Project Summary Immunotherapy has revolutionized the treatment of a variety of advanced cancers, especially immune checkpoint blockade (ICB). Although newly FDA approved antibodies that block CTLA-4 or PD-1 offer hope for some patients, not all patients respond, and others relapse after initial response. Consequently, there is a great importance in evaluating immune checkpoint blockade with other therapies that trigger immune activation. Based on extensive preclinical and clinical results from our group, we are focused on the combination of immune checkpoint blockade with hypofractionated radiotherapy (HFRT). The rationale for this approach is emerging evidence from our group and others that irradiation of tumors can stimulate the immune system, perhaps by releasing tumor-associated antigens which may allow for better response to the immunomodulatory agents. In a previous phase I trial of ipilimumab with HFRT in patients with advanced melanoma, we observed some cases of deep objective responses, reminiscent of prior case reports, but only in 18% of patients. In extensive preclinical experiments in which we modeled the interactions of radiation and ICB, we found across multiple histologies (breast and pancreatic cancer, melanoma) that blockade of CTLA-4 and PDL-1/PD- 1 in combination with HFRT achieved major tumor regressions and complete responses in mice without major toxicity (Tyman-Saint Victor, Nature, 2015). Both CTLA-4 and PD-L1 blockade are required for optimal therapy as each modality non-redundantly improves response and immunity. PD-L1 blockade is especially important to overcome immune resistance, as our data show that efficacy of HFRT plus CTLA-4 blockade alone is limited by PD-L1 expression in the tumor, a finding validated in samples from our initial trial. Based on these pre-clinical data, we propose two immediate clinical trials in collaboration with Core C combining agents that block CTLA-4 (ipilimumab or tremelimumab) or PD- 1/PD-L1 (nivolumab or durvalumab) with HFRT: (Aim 1) a phase 2 randomized trial of ipilimumab and nivolumab with or without HFRT in patients with metastatic melanoma, and (Aim 2) a phase 1 trial of tremelimumab and durvalumab in patients with metastatic pancreatic, lung, and breast cancer. In Aim 3, we will apply a comprehensive plan for immune assessment with collaborations across all Projects and Cores in this P01 to determine the immunological mechanisms of our approach. The expected clinical and immunological findings will significantly advance the development radiation and immune checkpoint therapy for patients. 1