Harnessing Oncolytic Extracellular Vesicles for Tumor Cell‐Preferential Cytoplasmic Delivery of Misfolded Proteins for Cancer Immunotherapy

Abstract In this study, extracellular vesicles (EVs) are reimagined as more than just a cellular waste disposal system and are repurposed for cancer immunotherapy. Potent oncolytic EVs (bRSVF‐EVs) loaded with misfolded proteins (MPs) are engineered, which are typically considered cellular debris. By impairing lysosomal function using bafilomycin A1 and expressing the respiratory syncytial virus F protein, a viral fusogen, MPs are successfully loaded into the EVs expressing RSVF. bRSVF‐EVs preferentially transplant a xenogeneic antigen onto cancer cell membranes in a nucleolin‐dependent manner, triggering an innate immune response. Furthermore, bRSVF‐EV‐mediated direct delivery of MPs into the cancer cell cytoplasm initiates endoplasmic reticulum stress and immunogenic cell death (ICD). This mechanism of action leads to substantial antitumor immune responses in murine tumor models. Importantly, when combined with PD‐1 blockade, bRSVF‐EV treatment elicits robust antitumor immunity, resulting in prolonged survival and complete remission in some cases. Overall, the findings demonstrate that utilizing tumor‐targeting oncolytic EVs for direct cytoplasmic delivery of MPs to induce ICD in cancer cells represents a promising approach for enhancing durable antitumor immunity.