Oncolytic Virus‐Engineered Extracellular Vesicles Rewire Glioblastoma Immunity
Klaudia Kiel, Arpita Balakrishnan, Aleksandra Cabaj, Luiza Stanaszek, Maciej Przybylski, Dawid Stępnik, Marta Małuszek, Christopher Jannotta, Chiara Zoe Linke, Joanna Suszyńska‐Zajczyk, Monika Knaur, Agnieszka Szczypiorowska, Szymon Hryhorowicz, Bianka Świderska, Katarzyna Piwocka, Alexander Lee Ling, E. Antonio Chiocca, Agnieszka Bronisz, Jakub GodlewskiABSTRACT
Immunotherapy with oncolytic Herpes simplex virus type 1 (oHSV‐1) shows promise for glioblastoma; however, clinical benefit remains uncommon due to tumour‐ and host‐mediated immunosuppression, which limits immune targeting of residual glioblastoma stem‐like cells (GSCs). To address this barrier, extracellular vesicles (EVs) harvested from oHSV‐1‐infected GSCs (EVOV) were evaluated for their immunomodulatory capacity by priming peripheral blood mononuclear cells (PBMCs) ex vivo and in vivo . In GSC‐PBMC organoids analysed by single‐cell RNA sequencing, IFN‐response and antigen‐presentation programs were induced in both immune and tumour compartments, despite the GSC state. EVOV organoid transcriptome associated with patient tumour antigen‐processing programs linked to improved clinical outcome, and EVOV immunopeptidomics identified candidate biomarker peptides that were also detected in patient immunopeptidomes from the clinical‐trial cohort. In PBMC‐engrafted NOD scid gamma mice bearing intracranial GSC xenografts, EVOV delivery increased circulating interferon (IFN) levels and enhanced systemic activation of T‐cell subsets, while spatial transcriptomics revealed recruitment of peripheral immune cells to the tumour, accompanied by reprogramming of GSC states. Blocking vesicle‐associated major histocompatibility complex I (MHC‐I) attenuated EVOV‐driven PBMC cytotoxicity against GSCs, supporting an MHC‐I peptide‐dependent mechanism of immune priming and a vaccine‐ or adjuvant‐like, non‐infectious, vesicle‐based immunotherapeutic strategy.