Click Chemistry-Mediated Functionalization of Engineered Oncolytic Extracellular Vesicles Promotes Coordinated T- and B-Cell Antitumor Immunity

Engineered extracellular vesicles (EVs) are promising therapeutic carriers for cancer treatment, but preserving intrinsic therapeutic activity while incorporating multiple immunomodulatory functions remains a challenge. Here, we developed a modular engineering strategy that combines the genetic engineering of EV-producing cells with bioorthogonal click chemistry to construct multifunctional oncolytic extracellular vesicles (OEVs). Azide-functionalized OEVs were generated from CD40L-expressing cells infected with a capsid-engineered oncolytic adenovirus. A toll-like receptor 9 (TLR9) agonist was subsequently conjugated to the OEV surface through click chemistry, integrating immune stimulation while retaining the oncolytic activity. The engineered OEVs efficiently infected tumor cells, induced antigen-presenting cell activation, enhanced intratumoral CD8+ T-cell responses, promoted B-cell activation and tumor-specific antibody production, and improved the therapeutic efficacy of the immune checkpoint blockade. These findings demonstrated that combining genetic engineering with bioorthogonal chemistry provided a strategy for constructing multifunctional oncolytic EVs with coordinated cytotoxic and immunomodulatory activities, offering an approach to next-generation cancer immunotherapies.