Receptor-mediated transcytosis of nanobodies targeting the heparin-binding EGF-like growth factor in human blood-brain barrier models

Transport of molecules into the brain is regulated by the blood-brain barrier (BBB). Receptor-mediated transcytosis (RMT) is a targeted vesicular transport mechanism of brain endothelial cells that can be employed to specifically transport large therapeutic molecules into the brain. ProHB-EGF is the transmembrane precursor of the heparin-binding EGF-like growth factor (HB-EGF) present on the intraluminal side of the brain endothelial cells. This molecule is characterized as an internalizing transport receptor with so far no discovery of endogenous ligands. In this study, we describe the selection and characterization of two nanobodies (named F12 and H7) with high binding affinity for proHB-EGF and their BBB transcytosis potential were tested in vitro. For the human BBB model, we found that a polarized co-culture environment was crucial for the expression and cell surface display of proHB-EGF. The ability of F12 and H7 to pass the BBB via RMT was demonstrated in both a primary human brain microvascular endothelial cell-based BBB model and a human induced pluripotent stem cell (hiPSC)-derived iBBB model. Our studies demonstrate that the proHB-EGF targeting Nbs are promising BBB shuttle molecules for delivery of therapeutic molecules into the brain.