Exosomes, also known as small extracellular vesicles, play crucial roles in cancer progression, inflammation, and tissue regeneration, serving as emerging platforms for diagnostics and therapeutic delivery. However, specific and high-affinity binders for non-destructive exosome labeling remain essential due to their complex and heterogeneous nature. Here, we report a nanobody (2E-Nb) identified by phage display that enables precise targeting of CD63, a tetraspanin highly enriched on exosomal membranes, allowing rapid and stable exosome surface modification. Six unique nanobody sequences were isolated, among which 2E-Nb exhibited superior solubility and binding activity. Structural modeling revealed that aromatic and basic residues in the 2E-Nb CDR3 loop form complementary electrostatic and π-π interactions with the CD63 extracellular domain (CD63-ECD). Bio-layer interferometry (BLI) confirmed high-affinity binding with a dissociation constant (KD) of 4.81 × 10− 8 M, characterized by fast association and moderate dissociation kinetics. Confocal microscopy verified the co-localization of 2E-Nb with CD63 in wild-type but not CD63-knockout H293T cells, confirming target specificity. Furthermore, NanoFCM analysis demonstrated that 2E-Nb effectively recognized native exosomal CD63 isolated from H293T cells and mediated efficient fluorescent labeling. Collectively, our findings establish 2E-Nb as a high-affinity, non-destructive tool for exosome labeling and engineering. This nanobody-based strategy provides a convenient and scalable platform for targeted exosome modification, advancing both fundamental exosome research and translational applications.
Chen et al. (Thu,) studied this question.