FerryCalin: an engineered lipocalin protein directed against the transferrin receptor with potential for brain drug delivery.

Abstract

The transferrin receptor (TfR) mediates transcytosis across the blood-brain barrier (BBB), which offers a promising approach for the non-invasive delivery of therapeutics into the brain parenchyma. Employing the recombinant homodimeric murine TfR ectodomain, prepared in a biochemically functional state, we have selected a cognate Anticalin via phage display and bacterial cell surface display from a random library based on the human lipocalin 2 (Lcn2). After affinity maturation, several engineered lipocalin were identified that bind murine TfR in a non-competitive manner with the natural ligand (transferrin•Fe3+), among those an Anticalin - dubbed FerryCalin - exhibiting a dissociation constant (KD) of 3.8 nM. Epitope analysis using the SPOT technique revealed a sequential epitope in a surface region of TfR remote from the transferrin-binding site. Due to their fast konrate and short complex half-life, as evidenced by real-time surface plasmon resonance (SPR) measurements, FerryCalin, or one of its related mutants, shows characteristics as a potential vehicle for the brain delivery of biopharmaceuticals.