Background Glioblastoma is the most common primary brain malignancy in adults, exhibiting aggressive behavior and an immunosuppressive tumor microenvironment that impedes therapeutic efficacy, with a 5-year survival of 6.8%. There are currently a variety of immunotherapeutic treatments being explored as possible strategies for inducing anti-tumor immune responses in glioblastoma and other high-grade gliomas (HGG), many of which involve directing immune responses against tumor antigens. Though significant progress has been made in the field mapping landscapes of tumor-associated antigens and neoantigens across cancers and patients, less is known about how tumor antigen landscapes change after administration of cancer immunotherapy. Oncolytic viruses form a class of immunotherapy capable of releasing tumor antigens and have demonstrated potential in solid tumors. Thus, in this work we sought to define the extent to which oncolytic virotherapy alters the composition of tumor antigen landscapes in syngeneic mouse models of high-grade glioma.

Methods Using the CT2A and GL261 models of HGG, we intracranially implanted tumors into C57BL/6 mice and treated them with vehicle control or oncolytic herpes simplex virus (oHSV) C134, which our group developed and is currently under study in a phase I clinical trial for glioblastoma (NCT03657576). We then harvested tumors at multiple post-treatment timepoints, performed RNA sequencing and whole-exome sequencing, and employed the Personalized Variant Antigens by Cancer Sequencing (pVAC-Seq) computational pipeline to predict antigens across the genomes of these tumors. We also performed immunophenotyping of these tumors using single cell RNA sequencing.

Results Here we present evidence that oHSV treatment decreases the abundance of a subset of neoantigens with high predicted binding affinity to MHC (ic50 <500 nM) in multiple models of HGG, consistent with antigen-specific immune killing of tumor cells expressing such antigens.

Conclusions Taken together with single cell RNA sequencing data revealing C134-induced increases in gene expression signatures of antigen presentation and response, these data suggest oHSV therapy poises the tumor immune microenvironment for recognizing and responding to multiple tumor antigens and may cause induction of immune responses that reduce or eliminate cells containing these antigens from the tumor. This work has the potential to reveal novel oHSV-modified HGG antigen targets and advances the understanding of how oHSVs may be harnessed to improve therapeutic outcomes for patients with glioblastoma.

Ethics Approval All animal experiments were approved by the Nationwide Children’s Hospital Institutional Animal Care and Use Committee (AR16-00057).