Abstract 6654: Oncolytic virotherapy targeting high-grade glioma tumor antigens
Authors: Hedberg Jack , Martin Alexia , Kim Doyeon , Navarro Jason , Rivaldi Adithe , Kim Yeaseul , Hernandez-Aguirre Ilse , Garfinkle Elizabeth , Miller Katherine , Cassady Kevin , Mardis Elaine 
Published: March 2024

Abstract
Glioblastoma, the most common primary brain malignancy in adults, is an aggressive cancer with five-year median survival of ~6.8%. New treatment options are urgently needed for glioblastoma and other high-grade gliomas (HGGs). Oncolytic viruses (OVs) are an emerging class of cancer therapy currently being evaluated as treatments for a variety of malignancies, including glioblastoma. Our labs developed OV C134, now under evaluation in a phase I clinical trial of recurrent glioblastoma (NCT03657576). In addition to directly infecting and lysing tumor cells, OVs are capable of stimulating anti-tumor immune responses. We previously showed in the CT2A syngeneic mouse model of HGG that treatment with a version of C134 engineered to express the shared tumor antigen Ephrin-A2 (EPHA2; commonly upregulated in glioblastoma) significantly increased survival and produced EPHA2-specific T cell responses. This finding led us to hypothesize that C134 could be further engineered to stimulate antigen-specific immune responses against tumor-specific neoantigens. We first assessed the impact of C134 treatment on immune cell dynamics in HGGs, by performing single cell RNA sequencing of CD45+ immune cell infiltrate from CT2A HGGs treated with C134 or saline (control). Clustering of these data identified 17 distinct immune cell types, and differential expression analyses revealed several C134-induced changes. These included increased expression of genes involved in antigen presentation and STAT signaling in microglia and macrophage subsets, the proliferation marker Mki67 in germinal center B cells, the chemokine Ccl12 from microglia, and tissue-resident memory marker genes Fos and Jun in T cells. These data suggest C134 treatment poises the tumor immune microenvironment for antigen presentation and antigen-specific immune responses. Next, we evaluated CT2A to identify candidate tumor neoantigens by isolating DNA and RNA from orthotopic tumors and DNA from tail snips of mice treated with saline or C134. We carried out RNA sequencing (tumors) and whole exome sequencing (tumor and normal), and employed the Personalized Variant Antigens by Cancer Sequencing (pVAC-Seq) computational pipeline. This pipeline takes somatic variant calls from the exome comparison of tumor to normal DNA, validates RNA expression of called variants, and feeds corresponding peptide sequences into algorithms that predict the binding of variant-containing peptides to major histocompatibility complex (MHC), thereby assessing for potential neoantigens. Our pVAC-Seq analysis revealed a conserved set of 1,264 tumor-exclusive variants expressed at the RNA level with high (ic50&lt;500nm) predicted binding to mouse MHC. I will present details of C134-induced immune remodeling in syngeneic HGG models, and describe a plan to engineer C134 to express a prioritized set of neoantigens to evaluate how efficacy from OV therapy may be further enhanced in a patient-specific manner.
Citation Format: Jack Hedberg, Alexia Martin, Doyeon Kim, Jason Navarro, Adithe Rivaldi, Yeaseul Kim, Ilse Hernandez-Aguirre, Elizabeth Garfinkle, Katherine Miller, Kevin Cassady, Elaine Mardis. Oncolytic virotherapy targeting high-grade glioma tumor antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6654.