introduced into a single vector for treatment efficacy studies. Analysis of human clinical trial data has allowed Project 2 to define a TME profile characteristic of rQNestin34.5v2 responders, demonstrating a correlation between patient response and increased TCR diversity. In Aim 3, we will test the hypothesis that rQNestin34.5v.1 and armed derivatives will increase the accumulation of effector T cell populations and induce clonal expansion of T cells that recognize viral- and tumor-specific antigens. Aim 3 analyses will compare the armed vectors generated in Aim 2 to those generated by Projects 3 and 4 and will allow us to establish correlations across multiple oHSV variants with respect to clonal T cell expansion and animal survival. The proposed vector modifications may substantially improve rQNestin34.5v.1 performance and effectively increase the number of tumors respon...