Modularly designed hybrid vesicles as dual-adjuvant nanovaccines for cancer immunotherapy.

Natural extracellular vesicles (EVs) and extruded membrane vesicles (MVs) have emerged as a promising platform for cancer vaccination by co-delivering tumor antigens and adjuvant. However, conventional designs rely on a single class of adjuvant, which often fail to adequately activate dendritic cells (DCs), resulting in inefficient cross-priming and weak cytotoxic T lymphocyte (CTL) activation. To address this, we designed a dual-adjuvant nanovaccine by modularly fusing interferon-alpha (IFNα)-displaying bacterial MVs (IFNα-BMVs) with programmed cell death protein 1 (PD1)-displaying cancer MVs (PD1-CMVs) to potentiate antitumor immune responses. In this design, IFNα-BMVs deliver dual-adjuvant pathogen-associated molecular patterns (PAMPs) and IFNα to promote DC maturation and cross-priming, thereby activating CTL. Concurrently, PD1-CMVs provide tumor-associated antigens (TAAs) and utilize membrane-anchored PD1 as a decoy receptor for targeted PD-L1 blockade, thus preventing CTL exhaustion. More importantly, this IFNα and PD1 co-displaying hybrid MVs (IP-HMVs) nanovaccine significantly upregulated costimulatory molecules and antigen-presenting molecules, thereby promoting DC maturation and CTL infiltration. Furthermore, RNA sequencing analysis validated that IP-HMVs robustly activated canonical antigen presentation pathways. In both subcutaneous and metastatic 4T1 tumor models, IP-HMVs significantly suppressed tumor progression and extended median survival to 35 days. The modular design offers a generalizable framework for developing next-generation cancer vaccines.