Fc-fusion proteins and FcRn: structural insights for longer-lasting and more effective therapeutics

Abstract

Nearly 350 IgG-based therapeutics are approved for clinical use or are under development for many diseases lacking adequate treatment options. These include molecularly engineered biologicals comprising the IgG Fc-domain fused to various effector molecules (so-called Fc-fusion proteins) that confer the advantages of IgG, including binding to the neonatal Fc receptor (FcRn) to facilitate in vivo stability, and the therapeutic benefit of the specific effector functions. Advances in IgG structure-function relationships and an understanding of FcRn biology have provided therapeutic opportunities for previously unapproachable diseases. This article discusses approved Fc-fusion therapeutics, novel Fc-fusion proteins and FcRn-dependent delivery approaches in development, and how engineering of the FcRn-Fc interaction can generate longer-lasting and more effective therapeutics.

Keywords: Fc-fusion proteins; IgG; mucosal drug delivery; neonatal Fc receptor; therapeutic antibody.

Figure 1

Crystal structure of FcRn and the FcRn–IgG Fc complex. (a) FcRn is a heterodimeric molecule consisting of a heavy chain with the three extracellular domains (shown in brown) that non-covalently associate with beta-2-microglobulin as a light chain (blue). Although the tertiary structure of FcRn resembles an MHC class I molecule, the binding sites where peptides bind to MHC class I molecules are inaccessible in FcRn. Human FcRn and human β2m are shown. (b) At acidic pH, FcRn (brown) binds to the C_H2-C_H3 hinge region of IgG (green). Critical amino acids in FcRn for the binding to the Fc fragment are highlighted in yellow. Rat FcRn and a rat Fc fragment are shown. Crystal structures were generated with Cn3d based on the protein data bank (PDB) ID 1EXU (West and Bjorkman, 2000) and 1FRT (Burmeister et al., 1994b).

Figure 2

Contribution of the hematopoietic and parenchymal compartment for the protection of monomeric IgG. To assess the contribution of hematopoietic and parenchymal cells to IgG protection, wild-type (WT) and Fcgrt^−/− (KO) mice were lethally irradiated and substituted with bone marrow from either WT or Fcgrt^−/− mice, and the percentage of a remaining monomeric NIP-OVA mouse IgG was measured over time. Mice in which FcRn was expressed in the hematopoietic compartment (WT→KO) exhibited a higher percentage of remaining IgG compared to mice in which FcRn was limited to the parenchymal compartment (KO→WT), indicating that the contribution of the hematopoietic compartment for IgG protection is greater than that of the parenchymal compartment. Based upon data previously reported by Qiao et al. (2008).

Figure 3

FcRn-dependent recycling and protection of monomeric IgG versus degradation and potentially presentation of complexed IgG. (a) In endothelial cells, IgG molecules or IgG Fc-fusion proteins can be internalized by fluid-phase processes such as pinocytosis. FcRn is present within EEA-1 positive early endosomes (Ober et al., 2004b), where acidic pH allows interaction between FcRn and the Fc region of IgG molecules. In nonpolarized endothelial cells, FcRn co-localizes with Rab4⁺ and Rab11⁺ vesicles in both fusion and fission events with the sorting endosome (Ward et al., 2005). Exocytosis of FcRn–IgG complexes is associated with a compartment positive for Rab11, but not Rab4 (Ward et al., 2005) and occurs via classical exocytosis, in which the exocytic vesicle fuses completely with the cell membrane and a “prolonged-release” where the vesicle only partially fuses with the cell membrane in repetitive cycles (Ober et al., 2004a). The slower-release mode is characterized by periodic, stepwise release of IgG, rather than the rapid burst that is observed for complete fusion events. In both processes, neutral pH of the bloodstream facilitates dissociation of IgG from FcRn and its release into the systemic circulation. In contrast, proteins not bound to IgG or unbound effector molecules are directed into LAMP-1⁺ lysosomes and subsequent undergo degradation. (b) FcRn-dependent degradation of IgG-immune complexes. Classical FcγRs on the surface of APCs can bind IgG containing immune complexes, leading to their internalization. In the acidic environment of the endosome, generated, at least in part, by the actions of V-ATPase, the immune complex is released from the internalizing FcγR and subsequently bound by FcRn. FcRn then directs the antigen within the immune complex into antigen processing pathways conducive to the generation of epitopes for loading onto both MHC class I and MHC class II molecules. Thereby, the same APC can effectively prime both CD4⁺ and CD8⁺ T cells in FcRn-dependent pathways.

Figure 4

Structure of IgG Fc-fusion proteins and mutations in the Fc region to modify FcRn binding. In typical IgG fusion proteins, the C-terminus of the effector molecule is fused to the N-terminus of the hinge region, followed by the CH2 and CH3 domain of human IgG. The effector molecule can be fused as a monomer (i) or a dimer (ii). Apart from full receptors or ligands as the effector molecules, bioactive peptide sequences (iii) have been successfully generated as Fc-fusion proteins. In order to generate improved therapeutics, various amino acid mutations, singly or in combination, have been introduced into the Fc region of human IgG that allow for enhanced binding to FcRn, thereby extending the therapeutic half-life of the fusion proteins. Black labeled residues: mutations with enhanced FcRn binding, grey labeled residues: mutations with decreased FcRn binding. For details see Supplementary Table 2.

Figure 5

FcRn-mediated transcytosis across polarized epithelial monolayers. In polarized epithelia such as intestinal epithelial cells, IgG molecules and Fc-fusion proteins can be recycled from the basolateral and apical membrane, from which FcRn rapidly directs IgG molecules into the apical and basal early endosomes (AEE and BEE) and the common recycling endosome (CRE). As such, FcRn is a receptor capable of bidirectional transcytosis. From the CRE, the actin motor myosin Vb and the GTPase Rab25 regulate a sorting step that specifies transcytosis in both directions without affecting recycling (Tzaban et al., 2009). Rab11a as a further regulatory component of the RE regulates a sorting step for recycling FcRn to the basolateral membrane, but it is dispensable for transcytosis (Tzaban et al., 2009). Thus the recycling and transcytosis pathway for IgG are distinct in polarized cells and display an inherent polarity.