Summary The critical role of the innate immune system in priming CD8+ T cells to generate tumor specific responses underscores a potentially important clinical strategy for the development of next-generation immunotherapies. In this proposal, we pursue the hypothesis that small molecules identified in a high throughput screen for innate immune agonists can significantly improve the therapeutic efficacy of the oncolytic NDV (NewCastle Disease Virus) through activation of antigen presenting cells (APCs), and enhancement of anticancer immune responses. To address this, we propose specific aims that focus on hit validation through both ex vivo and in vivo characterization of compounds and assessing the in vivo efficacy in a B16-F10 mouse melanoma model. First, utilizing orthogonal assays, we will validate the immunopotentiation properties of compounds in ex vivo human primary monocyte derived dendritic cell (MDDC). Specifically, we propose to assess compound effects on MDDC activation and maturation through examination of transcriptional profiles, expression of DC activation markers, and cytokines being secreted upon compound treatment. Validated hits will then be clustered based on their activities, and the most potent compounds from each cluster will be carried forward to anti-tumor efficacy testing in a mouse melanoma model. Compounds will be added either alone or together with intratumoral injection of oncolytic NDV. Compound that synergize and enhance the anti-tumor activity of NDV, provide durable protection, and abscopal activity will be prioritized for subsequent early lead optimization. Based on data generated from these hit validation approaches, we propose to delineate ex vivo immune signatures that can be used as surrogates for in vivo efficacy. The immune signatures elicited by selected hits within both MDDCs (ex vivo) and the tumor microenvironment (TME - in vivo) will be integrated to construct a computational model to assess correlative signatures that are able to link MDDC molecular and phenotypic responses to in vivo efficacy. Similar analysis will be conducted on immune activation readouts in the TME. This will be a reiterative process, with information obtained from the experimental studies will be utilized to refine predictions of ex vivo and in vivo biomarkers that correlate with efficacy. The information gained from this proposed study upon completion will significantly facilitate further hit-to-lead and lead optimization activities during subsequent phases of the drug development process.