Targeting FcRn for the modulation of antibody dynamics

Abstract

The MHC class I-related receptor, FcRn, is a multitasking protein that transports its IgG ligand within and across cells of diverse origins. The role of this receptor as a global regulator of IgG homeostasis and transport, combined with knowledge of the molecular details of FcRn-IgG interactions, has led to opportunities to modulate the in vivo dynamics of antibodies and their antigens through protein engineering. Consequently, the generation of half-life extended antibodies has shown a rapid expansion over the past decade. Further, FcRn itself can be targeted by inhibitors to induce decreased levels of circulating IgGs, which could have applications in multiple clinical settings. The engineering of antibody-antigen interactions to reduce antibody-mediated buffering of soluble ligand has also developed into an active area of investigation, leading to novel antibody platforms designed to result in more effective antigen clearance. Similarly, the target-mediated elimination of antibodies by internalizing, membrane bound antigens (receptors) can be decreased using novel engineering approaches. These strategies, combined with subcellular trafficking analyses of antibody/antigen/FcRn behavior in cells to predict in vivo behavior, have considerable promise for the production of next generation therapeutics and diagnostics.

Keywords: Antibody engineering; FcRn; IgG; Pharmacokinetics; Therapeutic antibodies; pH-dependence.

Fig. 1

Schematic representation of the different subcellular trafficking pathways taken by FcRn-positive TCs in endothelial cells. For further details, see Gan et al., (2013).

Fig. 2

Three dimensional structure of the FcRn:Fc-YTE complex (Oganesyan et al., 2014). The X-ray crystallographic structure of the FcRn:Fc-YTE complex is represented as a ribbon diagram with the Cα chains of FcRn, β₂-microglobulin and Fc-YTE colored in green, cyan and blue, respectively. The inset displays an enlarged view of the residues at the interface of the FcRn:Fc-YTE interaction. Salt bridges and hydrogen bonds between the indicated residues of FcRn and Fc-YTE are identified with a cutoff distance of 3.6 Å and are displayed using dotted yellow lines. Fc-YTE residues (Ile253, His310 and His435) playing a crucial role in binding to FcRn are shown in purple and the altered residues that constitute the YTE mutant (Tyr252, Thr254 and Glu256) resulting in affinity enhancement to FcRn at acidic pH are shown in red.

Fig. 3

Schematic representation of the effect of FcRn inhibition by an Abdeg on the subcellular trafficking of IgG. In the absence of inhibition, FcRn:IgG complexes are efficiently recycled and IgG is exocytosed (left panel). In the presence of an Abdeg, which binds to FcRn with increased affinity at pH 6.0-7.4, the interaction of wild type IgG with FcRn in endosomes is blocked (right panel). Consequently, increased levels of IgG enter lysosomes and are degraded.

Fig. 4

Schematic representation showing how the pH dependent binding of an antibody to a soluble antigen (higher affinity at pH 7.4 than at pH 6.0) increases antigen degradation. Antigen in complex with the pH-independent antibody is taken up into the cell and sorted to recycling TCs through binding to FcRn (left panel). The antigen:pH-independent antibody complex is exocytosed at the cell membrane. By contrast, pH-dependent antibodies dissociate from antigen in early endosomes, resulting in lysosomal delivery of the antigen (right panel). The pH-dependent antibody is recycled by FcRn for re-use.

Fig. 5

Fluorescence microscopy analyses of the endosomal trafficking behavior of antigen (IL-6) in the presence of antibodies that differ in their pH dependence for binding to antigen. Panels present representative images of endothelial cells (HMEC-1) co-transfected with FcRn-GFP and human β₂-microglobulin. Following transfection, cells were pulsed with antigen:pH-independent antibody complexes (left panels) or antigen:pH-dependent antibody complexes (right panels). Whole cell images are shown in the upper panels, with expansions of cropped regions to present individual endosomes. For the pH-independent antibody, antigen (IL-6), FcRn and IgG are colocalized on the limiting membrane of the endosome (lower left panels). By contrast, antigen dissociates into the endosomal vacuole in the presence of the pH-dependent antibody (lower right panels). In the expanded images, individual fluorophores are represented in black and white whereas for the overlays, antigen (Alexa 555), FcRn (GFP) and IgG (Alexa 647) are pseudocolored red, green and blue respectively. Background-subtracted fluorescence intensities along the dotted lines in the overlays are shown in the fluorescence intensity plots. The scale bars for the complete cell images and the cropped endosomes represent 4 and 0.5 µm, respectively.