Please use this identifier to cite or link to this item: https://scholarbank.nus.edu.sg/handle/10635/134869
Title: ENHANCING THE EFFICACY OF RECOMBINANT ANTIBODIES BY MANIPULATING THE GLYCOSYLATION PATHWAY IN CHO CELLS
Authors: CHAN KAH FAI
Keywords: Chinese hamster ovary (CHO) cells, GDP-fucose transporter (SLC35C1), Genome editing technologies, Glutamine synthetase, antibody glycosylation, ADCC
Issue Date: 20-Oct-2016
Citation: CHAN KAH FAI (2016-10-20). ENHANCING THE EFFICACY OF RECOMBINANT ANTIBODIES BY MANIPULATING THE GLYCOSYLATION PATHWAY IN CHO CELLS. ScholarBank@NUS Repository.
Abstract: Antibody glycosylation affects Fc-mediated effector functions such as antibody dependent cellular cytotoxicity (ADCC). It is known that lack of core α1,6-fucose on IgG1 Fc N-glycan enhances ADCC efficacy by up to 100-fold. But knowledge of antibody efficacy beyond the lack of fucose remains limited. Recombinant therapeutic IgGs produced from Chinese Hamster Ovary (CHO) cells are fucosylated and highly heterogeneous with variable galactosylation. This affects batch-to-batch consistency and drug efficacy. Using genome-editing technologies and FACS approach, I have successfully engineered a CHO mutant cell line deficient in GDP-fucose transporter, CHO-gmt3 and confirmed that antibodies produced by CHO-gmt3 are completely fucose-free. Importantly, removal of GDP-fucose transporter function in CHO cells does not affect cell growth and antibody productivity. Through lectin selection and genome editing technologies, a panel of different CHO glycosylation mutant cell lines was generated to produce antibodies with more homogeneous and defined glycoforms. The glutamine synthetase (GS) gene was further inactivated by TALENs in these mutants to generate GS system for antibody expression. Detailed glycan characterization of rituximab produced by the various CHO mutants provides an in-depth verification on the antibody glycan structures present. In vitro ADCC activities of CHO mutant-produced rituximab against human B lymphoma cell line, WIL2-S were then assessed. Data from this comprehensive study has provided further insight into the glycan structure and sugar requirement for ADCC effector function. Rituximab produced by two CHO mutant cell lines, CHO-gmt3 and CHO-gmt9 showed highest enhanced in vitro ADCC activity.
URI: http://scholarbank.nus.edu.sg/handle/10635/134869
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