Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcy receptor binding and antibody-dependent cell-mediated cytotoxicity activities

The presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward FcyRIIIa as well as their antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, the quantitative nature of this structure-function relationship remains unclear. In this study, the in vitro biological activity of an afucosylated antiCD20 antibody was fully characterized. Further, the effect of fucose reduction on Fc effector functions was quantitatively evaluated using the afucosylated antibody, its regular fucosylated counterpart and a series of mixtures containing varying proportions of regular and afucosylated materials. Compared with the regular fucosylated antibody, the afucosylated antibody demonstrated similar binding interactions with the target antigen (CD20), C1q and FcyRIa, moderate increases in binding to FcyRIIa and IIb, and substantially increased binding to FcyRIIIa. The afucosylated antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity. Based on EC50 values derived from dose- response curves, our results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcyRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the extent of ADCC enhancement due to fucose depletion was not affected by the FcyRIIIa genotype of the effector cells.