7. Project Summary/Abstract In a collaborative research project that I initiated between the labs of Prof. Paul Sondel and Prof. Paul Harari during my residency training, we explored a novel approach to augmenting anti-tumor immune response by combining two established cancer treatments, radiation therapy (RT) and tumor-specific antibodies (mAbs). In single-tumor murine models of melanoma, neuroblastoma, and head and neck squamous cell carcinoma we observed a cooperative anti-tumor interaction between local RT and intratumoral (IT) injection of tumor-specific mAb resulting, in part, from enhanced antibody-dependent cell-mediated cytotoxicity. To further augment this response, we investigated combined RT and IT-immunocytokine (IC), a fusion-protein linking tumor-specific mAb to IL2. With this we observed a greater anti-tumor immune response resulting in complete regression of established (~5 week post engraftment) tumors in most animals and a memory T cell response that rejected re-challenge with similar tumor cells. This demonstrated that combined RT + IT-IC can elicit a potent “in situ” tumor vaccine response. T cell checkpoint blockade is becoming a standard of oncologic care in certain cancer settings and we therefore tested the benefit of adding local RT and IT-IC to systemic checkpoint blockade in our murine melanoma model. In mice bearing large primary tumors (~ 7 week post engraftment) or microscopic distant sites of disease (IV injected on the day of RT), the triple-combination of primary tumor RT followed by IT-IC injection of this tumor and systemic T cell checkpoint blockade with anti-cytotoxic T- lymphocyte antigen-4 (CTLA-4) antibody improved animal survival compared to combinations of any two of these three interventions. However, in the presence of established (~3-week post engraftment) distant sites of disease we have now observed a key limitation to our in situ vaccination strategy. In the presence of an un- radiated second macroscopic tumor, the combination of primary tumor RT + primary tumor IT-IC injection is no more effective than primary tumor RT alone. This suggests that an untreated, distant second tumor may suppress the generation of an anti-tumor immune response in the primary tumor following RT + IT-IC. We describe this as “concomitant immune tolerance” (CIT). Here I hypothesize that 1) in situ vaccination may be achieved using local RT and IT injection of tumor- specific mAb with IL2, 2) regulatory T cell (Tregs) harbored in an established, macroscopic, distant tumor site may exert CIT, and 3) delivering external beam RT (EBRT) and IT-IC to a mouse’s primary tumor, together with IV 131I-NM404, a molecular targeted radiotherapy (MTRT), will eliminate CIT and enable an effective in situ vaccine response to eradicate all tumors in mice with primary and distant sites of disease. I propose to test these hypotheses in my independent research lab using establish...