PROJECT SUMMARY Recent advances in cancer immunotherapies have revolutionized lung cancer treatment paradigms. However, many patients derive little or no benefit from FDA approved immune checkpoint inhibitors (ICI). A host of clinical studies have shown that pre-existing tumor-reactive T cells are critically important to achieve benefit from ICI. Therefore, augmenting the number and functionality of tumor-reactive T cells could pave the way for more effective cancer treatments. Oncolytic viruses (OVs) have been developed for their ability to specifically replicate in cancer cells leading to cancer cell lysis but not cytotoxicity towards normal cells. Recent studies indicate that stimulation of host anti-tumor immunity is also a key mechanism of action of OVs. Using a novel conditionally replicative adenovirus type 5 that expresses two powerful innate immunity stimulators, the studies proposed here can lead to the development of highly effective OV-based therapeutic strategies to kill tumor cells and simultaneously enhance an anti-tumor T cell response. Engagement of CD40 expressed on dendritic cells (DCs) by CD40 ligand (CD40L) leads to acquisition of crucial cross-priming function to activate CD8 T cells. MEM40 is a chimeric CD40L engineered for stable cell surface expression. In addition to pro-inflammatory and anti-viral functions, type 1 IFNs also function as crucial activators of DCs. Our preclinical studies in mouse melanoma and lung tumor models indicate that intralesional administration of MEM40 + IFNβ expressing oncolytic adenovirus (MEM-288) we developed induces a robust systemic T cell response that in synergy with ICI is capable of significantly curtailing abscopal tumor growth. A key effect of CD40L and IFNβ expression in the tumor microenvironment (TME) was the enhancement of DC activation and migration to draining lymph nodes to induce T cell priming. These findings prompted us to develop a novel conditionally replicative oncolytic adenovirus (MEM-288) that expresses a membrane-stable CD40L (MEM40) and IFNβ. A phase 1 first-in-human dose escalation clinical trial of single agent MEM-288 is nearing completion while a combination trial of MEM-288 with ICI in NSCLC patients is expected to begin in Fall 2023. A primary goal of studies proposed here is to define MEM-288 clinical activity in NSCLC patients from both trials and assess immune stimulatory effects of MEM-288 in the tumor microenvironment (TME) and ability to generate a systemic anti-tumor T cell response. Two Specific Aims are proposed: Aim 1: Defining impact of in situ activation of CD40 and type 1 IFN signaling on DC and T cell responses in tumor models. Aim 2: Impact of oncolytic virus (MEM-288) induced CD40 and type 1 IFN signaling on TME remodeling and systemic T cell responses in NSCLC patients.