High affinity single-chain variable fragments are specific and versatile targeting motifs for extracellular vesicles

Abstract

Exosomes are extracellular vesicles that mediate cell-to-cell communication by transferring biological cargo, such as DNA, RNA and proteins. Through genetic engineering of exosome-producing cells or manipulation of purified exosomes, it is possible to load exosomes with therapeutic molecules and target them to specific cells via the display of targeting moieties on their surface. This provides an opportunity to exploit a naturally-occurring biological process for therapeutic purposes. In this study, we explored the potential of single chain variable fragments (scFv) as targeting domains to achieve delivery of exosomes to cells expressing a cognate antigen. We generated exosomes targeting the Her2 receptor and, by varying the affinity of the scFvs and the Her2 expression level on recipient cells, we determined that both a high-affinity anti-Her2-scFv (K≤ 1 nM) and cells expressing a high level (≥10 copies per cell) of Her2 were optimally required to enable selective uptake. We also demonstrate that targeting exosomes to cells via a specific cell surface receptor can alter their intracellular trafficking route, providing opportunities to influence the efficiency of delivery and fate of intracellular cargo. These experiments provide solid data to support the wider application of exosomes displaying antibody fragments as vehicles for the targeted delivery of therapeutic molecules.

Potential roles of extracellular vesicles in the pathophysiology, diagnosis, and treatment of autoimmune diseases

“…On the other hand, Kumar et al demonstrated an approach whereby EVs are encapsulated by a 10 nm thick protective film formed by a supramolecular complex of ferric ions and tannic acid to achieve non-essential EVs loss, and the protective film can be fused with other molecules as targeted delivery while the protective film is also subject to controlled degradation [62]. Longatti et al published a method for specifically targeting exosomes membrane, with a single-chain variable fragment of antibody [63]. Recently, Jia G et al demonstrated a method for treating glioma cells, using a click chemistry method to engineer exosomes with neuropilin-1 peptide and load them with curcumin [60].…”

Bioengineered Polyhydroxyalkanoates as Immobilized Enzyme Scaffolds for Industrial Applications

“…Extracellular membrane vesicles are lipid membrane-derived compartments and are found in all domains of life (Raposo and Stoorvogel, 2013;Gill et al, 2018;Lee, 2019). Their sizes are in the range of 20-1000 nm in diameter (van der Pol et al, 2012;Gill et al, 2018), and they mainly serve as carrier vehicles to mediate cell-to-cell communication by transporting biological cargo as, for example, DNA, RNA, and proteins (Burkova et al, 2018;Longatti et al, 2018). The classification of these functionally and structurally diverse EMVs, including the bacterial outer membrane vesicles, microvesicles, and exosomes, has been thoroughly reviewed (van der Pol et al, 2012;Cocucci and Meldolesi, 2015;Gill et al, 2018;Greening and Simpson, 2018).…”

“…In addition, EMVs are relatively stable in ambient environments and can be manufactured cost-effectively (Collins, 2011). However, isolation and purification of EMVs still require expensive and laborious ultracentrifugation steps, which potentially impact the structural integrity of EMVs and which prohibit industrial scale production Longatti et al, 2018;Bruce et al, 2019).…”

Gene Editing by Extracellular Vesicles

“…Distribution and uptake of EVs may be also altered by exposing different peptides on the EV surface, such as arginine-rich micropinocytosis-inducing peptide [ 101 ]; adding cell-penetrating peptides; activating receptors on the surface of EVs [ 194 ]; conjugating specific targeting moieties [ 195 ]; displaying vesicular stomatitis virus (VSV) G protein (VSV-G) for increased tropism [ 196 ]; or displaying targeting nanobodies/antibodies as highlighted above. Importantly, display of certain moieties on the EV surface can improve the release of active payloads into target cells, as was shown for surface antibodies that shifted EV uptake from caveolae-mediated endocytosis to micropinocytosis, thereby avoiding the endolysosomal pathway and improving efficacy of the intracellular cargo delivery [ 197 ]. Directing EVs into pre-determined locations in the body was also achieved by utilizing aptamers, nucleic acids specifically recognizing target molecules.…”