There are thousands of patients with glioblastoma (GBM) being diagnosed each year and despite current standard therapy of maximal surgical resection, radiation, and chemotherapy, have a median survival of less than 15 months. While immune checkpoint inhibitors (ICIs) have led to significant improvements in survival in a number of systemic cancers, their effectiveness has been less dramatic in GBM. Predictive metrics to determine which patients will better respond to ICIs, and effective immunotherapy targets are lacking in GBM. The overall objective of this application is to identify immunogenomic determinants of response to ICIs in order to identify patients who are most likely to benefit from these therapies. The central hypothesis is that the immunogenicity of GBM neoantigens, and temporal changes in neoantigen-specific immune responses during ICI therapy are correlated with clinical response. The rationale for this project is that better identification of patients who will respond to ICIs will improve the efficacy of targeted immunotherapy and allow for development of novel personalized therapies. This proposal will leverage tumor and matched blood specimens from a NIH-funded trial studying the efficacy of combination ICIs in GBM to pursue the following specific aims: 1) identify neoantigens that elicit expansion of neoantigen-specific T cell clones and determine the immunogenicity of lost or gained neoantigens during IC blockade, (2) identify shared neoantigen-specific T cell clones between the blood and tumor and quantify the temporal changes of these clones with IC blockade, and (3) determine the association of the diversity and proliferation of these shared clones with response to ICIs. Putative neoantigens will be identified through whole exome sequencing and application of a neoantigen prediction platform of pre-treatment tumors and their immunogenicity will be assessed by evaluating the expansion of neoantigen-specific T cell clones using next-generation sequencing of the TCR-Vβ CDR3 region following in vitro peptide stimulation. Utilizing this novel assay and bioinformatics platform, the TCR-Vβ CDR3 region will be used as a barcode to identify shared neoantigen-specific T cell clones between the blood and tumor, track and quantify the proliferation or depletion of these clones with IC blockade, and determine the association of the diversity and proliferation of these clones with response to ICIs. The research proposed in this application is innovative in that it uses a novel, quantitative, sensitive, and specific technique to determine immunogenicity of neoantigens at a frequency undetectable by conventional assays. The proposed research is significant because it will identify which neoantigens are most strongly associated with T cell proliferation and identify immunogenomic biomarkers of ICI response. Such knowledge will allow for selection of patients most likely to benefit from ICI therapy and offer new targets for novel personalize...