title: Inhibition of glycosylation on a camelid antibody uniquely affects its FcγRI binding activity.
abstract: Glycoengineering of mAbs has become common practice in attempts to generate the ideal mAb candidate for a wide range of therapeutic applications. The effects of these glycan modifications on the binding affinity of IgG mAbs for FcγRIIIa and their cytotoxicity are well known. However, little is understood about the effect that these modifications have on binding to the high affinity FcγRI receptor. This study analyzed the effect of variable N-glycosylation on a human-llama hybrid mAb (EG2-hFc, 80kDa) binding to FcγRI including a comparison to a full-sized IgG1 (DP-12, 150kDa). This was achieved by the addition of three glycosylation inhibitors (swainsonine, castanospermine, and kifunensine) independently to Chinese hamster ovary (CHO) cell cultures to generate hybrid and high mannose glycan structures. Biophysical analysis by circular dichroism, dynamic light scattering and analytical ultra-centrifugation confirmed that the solution-behaviour of the mAbs remained constant over multiple concentrations and glycan treatments. However, changes were observed when studying the interaction of FcγRI with variously glycosylated mAbs. Both mAbs were observed to have a decreased binding affinity upon treatment with swainsonine which produced hybrid glycans. Following de-glycosylation the binding affinity for EG2-hFc was only marginally reduced (6-fold) compared to a drastic (118-fold) decrease for DP-12. In summary, our data suggest that the relatively low molecular weight of chimeric EG2-hFc may contribute to its enhanced stability against glycan changes making it a highly suitable mAb candidate for therapeutic applications.
authors.affiliation: Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
authors.forename: Natalie
authors.initials: N
authors.lastname: Krahn
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
authors.forename: Maureen
authors.initials: M
authors.lastname: Spearman
authors.affiliation: Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
authors.forename: Markus
authors.initials: M
authors.lastname: Meier
authors.affiliation: Department of Chemical Engineering, École Polytechnique de Montréal, Montréal, Québec H3T 1J4, Canada.
authors.forename: July
authors.initials: J
authors.lastname: Dorion-Thibaudeau
authors.affiliation: Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
authors.forename: Matthew
authors.initials: M
authors.lastname: McDougall
authors.affiliation: Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta T1K 3M4, Canada.
authors.forename: Trushar R
authors.initials: TR
authors.lastname: Patel
authors.affiliation: Department of Chemical Engineering, École Polytechnique de Montréal, Montréal, Québec H3T 1J4, Canada.
authors.forename: Gregory
authors.initials: G
authors.lastname: De Crescenzo
authors.affiliation: National Research Council of Canada, Montréal, Québec H4P 2R2, Canada.
authors.forename: Yves
authors.initials: Y
authors.lastname: Durocher
authors.affiliation: Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
authors.forename: Jörg
authors.initials: J
authors.lastname: Stetefeld
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada. Electronic address: butler@cc.umanitoba.ca.
authors.forename: Michael
authors.initials: M
authors.lastname: Butler
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D016466:CHO Cells; D002161:Camelids, New World; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary; D017452:Receptors, IgG / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D017452:Receptors, IgG
keywords: Analytical ultracentrifuge; CD spectroscopy; Dynamic light scattering; Glycosylation inhibitor; Monoclonal antibody; N-linked glycosylation; Surface plasmon resonance