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

Stefan Vogt,Gerhard Stadlmayr,Florian Rüker,Gordana Wozniak-Knopp,Jørgen Kjems,Madhusudhan Reddy Bobbili,Katharina Stadlbauer,Johannes Grillari

doi:10.1002/jev2.12139

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.

Highlights

In recent years, extracellular vesicles (EVs) have gained recognition as elaborate drug delivery vehicles (Yamamoto et al, 2019)
We have attempted to improve the biophysical properties of CD81 large extracellular loop (LEL) and constructed mutants that exhibit in one case an extremely high level of thermostability and in one case reversible thermal denaturation (Vogt et al, 2018)
As these should be more permissive for mutagenesis required to introduce a novel antigen binding site, we have identified amino acid residues that could form a novel antigenbinding surface based on the above mutants

Summary

Introduction

Extracellular vesicles (EVs) have gained recognition as elaborate drug delivery vehicles (Yamamoto et al, 2019). Rashed et al, 2017), aging and senescence (Terlecki-Zaniewicz et al, 2019; Weilner et al, 2016), and further, to support processes such as regeneration after injury (Taverna et al, 2017) and limitation of tumorigenesis and malignant transformation (Wolfers et al, 2001) These properties have rendered them interesting for biotechnological applications, which has resulted in accelerated development of methods for their production, including optimization of source cells and respective culture media (Patel et al, 2018; Roura & Bayes-Genis, 2019), improved purification processes (Zhang et al, 2019), standardization of protocols for characterization (Coumans et al, 2017) and stable storage of EV preparations (Jeyaram & Jay, 2018). Sophisticated protocols have been devised to enable optimal enclosure of the drug of interest into the vesicles (Sutaria et al, 2017), employing a spectrum of strategies, including simple diffusion, lipofection, electroporation and sonication and endogenous loading methods related mostly to RNA-cargo (overexpression in host cells (Hung & Leonard, 2015), extrusion of host cells (Lunavat et al, 2016), vexosome hybrid vehicles (Maguire et al, 2012) and introduction of EXOmotif to bias the miRNA loading into the EVs (Villarroya-Beltri et al, 2013))

Methods

Results

Conclusion