SUMMARY: PROJECT 1 Through our pioneering work with the anti-glioma oncolytic virus Delta-24-RGD, it is now recognized that oncolytic viruses are potent anti-cancer immunotherapeutic agents, acting not only by direct oncolysis but also by inducing an anti-glioma immune response. This paradigm-shifting concept implies that oncolytic viruses, including Delta-24-RGD, alter the immune tumor microenvironment (TME) and may therefore induce synergistic effects when combined with other immunotherapy strategies. However, there is a significant gap in knowledge regarding the ideal agent to use in combination with Delta-24-RGD. In this content, we have recently engineered natural killer (NK) cells (called eCB-NK-TF-/GC- cells) for glioblastoma (GBM) which are resistant to tumor- and corticosteroid-induced immunosuppression by inactivation of TGFβ-Receptor 2 and the glucocorticoid receptor 2, respectively. In our current SPORE, we initiated a dose escalation/window-of-opportunity clinical trial of eCB- NK-TF-/GC- cells as a single agent in patients with recurrent GBM. In parallel pilot experiments exploring the interaction of Delta-24-RGD and NK cells, we have shown that treatment with Delta-24-RGD increased the recruitment, anti-cancer activity, survival, and memory of endogenous NK cells. Importantly, treatment with Delta-24-RGD followed by eCB-NK-TF-/GC- extended the survival of glioma-bearing mice compared with controls. Based on these preliminary data, we hypothesize that Delta-24-RGD treatment will actively contribute to enhancing the activity and memory of off-the-shelf eCB-NK-TF-/GC- cells resulting in strong anti-tumor activity without undue toxicity in patients with GBM. To test this hypothesis we will: Leverage the post-treatment biological specimens obtained from the window-of-opportunity arm of our ongoing Phase 1 clinical trial to evaluate the fate of eCB-NK-TF-/GC- cells as a single agent and to determine the associated immune changes in the TME of patients with recurrent GBM (Aim 1); Explore the combination of Delta-24-RGD and eCB-NK-TF- /GC- cells in a variety of preclinical animal models to evaluate the impact on the TME in GBM (Aim 2); And execute a phase I clinical trial with a window-of-opportunity arm of intratumorally administered Delta-24-RGD in combination with eCB-NK-TF-/GC- cells in patients with recurrent GBM (Aim 3). The samples obtained from the combination trial (Aim 3) will be compared with the samples from the single agent trial (Aim1) to assess the ability of Delta-24-RGD to alter the TME and enhance the activity of eCB-NK-TF-/GC- cells in patients. Our SPORE team has a consistent track record of translating therapies from the laboratory to the clinical setting, and this project is the next critical step in achieving our long-term goal of making viro-immunotherapy a standard treatment for malignant glioma.