PROJECT SUMMARY – PROJECT 2 Glioblastoma (GBM), arguably the deadliest of all cancers, has remained impervious to treatments, including immunotherapies that have seen evidence of success in other cancers. The profound immunosuppressive microenvironment in GBM thwarts immune activating stimuli like immune checkpoint blockade and significantly limits activated T cell tumor infiltration. In fact, GBM has been described as an immune-desert. As part of this Program Project, we plan to analyze the immune infiltration in clinical specimens from human GBMs injected with a novel oncolytic virus (NCT03152318, clinicaltrial.gov). Oncolytic viruses (OV) are a form of immunotherapy being investigated clinically against multiple cancers with one oncolytic Herpes Simplex Virus (oHSV) approved in the USA against melanoma and a different oHSV approved against GBM in Japan. In the previous funding period, we started and finished a phase 1 clinical trial of the novel oHSV, rQNestin34.5v.2, that accrued 50 human subjects with recurrent high-grade gliomas. Preliminary data from tumors after in situ administration of this oHSV shows increased TILs. In addition, we show that elevated T cell and/or B cell receptor (TCR/BCR) transcripts are associated with improved subject survival. Volumetric analyses of MRIs from subjects also show that growth rate changes correlate with increased response in treated patients. These published and preliminary data thus provide a conceptual framework justifying in situ administration of OVs to revert the immunosuppressive microenvironment of GBM into one whose cellular and molecular components become immuno-activating. Based on the above, we hypothesize that in situ oHSV administration profoundly changes the human GBM microenvironment into one that is more favorable for immunotherapy. We plan to utilize clinical GBM specimens obtained from the current clinical trial to validate the hypothesis, via the following Specific Aims: Aim 1. Validate the immune-activating changes in the human GBM microenvironment perturbed by oHSV in situ administration; Aim 2. Investigate subjects’ plasma proteome, serum cytokine/chemokine and MRI volumetrics as potential biomarkers of oHSV response and correlate with TCR/BCR transcript abundance; and Aim 3. Utilize human GBM patient derived cells and xenografts obtained from current clinical trial patients to characterize HLA-immunopeptidomes after oHSV infection. These aims, if successful, will support the development of oHSV-elicited tumor antigens to boost the immune response to oHSV infection and thereby improve patient outcomes. In addition, we will work with Project 4 to develop the studies needed to bring the novel oHSVs to future clinical trials and with Project 1 to add our discovered oHSV-tumor antigens to the preclinical therapeutic models with armed oHSV. With Project 3, we will measure immunosuppressive metabolites in biopsy specimens.