Immunogenomic Determinants of Immunotherapy Response in Glioblastoma

The clinical impact of immune checkpoint blockade (ICB) across solid tumors has not extended to glioblastoma (GBM), the most common and lethal primary brain malignancy in adults, where multiple phase III trials of anti-PD-1 therapy have failed to improve overall survival. The basis on which immunotherapy succeeds in some GBM patients and fails in others, and the strategies by which productive anti-tumor immunity might be generated in patients for whom ICB is insufficient, remain incompletely defined. Here, we describe three integrated studies: a multi-omic analysis of ICB-treated GBMs, a clinical trial of personalized neoantigen vaccination combined with anti-PD-1, and the development of a spatial transcriptomics platform that resolves neoantigen-directed immune interactions in situ. To define the immunogenomic features that govern ICB outcome in GBM, we profiled 181 ICB-treated tumors by whole-exome sequencing, bulk transcriptomics, and single-nucleus RNA sequencing, with paired pre-treatment and post-recurrence specimens. Established genomic biomarkers of ICB response, including tumor mutational burden, did not predict survival; baseline bulk transcriptional subtype did. The mesenchymal (MES) program was associated with prolonged survival following ICB but not standard chemoradiation, with MES-like malignant cells marked by high HLA class I expression and greater T cell infiltration. Longitudinal profiling identified a MES-to-non-MES transition as a trajectory of acquired resistance to ICB. To assess whether productive anti-tumor immunity can be generated in GBM through induction of de novo and amplification of pre-existing anti-tumor T cell responses, we conducted a phase I trial of personalized neoantigen vaccination (NeoVax) combined with pembrolizumab in 39 patients with newly diagnosed GBM, across four cohorts varying pembrolizumab timing and MGMT-stratified temozolomide. Vaccination induced broad neoantigen-specific T cell responses associated with longer overall survival, and putative vaccine-reactive clonotypes trafficked to and persisted within post-vaccination tumors across all cohorts. Earlier initiation of pembrolizumab relative to vaccine priming was associated with longer survival, and the MGMT-methylated, temozolomide-treated cohort exhibited a notably long median overall survival of 36.9 months. At the neoantigen level, post-vaccination tumors exhibited contraction of vaccine-targeted subclones, consistent with vaccine-driven immunoediting; at the cellular level, the surviving malignant compartment shifted toward greater immunogenicity, with loss of cold non-MES programs and upregulation of HLA class I on surviving tumor cells. Intratumoral vaccine-reactive T cells were predominantly CD4 tissue-resident memory cells localized to peri-necrotic, macrophage-rich niches characterized by interferon-driven chemokine signaling. Although these analyses provided evidence of vaccine-driven antigenic selection, existing immunogenomic technologies could not directly resolve the spatial relationships between neoantigen-specific T cells and their cognate tumor targets at single-cell resolution. We therefore developed Slide-GoTags, a droplet-based spatial transcriptomics platform that integrates targeted transcript genotyping and T cell receptor (TCR) sequencing with single-nucleus RNA sequencing. Application of Slide-GoTags to murine and human tumors revealed colocalization of clonally expanded, neoantigen-specific T cells with tumor cells expressing their cognate neoantigens within interferon-driven immunogenicity niches, providing a framework for the direct observation of productive tumor-immune engagement in situ. Together, these studies define determinants of response and resistance to immunotherapy in glioblastoma, demonstrate that personalized neoantigen vaccination can engage productive anti-tumor immunity within the immunologically cold GBM microenvironment, and establish a technological foundation for resolving the spatial architecture of neoantigen-directed immunity. MES-like features emerge as an important correlate of response across both ICB and combination neoantigen vaccination strategies, while baseline tumor-infiltrating T cells are no longer requisite when anti-tumor T cell responses are induced or amplified through vaccination. These findings advance the rational selection of patients for GBM immunotherapy, support the integration of personalized neoantigen vaccination into clinical practice, and provide tools for studying spatially resolved anti-tumor immunity across solid tumors.