Affibody Molecules Intended for Receptor-Mediated Transcytosis via the Transferrin Receptor

The development of biologics for diseases affecting the central nervous system has been less successful compared to other disease areas, in part due to the challenge of delivering drugs to the brain. The most well-investigated and successful strategy for increasing brain uptake of biological drugs is using receptor-mediated transcytosis over the blood-brain barrier and, in particular, targeting the transferrin receptor-1 (TfR). Here, affibody molecules are selected for TfR using phage display technology. The two most interesting candidates demonstrated binding to human TfR, cross-reactivity to the murine orthologue, non-competitive binding with human transferrin, and binding to TfR-expressing brain endothelial cell lines. Single amino acid mutagenesis of the affibody molecules revealed the binding contribution of individual residues and was used to develop second-generation variants with improved properties. The second-generation variants were further analyzed and showed an ability for transcytosis in an in vitro transwell assay. The new TfR-specific affibody molecules have the potential for the development of small brain shuttles for increasing the uptake of various compounds to the central nervous system and thus warrant further investigations.

Automated summary

The development of biologics for the central nervous system has faced challenges in drug delivery to the brain. Receptor-mediated transcytosis over the blood-brain barrier, particularly targeting the TfR, has been the most successful strategy. Affibody molecules selected for TfR showed promising binding to human TfR, cross-reactivity to the murine orthologue, and binding to TfR-expressing brain endothelial cell lines. Mutagenesis of affibody molecules led to the development of second-generation variants with improved properties, which demonstrated transcytosis ability in an in vitro assay. These TfR-specific affibody molecules have potential as brain shuttles and warrant further investigations.