Fine tuning expression levels of α-mannosidase II (MANII) and β-1,4-N-acetylglucosaminyl- transferase III (GNTIIII) in CHO cells for production of IgG1 antibody with tailored Fc-mediated effector functions

Abstract

Abstract Monoclonal antibodies (mAbs) eliminate cancer cells via various effector mechanisms including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which are determined by the structures of N-glycans on the Fc region of mAbs. Controlling the glycan structures on mAbs enables improving the therapeutic benefits of different effector functions. Traditional gene deletion or overexpression, which often result in only all-or-nothing changes, are not able to modulate the expression of multiple genes to defined ratios and levels. In this work, we developed a CHO cell engineering platform that allows precise modulation of multiple genes’ expression to custom design N-glycan profiles of mAbs for optimizing effector functions. A CHO targeted integration system with two independent landing pads was constructed, which allows expression of multiple genes at two pre-determined genomic sites. This platform was combined with internal ribosome entry site (IRES)-based polycistronic vectors to simultaneously fine-tuning the expression of α-mannosidase II (MANII) and chimeric β-1,4-N-acetylglucosaminyl-transferase III (cGNTIII) genes in CHO cells to produce mAbs carrying N-glycans with various levels of bisecting and non-fucosylated structures. These mAbs were confirmed to feature different degrees of ADCC and CDC, allowing mAbs with optimal effector functions to be identified. This platform was demonstrated as a powerful tool for producing antibody therapeutics with tailored effector functions via precise engineering of N-glycan profiles.