A C-terminal interdomain disulfide bond significantly stabilizes the Fc fragment of IgG

We describe the stabilization of human IgG1 Fc by an engineered interdomain disulfide bond at the C-terminal end of the molecule. Covalently interconnecting the C-termini of the CH3 domains led to an increase of the melting temperatures by 5.6 and 9.1 degrees C respectively as compared to CH3 domains in the context of the wild-type Fc. Combined with a recently described additional intradomain disulfide bond, both novel disulfide bonds led to an increase of the Tm by about 18.1 degrees C to 100.7 degrees C. The interdomain disulfide bond had no impact on the thermal stability of the CH2 domain. Far- and near-UV CD spectroscopy showed very similar overall CD profiles, indicating that secondary and tertiary structure of the Fc was not negatively affected. When introduced into an Fc fragment that had been engineered to bind to Her2/neu via a novel antigen binding site located at the C-terminus of the CH3 domain, the novel inter- and intra-domain bonds also brought about a significant increase in thermostability. Using them in combination, the Tm of the CH3 domain was raised by 18 degrees C and thus restored to the Tm of the wild-type CH3 domain. Importantly, antigen binding of the modified Fc was not affected by the engineered disulfide bonds. (C) 2012 Elsevier Inc. All rights reserved.