Akira Okazaki1, Emi Shoji-Hosaka1, Kazuyasu Nakamura1, Masako Wakitani1, Kazuhisa Uchida1, Shingo Kakita1, Kouhei Tsumoto2, Izumi Kumagai2 and Kenya Shitara1
1Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, Japan; Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Aoba-ku, Sendai 980-8579, Japan
Abstract: Lack of fucose on human IgG1 oligosaccharide improves its affinity for Fcy receptor Ilia (FcyRIIIa). To address the mechanisms of affinity improvement by the defucosylation, we used isothermal titration calorimetry (ITC) and biosensor analysis with surface plasmon resonance. ITC demonstrated that IgG1-FcyRIIIa binding was driven by favorable binding enthalpy (AH) but opposed by unfavorable binding entropy change (AS). Lack of fucose on IgG1 enhanced the favorable AH, leading to the increase in the binding constant of IgG1 for the receptor by a factor of 20 to 30. The increase in the affinity was mainly attributed to an enhanced association rate. A triple amino acid substitution in IgG1, S298A/E333A/K334A, is also known to improve IgG1 affinity for FcyRIIIa. ITC demonstrated that the amino acid substitution attenuated the unfavorable AS, resulting in a 3 to 4-fold increase in the binding constant. The affinity enhancement by the amino acid substitution was due to a reduced dissociation rate. These results indicate that the mechanism of affinity improvement by the defucosylation is quite distinct from that by the amino acid substitution. Defucosylated IgG1 exhibited higher antibody-dependent cellular cytotoxicity (ADCC) than S298A/-E333A/K334A-IgG1, showing a correlation between IgG1 affinity for FcyRIIIa and ADCC.
Key words: Isothermal titration calorimetry, surface plasmon resonance, antibody-dependent cellular cytotoxicity
Was this article helpful?