Abstract Extracellular vesicles (EV), facilitated with cancer-targeted antibody or peptide, have been demonstrated as an effective delivery vehicle of chemotherapy and gene therapy. This study aimed to develop and evaluate a targeted EV to deliver a newly identified natural cytotoxic marine compound verrucarin A (Ver-A) or combination with highly potent microtubule polymerization inhibitor mertansine (DM1). The tumor models, glioblastoma (GBM), triple-negative breast cancer (TNBC) and others, were used to validate the developed targeted therapy. First, the stirred-tank bioreactor-based EV biomanufacturing was optimized by developing a two-step purification procedure using size exclusion (300 kDa) and affinity (CD63) columns in liquid chromatography system. Second, both single and dual tumor-targeting monoclonal antibodies (mAbs), such as anti-SSTR2, CD276, EGFR and/or CD47 mAb either developed in house or used in clinics, were tagged on the surface of PEGylated EV to construct mAb-EV-drugs. The flow cytometry analysis, live-cell confocal images and IC50 assay revealed the effective surface binding, intracellular drug delivery and high anti-cancer cytotoxicity of mAb-EV-drugs in cancer cell lines, and the biodistribution study confirmed the tumor targeting in xenograft mouse models. Furthermore, the maximum tolerated dose study showed minimal systemic toxicity, as confirmed by hematoxylin and eosin staining of multiple important organs. Finally, the anti-tumor efficacy study demonstrated significant inhibition of tumor growth by mAb-EV-Ver-A or Ver-A/DM1 in cell line-derived xenograft models, patient-derived xenograft models, and/or immunocompetent xenograft models. This study suggested that the targeted EV is an efficient platform to deliver combined potent chemotherapies to treat cancer or tumor. Citation Format: Kai Chen, Yingnan Si, Seulhee Kim, Zhuoxin Zhou, Lufang Zhou, X. Margaret Liu. Targeted extracellular vesicle to deliver combined chemotherapies to treat cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5326.