Structural characterization of a human Fc fragment engineered for extended serum half-life

Abstract

The first three-dimensional structure of a human Fc fragment genetically engineered for improved pharmacokinetics properties is reported. When introduced into the C_H2 domain of human immunoglobulin G (IgG) molecules, the triple mutation M252Y/S254T/T256E (‘YTE’) causes an about 10-fold increase in their binding to the human neonatal Fc receptor (FcRn). This translates into an almost 4-fold increase in the serum half-life of YTE-containing human IgGs in cynomolgus monkeys. A recombinantly produced human Fc/YTE fragment was crystallized and its structure solved at a resolution of 2.5 Å using molecular replacement. This revealed that Fc/YTE three-dimensional structure is very similar to that of other human Fc fragments in the experimentally visible region spanning residues 236–444. We propose that the enhanced interaction between Fc/YTE and human FcRn is likely mediated by local effects at the substitutions sites. Molecular modeling suggested that potential favorable hydrogen bonds along with an increase in the surface of contact between the two partners may account in part for the corresponding increase in affinity.

Introduction

Control of the serum half-life of immunoglobulin G (IgG) molecules in mammals is achieved through the interaction of their Fc portion with the neonatal Fc receptor (FcRn). FcRn is structurally related to the class I major histocompatibility complex (Simister and Mostov, 1989, Burmeister et al., 1994). It recycles IgGs within endothelial cells and rescues them from a degradative pathway (Brambell et al., 1964, Junghans and Anderson, 1996, Ghetie and Ward, 2000, Roopenian and Akilesh, 2007). The pH dependent binding of IgGs to FcRn lies at the center of this salvage mechanism in which the corresponding association constants range from highest at acidic pH to lowest around neutral pH (Rodewald, 1976, Raghavan et al., 1995).

Various strategies have explored the effects of modulating the affinity of IgG molecules to FcRn on their serum persistence in vivo. In particular, several mutagenesis studies have targeted human Fc regions in an effort to decrease their binding affinity to human or murine FcRn at acidic pH. The serum half-lives of such engineered molecules were significantly reduced in mice expressing endogenous (Kim et al., 1999) or human (Petkova et al., 2006) FcRn. Conversely, various Fc mutations have been described which resulted in significant increases in human or mouse IgG Fc binding to mouse (Ghetie et al., 1997), rhesus monkey (Hinton et al., 2004, Hinton et al., 2005) and cynomolgus monkey (Dall’Acqua et al., 2006) FcRn. In this situation, the modified IgG molecules exhibited significantly improved serum half-life in the corresponding hosts. One particular set of substitutions, M252Y/S254T/T256E (referred to as ‘YTE’ thereafter), resulted in an about 10-fold pH dependent increase in the binding of various humanized IgGs to both human and cynomolgus monkey FcRn at pH 6.0 (Dall’Acqua et al., 2002, Dall’Acqua et al., 2006). When dosed in cynomolgus monkeys, the serum half-life of a YTE-modified humanized IgG was increased by nearly 4-fold when compared with its unmutated counterpart (Dall’Acqua et al., 2006). The introduction of YTE into therapeutic IgGs could potentially provide many benefits such as the ability to decrease their administration frequency or dosing requirements.

We set out to decipher the structural consequences of introducing YTE into a human Fc and the molecular mechanisms by which YTE enhances the interaction of human IgG molecules with human FcRn. For this purpose, we solved the X-ray crystallographic structure of a recombinantly produced human IgG1 Fc fragment containing the YTE set of substitutions. This constitutes the first three-dimensional structure of a human Fc fragment specifically engineered for increased binding to FcRn and improved pharmacokinetics properties.