Fucose removal from complex-type oligosaccharide enhances the antibody-dependent cellular cytotoxicity of single-gene-encoded bispecific antibody comprising of two single-chain antibodies linked to the antibody constant region.

Bispecific antibodies (bsAbs) have the potential to extend binding selectivity, increase avidity and exert potent cytotoxicity due to the combination of dual specificities. scFv 2 -Fc type of single-gene-encoded bispecific antibody, composed of two different single-chain Fvs and an Fc, has been reported to be capable of binding to different antigens. The aim of this study was to determine the effect of fucose removal on effector functions of scFv 2 -Fc since fucose depletion from oligosaccharide of human IgG1 and scFv-Fc results in significant enhancement ofADCC. We generated novel single-gene-encoded bsAb with dual specificity against tumor associated glycoprotein (TAG)-72 and MUC1 mucin as fucose-negative scFv 2 -Fc from α-1,6-fucosyltransferase knock-out CHO cells and a highly fucosylated scFv 2 -Fc comparator from parental CHO cells. Expression, assembly and the antigen-binding activity of the scFv 2 -Fc were not influenced by removal of fucose. The fucose negative scFv 2 -Fc bound with higher avidity to FcyRIIIa and enhanced ADCC compared to the highly fucosylated scFv 2 -Fc. These results demonstrate that ADCC-enhancement by removal of fucose is effective in not only whole IgG1 and scFv-Fc, but also scFv 2 -Fc targeting two different antigens, and thus increases the potential of fucose-negative scFv 2 -Fcs as novel therapeutic candidates.