Structural consequences of aglycosylated IgG Fc variants evolved for Fc gamma RI binding

Abstract

In contrast to the glycosylated IgG antibodies secreted by human plasma cells, the aglycosylated IgG antibodies produced by bacteria are unable to bind Fc gamma Rs expressed on the surface of immune effector cells and cannot trigger immune effector functions. To avoid glycan heterogeneity problems, elicit novel effector functions, and produce therapeutic antibodies with effector function using a simple bacterial expression system, Fc gamma RI-specific Fc-engineered aglycosylated antibodies, Fc11 (E382V) and Fc (E382V/M428I), containing mutations in the CH3 region, were isolated in a previous study. To elucidate the relationship between Fc gamma RI binding affinity and the structural dynamics of the upper CH2 region of Fc induced by the CH3 mutations, the conformational variation of Fc variants was observed by single-molecule Forster resonance energy transfer (FRET) analysis using alternating-laser excitation (ALEX). In sharp contrast to wild-type Fc, which exhibits a highly dynamic upper CH2 region, the mutations in the CH3 region significantly stabilized the upper CH2 region. The results indicate that conformational plasticity, as well as the openness of the upper CH2 region, is critical for Fc gamma R binding and therapeutic effector functions of IgG antibodies. (C) 2015 Elsevier Ltd. All rights reserved.