Abstract Glioblastoma (GBM) is the most common primary brain tumor, accounting for 54% of malignant glioma diagnoses. Over the course of the past 30 years, a diagnosis of GBM has remained fatal even after maximum surgical resection, radiotherapy (RT), and chemotherapy, with a median overall survival of 14.6 months. The immunosuppressive microenvironment of GBM is a major contributor to the poor patient outcome. Expression of IDO1, as well as the accumulation of tumor-infiltrating regulatory T cells contribute to the avoidance of immune surveillance. Although current immunotherapies have had some success in extending patient survival, combinatorial treatment approaches addressing both tumor growth and the potent immunosuppression may prove to be more effective. This work aimed to determine the efficacy of a novel, pharmaceutical-grade, blood brain barrier-penetrating small molecule IDO1 inhibitor, BGB-5777, in combination with PD-1 blockade and/or whole brain radiation in an immunocompetent mouse GBM model. METHODS/RESULTS: All mice were intracranially-engrafted 2×105 GL261 (syngeneic to B6 background) cells to recapitulate brain tumors. At 14 days post-intracranial injection (dp-ic.), mice were treated with IgG alone as a control (n = 7), 2Gy RT for 5 days (n = 8), 500 ug (loading dose), followed by three 200 ug maintenance doses given every 3 days, of PD-1 mAb (J43) (n = 8), or 100mg/kg BGB-5777 for 4 weeks (n = 10), with a median overall survival (OS) of 25, 25, 32, and 26.5 days, respectively. Mice treated with dual therapies including RT and PD-1 mAb (n = 10), RT and BGB-5777 (n = 8), or PD-1 mAb and BGB-5777 (n = 9), had a median OS of 30, 39, and 32 days, respectively. All mice treated with mono- or dual-therapy succumbed to tumor burden. In contrast, mice treated with concurrent RT, PD-1 mAb and BGB-5777 (n = 9) showed a significant increase in median OS to 53 days (P<0.0001) with 33% of mice demonstrating a durable survival benefit of more than 150 days. Importantly, mice treated with the triple therapy and co-administered CD4 (n = 9)-, but not CD8 (n = 9)- or NK1.1 (n = 9)-depleting mAb, significantly decreased median OS to 29 days (P<0.001) confirming the requirement of CD4+ T cells for efficacy of this immunotherapeutic approach. CONCLUSION: The data indicate that combining the standard of care agent, RT, with PD-1 blockade and the novel IDO1 inhibitor, BGB-5777, synergistically increases OS in a mouse GBM model. Unexpectedly, CD4+, rather than CD8+ T cells, are required for immunotherapeutic efficacy. Understanding how CD4+ T cells coordinate anti-GBM immunity, the kinetics of PD-1 and IDO1 expression after RT, and determining why CD8+ T cells are dispensable under these conditions, are current areas of active investigation in our laboratory. Ultimately, these data suggest that using radiation to induce potential immunogenicity and/or inflammation in GBM, while co-inhibiting immunosuppression, is a rational and potentially clinically-beneficial pursuit. Citation Format: Erik Ladomersky, Lijie Zhai, Galina Gritsina, Kristen L. Lauing, Matthew Genet, C. David James, Derek A. Wainwright. A novel IDO1 inhibitor combined with targeted immunotherapy durably increases survival in a mouse model of glioblastoma abstract. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B018.