Scalable Engineering of Bio‐Manufactured Extracellular Vesicles for Selective Delivery in Ovarian Cancer Patient‐Derived Models

ABSTRACT Ovarian cancer remains challenging to treat because of late diagnosis, heterogeneity, and poor response to existing therapies. Here, we present a streamlined and scalable extracellular vesicle (EV) engineering approach that enables efficient cargo packaging and enhances targeted cargo delivery to ovarian tumors in vivo. Systematic comparison of vesicle‐anchoring domains identifies an optimized scaffold for loading bioluminescent cargo into EVs. We characterized EV production from five cell lines commonly used for biopharmaceutical manufacturing and selected and stably engineered ExpiCHO cells as a robust, large‐scale source of engineered EVs (eEVs). To achieve molecular targeting, the transmembrane scaffold Δ688 PTGFRN is modified to display tissue‐targeting ligand Ephrin‐B2 (EB2) on the EV surface, exploiting its high‐affinity interaction with the Eph receptor B4 (EphB4), which is overexpressed in advanced ovarian carcinoma. Following systemic administration, these eEVs carrying bioluminescent cargo preferentially accumulate in EphB4‐positive cells in vivo and permit non‐invasive, spatiotemporal tracking via cargo‐mediated bioluminescence resonance energy transfer. In patient‐derived xenograft models with differential EphB4 expression, Ephrin‐B2–displaying eEVs show selective localization to EphB4‐positive tumors and report intratumoral cargo distribution. This work establishes a translational strategy for large‐scale eEV production, advancing EV‐based delivery platforms for precision targeting of EphB4‐expressing ovarian cancer.