PROJECT SUMMARY ? Project 1 We recently reported in Nature that patients with glioblastoma (GBM) randomized to receive a vaccine against Cytomegalovirus (CMV) major integument protein pp65 using a tetanus/diphtheria (Td) vaccine site preconditioning regimen had a statistically significant increase in progression-free survival (PFS) and overall survival (OS) in a small but randomized, blinded, and controlled trial. Half of the patients treated this way were still alive nearly 5 years later despite only 10% of patients typically surviving past 5 years. We targeted CMV because many different groups, including our own, had shown that CMV antigens (Ags), like the immunodominant pp65, are found in GBM, but not surrounding normal brain; this suggests CMV pp65 could be subverted as a highly immunogenic and often homogeneously expressed target for anti-tumor immunotherapy. In our preliminary study, combined with in-depth mechanistic studies in mice, we demonstrated that preconditioning the vaccination site with Td recall Ags increased DC migration to the draining lymph nodes (DLNs), which predicted PFS and OS. Mechanistic studies in mice revealed that the antitumor efficacy of these vaccines was dependent on the vaccinating Ag being present in the tumor, underscoring pp65 as a target in GBM. Efficacy was also dependent on a Td recall response and high systemic levels of the chemokine (C-C motif) ligand 3 (CCL3), which was the only immune mediator elevated in mice and patients. We believe these data warrant confirmation in our proposed Phase 2 trial with a larger series of patients. This will also allow us to confirm some of the mechanistic findings in human patients. However, systemic immunosuppression mediated in part by elevated levels of regulatory T cells (TRegs) in patients with GBM still likely limits vaccine efficacy. Recently, we and others have demonstrated that a clinical- grade antibody targeting CD27 specifically depletes TRegs in transgenic mice and humans. We have also demonstrated that, unlike clinical approaches targeting CD25 to deplete TRegs, that the anti-CD27 antibody simultaneously increases vaccine-induced immune responses. Moreover, it specifically coordinates CD4+ and CD8+ T cell responses leading to enhanced vaccine-induced immunogenicity and increased survival in mice with established orthotopic glioma. Overall, we hypothesize that Td preconditioning will increase DC migration, systemic CCL3, and OS, and that TRegs will be reduced while CMV vaccine responses are further enhanced when a novel anti-CD27 mAb is added to this regimen.