PROJECT SUMMARY The overarching goal of this project is to quantify the temporal kinetics of T-cell activation and infiltration during novel combination intratumoral oncolytic herpes simplex virus (oHSV) immunotherapy with advanced molecular immuno- positron emission tomography (PET) imaging in preclinical models of glioblastoma (GBM). Novel targeted oHSV immunotherapy has been shown to directly kill GBM tumor cells as the virus selectively replicates within and lyses malignantly transformed cells, however there is a knowledge gap in understanding the kinetics of the immune cell changes and how to harness those changes to improve therapeutic efficacy. As inflammation and pseudo progression are key characteristics of immunotherapy-induced tumor changes, standard imaging methods fail to provide reliable response assessments, which can sometimes take up to six months to reveal through clinical changes in tumor size. Advanced quantitative imaging strategies can provide spatial and temporal information on biological alterations prior to the clinically observed, downstream changes in tumor size. Immuno- PET imaging could stratify selection of patients who pursue immunotherapy and guide the timing and sequencing of combinatorial therapies. We have preliminary data showing that there are differential responses in CD8 expression in gliomas during oHSV therapy and that oHSV increases CD8 infiltration, as we can image these changes with PET. Further, preliminary evidence shows that granzyme B increases (as measured by GZP-PET) are related to overall tumor response. The overarching hypothesis is PET molecular imaging can guide targeted IL-12 and IL-18 oHSV to enhance therapeutic efficacy and extend survival in preclinical models of GBM. There are three specific aims to answer this hypothesis: Aim 1. Using advanced molecular immunoPET in GBM murine models with biological validation, determine if early T-cell kinetics of infiltration and activation are increased following oHSV therapy alone, or with oHSV expressing IL-12 and/or IL- 18. We will quantify longitudinal alterations in T-cell infiltration with [89Zr]-CD8-PET imaging of CD8+ cells and quantify longitudinal alterations in T-cell activation (granzyme B) with [68Ga]-GZP-PET imaging. Aim 2. Using advanced molecular immunoPET in GBM murine models, determine if secondary therapeutics (radiation therapy, or immunomodulatory immune checkpoint inhibitors) used in combination with oHSV IOT produce increased intermediate term T cell infiltration and activation and improve long-term tumor response. Aim 3. Determine if secondary boosts of oHSV IOT during imaging-identified timing windows of decreased T cell infiltration and activation can salvage therapeutic response. ImmunoPET imaging allows longitudinal quantification of underlying immunological kinetics during oncolytic herpes simplex virus (oHSV) immunotherapy (IOT) that will allow optimization of therapeutic regimens on a personalized basis.