The uptake, intracellular trafficking and recycling of FcRn-blocking therapeutics in human endothelial cells in vitro.

The neonatal Fc receptor (FcRn) recycles immunoglobulin G (IgG) in cells and is responsible for the long half-life of IgG relative to other plasma proteins. FcRn also recycles pathogenic IgG autoantibodies and is the target for several new targeted therapeutic agents in IgG autoantibody-driven disorders. These therapeutics include rozanolixizumab, a high-affinity monoclonal antibody that directly blocks the IgG binding site on FcRn in a pH-independent manner. This study explored the impact of these molecular characteristics on rozanolixizumab's cellular uptake, endosomal trafficking and recycling. Another anti-FcRn therapeutic, the Fc fragment MST-HN IgG Fc (an analog of efgartigimod), a competitive antagonist with weaker affinity and pH-dependent binding to FcRn, was included for comparison. Using high-content imaging methods in human umbilical vein endothelial cells (HUVECs), a time- and concentration-dependent uptake of fluorescently labeled rozanolixizumab into intracellular compartments was observed. Uptake was rapid, pH-independent, and competed out with unlabeled inhibitor, supporting a receptor-mediated mechanism. Conversely, uptake of MST-HN IgG Fc was slower and required higher concentrations to detect uptake, which was pH-dependent, not competed out with unlabeled inhibitor, and occurred with similar potency in cells that did not express FcRn, suggesting a receptor-independent mechanism such as fluid phase pinocytosis. Using Rab proteins associated with different endosomal compartments, FcRn inhibitors appeared to traffic through recycling compartments in a similar manner in HUVECs, and their return to the cell surface in FcRn-transfected cells occurred with similar kinetics. These data demonstrate the impact of different structural features of FcRn inhibitors on functional outcomes on cells in vitro.