Generation of FX^−/− and Gmds^−/−CHOZN host cell lines for the production of afucosylated therapeutic antibodies

Abstract

Antibody-dependent cellular cytotoxicity (ADCC) is the primary mechanism of actions for several marketed therapeutic antibodies (mAbs) and for many more in clinical trials. The ADCC efficacy is highly dependent on the ability of therapeutic mAbs to recruit effector cells such as natural killer cells, which induce the apoptosis of targeted cells. The recruitment of effector cells by mAbs is negatively affected by fucose modification of N-Glycans on the Fc; thus, utilization of afucosylated mAbs has been a trend for enhanced ADCC therapeutics. Most of afucosylated mAbs in clinical or commercial manufacturing were produced from Fut8 −/− Chinese hamster ovary cells (CHO) host cells, generally generating low yields compared to wildtype CHO host. This study details the generation and characterization of two engineered CHOZN® cell lines, in which the enzyme involved in guanosine diphosphate (GDP)fucose synthesis, GDP mannose-4,6-dehydratase (Gmds) and GDP-L-fucose synthase (FX), was knocked out. The top host cell lines for each of the knockouts, FX−/− and Gmds−/−, were selected based on growth robustness, bulk MSX selection tolerance, production titer, fucosylation level, and cell stability. We tested the production of

Strategies for controlling afucosylation in monoclonal antibodies during upstream manufacturing

“…Protein expression regulation reduces the cell line's ability to conduct fucosylation by targeting genes associated with core fucosylation. Several proteins in the fucosylation metabolic pathway are popular targets for down regulation or knockout including GDP‐mannose 4,6‐dehydratase (GMD), [ 27–29 ] GDP‐L‐fucose synthase (GFS), [ 17,28 ] and FUT8 [ 29,30 ] to generate products with reduced or negligible levels of fucosylation. Genetic engineering can target proteins either individually or in combination to improve inhibition of core fucosylation and increase levels of afucosylation.…”