FerryCalin: an Engineered Lipocalin Protein Directed against the Transferrin Receptor with Potential for Brain Drug Delivery

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 variants 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 (K_D) 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 the fast k_on rate 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.