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

• Published in: Chembiochem : a European journal of chemical biology 2023-05, Vol.24 (10), p.e202200795-n/a
• Main Authors: Nästle, Lea, Deuschle, Friedrich‐Christian, Morath, Volker, Skerra, Arne
• Format: Article
• Language: English
• Subjects: Animals; Binding sites; Biopharmaceuticals; Blood-brain barrier; Brain; Brain - metabolism; Cell surface; Drug delivery; Epitopes; Humans; Lipocalin; Lipocalins - genetics; Mice; Parenchyma; Phage display; Phages; protein engineering; Receptors; Receptors, Transferrin - chemistry; Receptors, Transferrin - metabolism; Surface plasmon resonance; TfR; Transferrin; Transferrin - chemistry; Transferrin - metabolism; Transferrins
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.202200795

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 (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 the fast kon 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. The transferrin receptor (TfR) mediates transcytosis across the blood–brain barrier, thus enabling the non‐invasive delivery of therapeutics into the brain, for example as fusion proteins with the single‐chain Fv of the antibody 8D3. As a robust alternative, we have developed a cognate Anticalin, dubbed FerryCalin, based on the human lipocalin 2, an abundant plasma protein. With its dynamic binding behavior and its epitope remote from the transferrin binding site, FerryCalin offers a potential vehicle for the brain delivery of biopharmaceuticals.