Background: Exosomes are cell-derived nanovesicles which have been proposed as potential drug carriers. Fusion of affinity ligands to exosome surface proteins has been shown to enhance uptake by target tissues, but requires genetic modification of parent cells. Similarly, direct chemical conjugation of affinity ligands can damage exosome structure and lacks reproducible surface modification. We present a novel bispecific antibody platform, which enables reproducible surface decoration of exosomes, increasing uptake by target cells and altering tissue distribution in animals. Methods: Exosome Targeting bispecific Antibodies (ExTAbs) were designed by fusing single-chain variable fragments (scFv) to the C-terminus of the heavy chain of a recombinant, anti-CD63 monoclonal antibody. ExTAbs were expressed in HEK cells, purified using affinity chromatography, and confirmed via SDS-PAGE and size exclusion HPLC. Binding affinities to CD63 and target antigens were determined using flow cytometry and/or ELISA. Decoration of exosomes was quantified via competitive-binding assay using radiolabeled 125 I-anti-CD63. For in vitro targeting experiments, fluorescent exosomes were decorated with ExTAbs or anti-CD63 as a non-targeting control. For in vivo biodistribution studies, radiolabeled exosomes were decorated with ExTAbs (or anti-CD63 as untargeted control) and injected intravenously in mice. Results: ExTAbs targeting mouse ICAM-1, PECAM-1, and CD98hc were synthesized with > 95% purity and demonstrated nanomolar binding affinity to both CD63 (K d = 4.47 ± 0.47nM) and their respective target antigens (e.g., PECAM-1 Kd = 4.63 ± 0.61 nM). Target cells showed an ~20-fold increase in uptake of targeted vs. untargeted exosomes following 30-minute incubation at 37C. Uptake of PECAM-targeted exosomes was significantly higher than untargeted exosomes in heavily-endothelialized organs (e.g., lung, kidney, and heart), including a 10-fold increase in lung uptake (14.8 ± 2.7 v.s. 1.4 ± 0.2 % ID/g, p = 0.001). Conclusion: CD63 bispecific antibodies enable straightforward and reproducible surface decoration of human exosomes. Targeted exosomes demonstrate increased uptake in target cells and organs.http://deepblue.lib.umich.edu/bitstream/2027.42/176966/1/Honors_Capstone_Report_-_Kaitlyn_Pierpont.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/176966/2/Design_Expo_Poster_-_Kaitlyn_Pierpont.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/176966/3/ACS_Spring_2023_ExTAbs_Presentation_-_Kaitlyn_Pierpont.ppt
