Targeting the apical domain of the transferrin receptor: Development of a new protein scaffold for cellular delivery

Human transferrin receptor 1 (TfR) is essential for cellular iron homeostasis by internalizing the iron carrier proteins transferrin and ferritin. It is also an entry point for various pathogens, such as South American hemorrhagic fever caused by arenaviruses and the malaria parasite Plasmodium vivax, which utilize TfR to gain access to cells. The receptor is additionally upregulated in many aggressive cancers and at the blood-brain barrier. Altogether, the TfR is a highly relevant target for many medical applications, and novel protein-interacting partners are sought after. A protein design strategy was explored here to develop a small protein that can be used for drug delivery across cell membranes, to investigate blood-brain barrier crossing, study endocytosis, or to block pathogen access to the apical domain. A computationally docked library of small protein scaffolds to the TfR apical domain, the native binding site of the Machupo arenavirus, was a starting point for the design and optimization. The best variants were expressed in a yeast surface display system and assessed for interaction with TfR by flow cytometry. One protein variant, which initially showed a low binding signal, was further optimized by directed evolution to bind to the target receptor at nanomolar concentration. The evolved construct, tagged with the enhanced green fluorescent protein (eGFP) and bacterially expressed, showed uptake similar to that of FITC-coupled transferrin in a cell-based assay. The designed protein can be utilized as a tool to target cell entry via TfR for drug delivery applications or as a foundation for developing antiviral therapeutics against arenaviruses.