OX40 ligand-engineered dendritic cell nanovesicles potentiate antitumor immunity through dual-mode T cell activation

The efficacy of classical dendritic cell (DC)-based cancer vaccines is limited by poor in vivo stability, inefficient lymph node delivery, and the immunosuppressive tumor microenvironment. Here, we developed a nanovaccine platform by engineering DC-derived nanovesicles (NVs) and established a membrane-anchoring strategy based on a recombinant cytolysin A (ClyA) fusion protein that efficiently inserts OX40 ligand (OX40L) from the extracellular side onto the surface of mature DCs and their derived NVs. The resulting E7-OX40L-NV, loaded with HPV16 E7 peptide, retained native and modified DC membrane proteins, exhibited efficient migration to draining lymph nodes, and showed good biocompatibility. In vitro, E7-OX40L-NV directly activated both naive and antigen-experienced antigen-specific CD8+ T cells and induced cytotoxic function. After uptake by DCs, the vesicles transferred peptide-MHC (pMHC) and OX40L to the DC surface, indirectly enhancing CD8+ T cell responses. In therapeutic TC-1 tumor models, E7-OX40L-NV induced potent antitumor immunity, including enhanced T cell effector function, reduced regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), increased intratumoral infiltration of functional T cells, remodeled T cell proliferation and exhaustion dynamics, and increased formation of long-lived memory precursor effector cells (MPECs), leading to significant tumor suppression and complete regression in a proportion of mice that rejected contralateral tumor rechallenge. Co-delivery of OX40L and antigen on the same NV was more effective than a physical mixture, underscoring the importance of spatial coordination. This ClyA-mediated membrane functionalization strategy provides a versatile tool for engineering cell-derived vesicles, and OX40L-engineered DC nanovesicles offer a promising platform for developing next-generation personalized cancer vaccines.