De novo Antigen Binding CD81 for Targeting Extracellular Vesicles

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ICE Krakow

ICE Krakow

ul. Marii Konopnickiej 17 30-302 Kraków


Reddy Bobbili, Madhusudhan (Ludwig Boltzmann Institute for Traumatology )


The research of extracellular vesicles (EVs) has boomed in the last decade, with the promise of them functioning as target-directed drug delivery vehicles, able to modulate proliferation, migration, differentiation, and other properties of the recipient cell that are vital for health of the host organism. To enhance the ability of their targeted delivery, we employed an intrinsically overrepresented protein, CD81, to serve for recognition of the desired target antigen. Yeast libraries displaying mutant variants of the large extracellular loop of CD81 have been selected for binders to human placental laminin, as an example target. Their specific interaction with laminin was confirmed in a mammalian display system. Derived sequences were reformatted to full-length CD81 and expressed in EVs produced by HeLa & WJ-MSC cells for laminin binders.
To assess the novel functionality of antigen-binding CD81 LEL variants, internalization of such EVs into laminin-secreting cell lines Huh-7 and NCI-N87 was compared with EVs derived from wild-type CD81-transfected production cell line, and further, their reactivity with several recombinant laminin isoforms was examined. The specificity of binding of laminin targeting EVs to their cognate antigen was tested under competitive conditions. Finally, the ability of laminin-targeting EVs to transfer cel-miR-39 to Huh-7 cell line was examined.
To our knowledge, this is the first example of harnessing an EV membrane protein as mediator of de novo target antigen recognition via in vitro molecular evolution, opening horizons to a broad range of applications in various therapeutic and regenerative settings. The advantage of the method presented here is that it can rapidly deliver binders to any antigen of choice, which can simply be ‘clicked’ into the full-length CD81, recombinantly expressed on the EV surface, enabling specific EV-mediated delivery to a large variety of cells and tissues.


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