An engineered CD81‐based combinatorial library for selecting recombinant binders to cell surface proteins: Laminin binding CD81 enhances cellular uptake of extracellular vesicles

Abstract

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 cells. These EVs were examined for the presence of the recombinant protein and were shown to exhibit an enhanced uptake into laminin‐secreting mammalian cell lines. For the best candidate, the specificity of antigen interaction was demonstrated with a competition experiment. 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 settings.

CD81-guided heterologous EVs present heterogeneous interactions with breast cancer cells

“…Several studies have reported tetraspanins as valuable tools for engineering and tracing cellsecreted EVs with fluorescent markers (Suetsugu, 2013) , (Yoshimura et al, 2016) , (Mathieu et al, 2021). Specifically, CD81 was fused with fluorescent reporters (Stickney et al, 2016) , (Zuppone et al, 2023) or used as a direct targeting moiety against human placental laminin (Vogt et al, 2021). In this work, CD81 fusion proteins were exploited to track the cell engagement of secreted, heterologous EV sub-populations.…”

Proteomic analysis of extracellular vesicles from tick hemolymph and uptake of extracellular vesicles by salivary glands and ovary cells

“…Laminins are major signaling and structural molecules of basement membranes and modulate several diverse cellular functions. These include maintaining tissue structure, adhesion and migration, differentiation and survival [ 66 , 67 ]. In oral squamous cell carcinoma (OSCC) LN1-1 cell-derived EVs, laminin-332 proteins were validated as highly expressed proteins, including laminin α3, β3 and γ2 [ 68 ].…”

Current and prospective strategies for advancing the targeted delivery of CRISPR/Cas system via extracellular vesicles

“…4A). Common transmembrane proteins include the lysosomeassociated membrane protein 2B (LAMP-2B) [124], tetraspanins [29,125,126], glycosyl-phosphatidyl-inositol (GPI) [127], C1C2 [128], and platelet-derived growth factor receptors (PDGFR) [31,129], among others (Fig. 4A).…”

“…As a viable and straightforward strategy, the tetraspanin superfamily (CD63/CD9/CD81) is often selected for EV modification with target molecules [29,125,136]. By conjugating CD63 with apoprotein-A1 and further bonding to scavenger receptors expressed on hepatocellular carcinoma cells, engineered EVs showed a twofold increase in uptake for targeted delivery [136].…”