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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\u2010anchoring 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\u2010scale source of engineered EVs (eEVs). To achieve molecular targeting, the transmembrane scaffold \u0394688 PTGFRN is modified to display tissue\u2010targeting ligand Ephrin\u2010B2 (EB2) on the EV surface, exploiting its high\u2010affinity 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\u2010positive cells in vivo and permit non\u2010invasive, spatiotemporal tracking via cargo\u2010mediated bioluminescence resonance energy transfer. In patient\u2010derived xenograft models with differential EphB4 expression, Ephrin\u2010B2\u2013displaying eEVs show selective localization to EphB4\u2010positive tumors and report intratumoral cargo distribution. This work establishes a translational strategy for large\u2010scale eEV production, advancing EV\u2010based delivery platforms for precision targeting of EphB4\u2010expressing ovarian cancer.<\/jats:p>","DOI":"10.1002\/advs.75415","type":"journal-article","created":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T13:41:26Z","timestamp":1776951686000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Scalable Engineering of Bio\u2010Manufactured Extracellular Vesicles for Selective Delivery in Ovarian Cancer Patient\u2010Derived Models"],"prefix":"10.1002","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6900-9551","authenticated-orcid":false,"given":"Nihar","family":"Godbole","sequence":"first","affiliation":[{"name":"Translational Extracellular Vesicles in Obstetrics and Gynae\u2010Oncology Group Frazer Institute Faculty of Health Medicine and Behavioural Sciences Royal Brisbane and Women's 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