Despite the great potential of antibodies as intracellular therapeutics, there is a significant, unmet challenge in delivering sufficient amounts of folded antibodies inside cells. We describe an all-protein self-assembled nanocarrier capable of delivering functional antibodies to the cytosol. By combining an α-helical peptide that self-assembles into a hexameric coiled-coil bundle and an Fc-binding Protein A fragment, we generated the Hex nanocarrier that is efficiently internalized by cells without cytotoxicity. Localization of multiple Fc-binding domains on the hexameric core allowed the Hex nanocarrier to tightly bind antibody with sub-nanomolar affinity regardless of pH and the antibody's origin... More
Despite the great potential of antibodies as intracellular therapeutics, there is a significant, unmet challenge in delivering sufficient amounts of folded antibodies inside cells. We describe an all-protein self-assembled nanocarrier capable of delivering functional antibodies to the cytosol. By combining an α-helical peptide that self-assembles into a hexameric coiled-coil bundle and an Fc-binding Protein A fragment, we generated the Hex nanocarrier that is efficiently internalized by cells without cytotoxicity. Localization of multiple Fc-binding domains on the hexameric core allowed the Hex nanocarrier to tightly bind antibody with sub-nanomolar affinity regardless of pH and the antibody's originating species. The size of the Hex nanocarrier ranges from 25 to 35nm depending on the antibody loading ratio. We demonstrated the capacity of the Hex nanocarrier to deliver functional antibodies to the cytosol by employing anti-β-tubulin or anti-nuclear pore complex antibody as cargo. The design of the Hex nanocarrier is modular, which enables functionalization beyond Fc-binding. We exploited this feature to improve the cytosolic delivery efficiency of the Hex nanocarrier by addition of an endosomolytic motif to the core. The modified Hex nanocarrier exhibited similar antibody-binding behavior, but delivered more antibodies to their cytosolic targets at a faster rate. This work demonstrates an efficient intracellular antibody delivery platform with significant advantages over existing approaches as it does not require modification of the antibody, is biodegradable, and has an antibody to carrier mass ratio of 13, which is greater than other reported antibody carriers.
