PROJECT 2: SUMMARY The glioblastoma (GBM) microenvironment is dominated by myeloid cell infiltrates. Results from multiple studies indicate these tumor-associated myeloid cells (TAMS) as supporting GBM growth. The goal of this project is to reprogram TAMS for immunologic anti-tumor activity. Stimulator of interferon genes (STING) is a widely expressed sensor of cellular stress that is activated by the presence of DNA in the cytoplasm. Distinct from most other immune agonists, STING activation re-educates tumor supportive M2 macrophage TAMS toward a proinflammatory anti- tumor M1 phenotype. Macrophage proinflammatory phenotypic conversion, in turn, promotes cytotoxic T cell infiltration of and activity against tumor. We have developed a high potency STING agonist, IACS-8803, with marked antitumor activity when tested in humanized mice bearing human GBM, and in canines with spontaneously arising high-grade gliomas. In addressing the clinical potential of this agonist in treating GBM, we will first determine its effect on interferon responses, using [18F]FLT PET, when IACS-8803 is administered to patients with recurrent tumor. This first-in-man Phase I clinical trial will inform regarding the range in IACS-8803 activity that is observed across the cohort of treated patients, with activity results compared against tumor molecular characteristics, and patient clinical data. The clinical trial will include analysis of several unique endpoints, among which are target engagement and longitudinal kinetics of IACS-8803 induced T cell chemokine expression such as CXCL10. In addition, a window-of-opportunity patient cohort will receive direct intratumoral administration of IACS-8803, and whose results will be compared against those from patients that have received systemic administration of standard-of-care therapeutics. The PET Imaging results will be analyzed with respect to inflammatory immune response in resected tumors from active vs. non-active tracer regions in post-STING treated subjects, using multiplex immunofluorescence, CyTOF, and/or mass cytometry. This clinical study will ultimately provide sufficient data to make a clear go/no go determination for later-stage clinical trials based on sufficient target engagement in the tumor microenvironment. While conducting this clinical trial, we will move forward with preclinical research by evaluating the efficacy of combined IAC-8803 + radiation in orthotopic models of GBM. Results from these preclinical studies will inform whether STING agonist and radiation treatment should be tested in patients with newly diagnosed GBM, and whose tumors have unmethylated MGMT promoter, and as such, do not require treatment with temozolomide.